Some Protein-Chemical Aspects of Tanning Processes

Some Protein-Chemical Aspects of Tanning Processes

BY K. H. GUSTAVSON Swedieh Tanning Research Inutituts, Stockholm, Sweden

CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. The Skin . . . . . . . . . . . . .

paqc 354

I I. Chemistry of Collagen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6. Coordinative Reactions..

........

. . . . . . . . . . . . . . . 368 . . . . . . . . . . . . . . .369 . . . . . . . . . . . . . . . . . . 378

1. Structure of Basic Chromic Salts of Importance in Tanning.. 2. Factors Governing the Tanning Effect.. . . . . . . .

. . . . . . 379

a. Nature of the Anion... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

381

b. Basicity of Chromic Salt.. .................... c. Concentration of Chromium Salt.. . . . . . . . d. Neutral Salt Effect.. . . . . . . . . . . . . . . . . . . . . . . . . e. Hydrogen Ion Concentration.. f. Influence of Previous History of 3. Nature of Chrome-Collagen Compound.. ........................ 388 4. Some Important Properties of Chrome Leather of Theoretical Interest 392

.

.......................

396

c. Degree of Stabilization. ....

VIII. Tanning Power of Aldehydes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Tanning with Formaldehyde. . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. General Aspects of the Reaction.. ..........................

353

406

354

K. H. QUBTAVSON b. Participation of Lysine Groups. . . . . . . . . . .

c. Reaction of Arginine Gtoups.. . . . . . . . . . . . . . . . d. Participation of Peptide and Other Groups. . . . . . e. Influence of Solvent. . . . . . . . . . . . . . . . . f. Ewald Reaction., . . . . . . . . . . . . . . . . . . . . . . . . . IX. Quinone Tannage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. General Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Page 406 . 407

.

402)

411 41 1 . 411 413 415

I. INTRODUCTION At an early stage of civilization, the art of tanning was discovered. By tanning skin is converted into a material possessing the valuable properties of resisting water and putrefaction and of remaining soft and pliable. By incorporation of tanning agents within the hide structure, suitably pretreated and conditioned, leather is formed. A great many substances of different types and chemical compositions possess tanning potency, e.g., vegetable tannins, unsaturated oils, aldehydes, and, preeminently, basic chromium salts. Theoretically, tanning connotes stabilization of proteins by their irreversible combination with tanning agents. Practically, tanning may be defined as a process making the putrescible proteins of hide microbiologically and hydrothermally resistant, preserving certain properties of the original fiber structure, and, further, imparting certain desirable mechanical properties to the hide. The main operations in the preparation of hide for tanning are briefly as follows. The first step is the removal of salt and other extraneous matter from the salted hide by soaking it in water, restoring the natural water content of the fresh hide. In the subsequent liming process, the epidermal and subcutaneous layers are removed and certain accessory proteins dissolved or modified. The final result is the leather-forming pelt, mainly containing collagen, which after further conditioning is made neutral and simultaneously brought to the state of minimum swelling. The pelt is then ready for the tanning proper. 1. The Skin Since animal skin is the basis of leather, some knowledge of its microstructure is essential for an appreciation of the complicated reactions involved in tanning. The skin protein chiefly concerned in ordinary tanning is collagen, which forms the main part of the skin undergoing tanning, the corium or dermis. The major part of the raw material is of bovine origin. The skin of mammals (211) consists of three distinctive layers: ( I ) epidermis, ( 2 ) corium or skin substance, and ( 3 ) subcutaneous tissue. The epidermis and subcutaneous tissue are removed in the preparatory processes. The remaining corium consists of bundles of collagen fibers interwoven in all directions. The collagen fibers of mammalian hide are arranged in bundles. Fibrils make up the fiber, which appears to be interwoven in all directions. In bovine skin the fiber

PROTEIN-CHEMICAL ASPECTS OF TANNINQ

355

bundles generally are 50-100 p thick, the fibers about 25 p, and the fibrils 1 p or less in diameter. The channels between the bundles are fairly wide, averaging a few microns. In the pretreatmerfts for conditioning the hide for tannage, the space may be narrowed or widened according to the properties desired. The removal of interfibrillar proteins, albumins, globulins, and mucoids in the pretreatment8 tends to increase the permeability of the skin. By swelling and by introduction of large molecules into thc structure in the form of tanning agents the size of the channels is also altered. The influence of the internal structure of skin is important in tanning processes, topochemical factors being prominent. In practical tanning, the additional complication of uneven distribution of the tanning agent throughout the interior of the hide is caused by the polydispersity of the solutions of many important tanning agents, e.g., the vegetable tannins. This complication may be largely eliminated in laboratory studies by increased subdivision of the substrate, the collagen being used in the form of hide powder or single fibrr bundles. Single bundles are preferably applied in studies of theoretical problems by means of physicochemical and mechanical methods, since the macroweave s t h c t u r e is thus eliminated. The implication of the twophase systems with accompanying difficulty of attaining true equilibria must be borne in mind.

2. General Structure of Fibrous Proteins

The classification of the proteins into two maiu groups, the globular and the fibrous, is made on the basis of the arrangement of the protein chains, the peptide chains of fibrous proteins being more or less extended and oriented in parallel, whereas the units of the globular proteins are folded, no preferred direction of orientation being evident. The properties and reactivity of fibrous proteins are primarily governed by their chemical composition, with due regard given t o the organization of the protein structures. This fact is clearly brought out by comparing collagen and its secondary product, gelatin, which differ markedly with respect to their physical properties although their compositions are identical. The different degrees of organization of the peptide chains and the higher units account for the differences between collagen and gelatin. The collagen units are arranged in parallel and stabilized by valency forces between adjacent peptide chains and micelles (cross links). In the conversion of collagen into gelatin, some of these cross links are broken, resulting in shortening and disorganization of the protein chains. The aspect of organization of proteins was early recognized by Jordan Lloyd (112). Attention was called t o the interrelationship of chemical composition, degree of stabilization of the units, and the physicochemical reactivity of proteins, particularly the degree of swelling (water imbibition) a t the p H of maximum swelling and at the p H of minimum swelling (isoclectric range). The data of Table I (112) illustrate this point for some typical fibrous proteins (silk fibroin, keratin, collagen, gelatin, and

356

K . H. QUSTAVSON

muscle protein). The acid-binding capacities of the proteins mentioned are included in order t o show that the degree of swelling by acids is not a simple function of the amount of acid-binding protein groups. It is TABLE I Organization of Proteins and Degree of Water Imbibition (Swelling) -.

Protein

Silk fibroin Keratin Collagen Gelatin Muscle protein

-

Total Total bases, dicarboxylic acids, % 1 11 15 15 17

15 17 17

20

bfilliequivalents HCI fixed per g. protein 0.1 0.8 0.9 0.9 1 .o

-

Degree of swelling, % water taken up PH 5 20

22

30

35 490

260

-

1300 300

6500 1400

evident that fibrous proteins, mainly built up from amino acids with nonpolar groups, possess low affinity for water, whereas proteins containing numerous polar groups take up water readily. However, collagen and keratin, with practically the same degree of polarity and acid-binding capacity, show great differences in water uptake and degree of swelling. The different internal organizations of these proteins explains their behavior. Keratin contains the covalent cystine bridge, which resists the action of acids, maintaining the rigid structure in spite of the electrostatic repulsions of those protein groups which are positively charged in acid solutions. In collagen, on the other hand, the negatively charged R groups, t o a large extent internally compensated by electropositive k groups, forming saltlike cross links, are discharged and the cross links broken. By this disorganization of the chains, some of the second type of cohesive forces of the collagen structure, the coordinate cross links on the peptide groups (hydrogen bonds), are probably ruptured. Water is added to the protein by polar forces and by association on the coordinate bci of the peptide groups set free by the contraction of the main chains. The effective stabilization of keratin is due t o the disulfide bridges. This is evident from the fact that, on breaking the covalent bridge, the resulting products, the keratoses, swell in acid solution t o a degree comparable to that of collagen.

11. CHEMISTRY OF COLLAQEN 1. General Aspects

The amino acid composition of collagen (and gelatin) is more completely known than the compositions of most proteins. The early work

357

PROTEIN-CHEMICAL ASPECTS OF TANNING

was chiefly carried out on gelatin (10,28). In recent years determinations of the amino acid composition of collagen of known previous history and high degree of purity have been made by specific methods (24). The present discussion of the behavior of collagen as a dipolar structure was originally based upon the data of the amino acid composition of native collagen given by Chibnall in 1946 (24). Since this chapter was written, revised values obtained by the Cambridge School of Biochemistry in collaboration with the British Leather Manufacturers' Research Association have been published. Bowes and Kenten (14a) have tabulated the most probable values of the amino acid composition of native collagen of ox hide which had received no alkaline or enzymic treatment (Table 11). The total nitrogen content was 18.6%. The total nitrogen and nmide contents of this pure preparation of TABLE I1 Amino Acid Composition of Native Unlimed Collagen

Amino acid Amino nitrogen Glycirie Alanine Leucine Isoleucine Valine Phenylalanine Tyrosine Tryptophan Serine Threonine Cystine Methionine Proline Hydroxyproline Lysine Hydroxylysine Arginine Histidine Aspartic acid Glutamic acid Amide nitrogen Total found

1

0

b

N as % G. residues protein N per 100 g.

Mmol. per g.

Assumed Apparent number of minimum residues

mol. wt.

2.5 26.3 8.0

0.33 3.50 1.06

-

-

19.9 7.6

136 41

38,880 38,580

3.2

4.8

0.42

17

39,950

2.2 1.9 0.6 0.0 2.5 1.5 0.0 0.4 9.9 8.Oa 4.7 1.2 15.3 1.2 3.6 5.8 3.5 99.8

2.9 3.7 1.3 0.0 2.7 2.0 0.0 0.7 12.7 12.1 4 .O 1.1 7.9 0.7 5.5 10.0

0.29 0.25 0.08 0.00 0.33 0.20 0.00 0.05 1.32 1.07 0.31 0.08 0.51 0.05 0.47 0.77 0.47 10.76'

11 10 3

37,620 39,600 37,620

8

39,130 40,160

Determined on gelatin. Excluding amide nitrogen.

-

-

99.6*

13 -

2 51 41 12 3 20 2 19 30 18 419

-

-

37,640 38,760 38,580 38,400 37,680 39,380 37,640 39,750 38,910 38,740 38,730 (mean)

358

K. H. GUSTAVSON

native collagen are higher than those previously reported. Since these figures account for over 99% of the total nitrogen of collagen, it appears as the analysis of collagen is virtually complete. The amino acid composition of gelatin is nearly the same as that of collagen; the main difference is the low content of amide nitrogen of gelatin (0.5%). The new values of dicarboxylic acids were obtained by microbiological assays and the remainder by the chromatographic technique. In comparing these data t o the older values, the main differences are found in the contents of glutamic and aspartic acids, which are nearly twice as large as the earlier values. This will radically affect the proportion of basic (cationic) t o acidic (anionic) groups changing the ratio from 1.5:1 to 1 : 1.3. The adjusted figures of the dicarboxylic acids, and the resulting ratio of basic to acidic groups with due consideration of the deamidation of collagen taking place in the alkali treatment, remove the paradox that gelatin and limed collagen are isoelectric in the acid p H range, although according t o the old values they should contain a large excess of cationic groups. On the other hand the new data make the discrepancy between the figures obtained for basebinding capacity of collagen and its content of free carboxyl groups still wider. The minimum molecular weight calculated from the figures of Table I1 is about 39,000 and the average residue weight as 92.6, which figure exactly agrees with thc value obtained by Chibnall from the nitrogen distribution (23). Collagen contains a well-balanccd proportion of posit ivcly and negatively charged polar groups, securing a fair degree of ionic reactivity. This function is particularly important for its behavior in the preliminary processes of tanning and in the tanning processes proper. Characteristic for the collagen group of proteins are the large content of nonpolar amino acids, glycine and alanine, composing about a third of the total and, above all, the prominence of prolinc and hydroxyproline and the paucity of aromatic residues. The large number of prolines, built into the chains links, introduces a rcguliLr intcrrupwith the formation of -("()-If= tion of the regular -CO-NfIlinks formed hy the remainder of the amino acid residues. I t is also interesting to note that hydroxyl groups, mainly contributed by hydroxyproline and serine, form nearly one-half the total number of polar groups. The function of these groups, containing residual negative charge on oxygen (183), in the reactions of tanning agents with collagen has not yet been considered. Probably they are of importance as electrostatic centers and as hydrogen-bonding loci in coordination of vegetable tannins and chromic salts in these tannages. From investigations of collagen by X ray and electron microscope the

PROTEIN-CHEMICAL ASPECTS OF TANNING

359

presence of protein chains aligned in parallel has been experimentally proved; they are nearly completely stretched. The X-ray analysis shows a meridional spacing of about 2.9 b, corresponding to the average length of an amino acid unit in the length of the chain (3). Since the length of one residue of a fully stretched protein chain (silk fibroin) is 3.5 b (156), it is evident that the main chain of collagen is slightly contracted. This curling of the chains is probably caused by the presence of large proline and hydroxyproline residues and is thus of steric nature (3). The long spacing of about 640 b reported by Rear (6) may be taken as a measure of the over-all length of the molecule. Two equatorial spacings are of special significance: a regular lateral spacing of about 11-15 b, according to the degree of hydration of the fiber, and a less regular one of about 4.5 A, corresponding to the distance between side chains and between backbones, respectively (2). In the electron microscope collagen shows a banded structure (98,162, 179,180). Two distinct phases of collagen, one fully stretched and one with contracted chains, are indicated. The average spacing from one dark band to the next is 645 A, closely corresponding to the long meridional spacing found by X-ray analysis (6). The unexpected discovery of the large extensibility of collagen fibrils (179), not shown by bundles of fibers, is in line with the presrnce of folded sections in the chains. The possibility of the presence of different chains, each with a certain amino acid pattern associated by means of cross links-the molecular grid type of structure, a s indicated in insulin and other proteins according to Chibnall and coworkers (23)-is also of interest in this connection. The following presentation will be based upon the established concept that collagen contains parallel protein chains of the general form: -C €1--N H-CO-C H-

I

Ri

I

R*

The cohesion of the higher units is due to the attraction of oppositely charged side chains and to hydrogen bonds acting between prptide groups of adjacent chains. I n Astbury’s (3) model of collagen, obtained by interpretation of X-ray data and from figures of amino acid composition, the molecule is considered to be composed of parallel chains, practically fully extended, in which glycine rrsidues represent every third residue, proline and hydroxyproline anothcr third, the rest of the residues accounting for the remaining third. The presence of imino links in every third residue (proline and hydroxyproline being built into chain forming =N-COlinks) is considered to limit the rotational possibility about these atoms, leading to a spiral or slightly folded configuration of tlie chains a t these points. The bulky proline groups arc considered to be

360

K. H. OUSTAVSON

held on one side of the chain and the remaining R groups on the opposite side (3). A general concept may also be based upon Huggins’ model of collagen (109,168) with a spiral configuration of the main chain, the side groups projecting alternately above and below the main chain. The chains are considered to be grouped into layers, the individual chains held together mainly by means of hydrogen bonds, supplemented by electrostatic attraction of directed and undirected valency forces. Astbury’s (2) deductions led to the concept of periodicity of the protein chains. This hypothesis was further advanced by Bergmann and Niemann (11). The isolation of a tripeptide lysylprolylglycine from gelatin by Grassmann and Riederle ( 5 2 ) strengthened the periodicity claim. However, Chibnall and his school (23) have proved the limitations of the simple whole number rule applied to analytical data of proteins of known composition. The weakness of the periodic concept from the statistical and probability point of view has also been demonstrated (163) and further data obtained by the chromographic method of Martin and Synge (144,145) do not support the original postulate of periodicity. However, a certain sequence of residues seems probable. For a more precise theory information is needed regarding the general sequence of the amino acids in the subunits, the location of the reactive R groups and their spatial relation. However, these fine structural details are unknown. Additional evidence for the lattice structure of collagen is furnished by the birefringence of the fibers. The collagen fiber shows positive double refraction (119). By the incorporation of various tanning agents into the structure, the degree and sign of the birefringence are in many instances altered (121). Some tanning agents, e.g., the vegetable tannins may cause a shift from positive to negative values. Marriott (142) has found some relationship between the value of the birefringence of vegetabletanned collagen fibers (leather) and their abrasional resistance. Further, Kiintzel (121) asserts that it is possible from the study of the birefringence of tanned fibers to prove the occurrence of intra- or intermicellar types of combination of tanning agents with collagen. Thus, Kiintzel concludes, from the inversion in sign of the birefringence of collagen upon vegetable tannage, t h at the tannins are incorporated intramieellarly (121,126). The type of micellar reactions is important in tannage, particularly in chrome tanning. Electron microscope studies may give needed information (see, e.g., 122 and 162).

2. Stabilization of Collagen The cohesive forces of mammalian collagen, inter- and intramicellar, are generally considered to be of two main types, directed valency and undirected valency. The former consists of: (1) electrovalency, located

PROTEIN-CHEMICAL ASPECTS OF TANNINQ

361

a t polar R groups of the chains with opposite charge, forming a saltlike cross link, and ( 2 ) coordinate valency between the carbonyl groups of one chain and the imino group of an adjacent chain’s peptide group (hydrogen bond). The undirected valency is of the nature of electrostatic attraction or van der Waals forces. An evaluation of the relative importance of the two types of directed valency linkages may be obtained by measuring the hydrothermal stability of collagen (its shrinkage temperature) (68), after treatment with agents which are specific for the groups involved. Thus, P-naphthalenesulfonic acid will inactivate the basic groups completely, thereby eliminating the saltlike cross links, without swelling collagen and without interfering with the coordinate activity of the peptide groups (71). The change of shrinkage temperature caused by such inactivation may be applied for the evaluation of the degree of cohesion of the collagen lattice due to electrovalent interaction of polar R groups. The shrinkage temperature of mammalian collagen (65-68”C.) is decreased 10-12°C. by this treatment (71). Further, by pretreatment of bovine collagen in solutions of agents with a specific action on peptide groups and their coordination centra, as, for example, concentrated solutions of urea, shrinkage occurs a t room temperature. This means a lowering of the shrinkage temperature of some 40°C. (71,llG). Accordingly, it is evident that the main stabilization forces of mammalian collagen belong to the type of hydrogen bonds. The data indicate that they are responsible for at least three-fourths of the internal cohesion, the rest being supplied by the saltlike cross links. (See further Section 7.) The tensile strength of the fiber bundles apparently is not simply a function of the strength of the bonds of the molecular units (93), the cohesion of the fibers being an additional factor. Hence, a direct comparison of the shrinkage temperature and the mechanical strength of fibers appears not to be permissible. (Cf. 93 and 101.) In fibers in equilibrium with water, its dipole nature will probably influence the internal cohesion, especially the strength of attraction between oppositely charged R groups, affecting the strength of the wet and dry fibers. The distance between the ionic groups is also widened by hydration of collagen (1 15). The fiber strength generally decreases with increased moisture content. Exceedingly drastic drying may lead to markedly increased strength, probably caused by conversion of ionic cross links to covalent links. Some water of constitution (“bound water”), which amounts to about 20% of the weight of collagen (107), must be lost in drying collagen to 12% water content. It is not known if this water is withdrawn from the polar groups or from the peptide groups. It has been assumed by some workers (108) that the water molecule forms the link between

362

K.

n.

GUSTAVSON

internally compensated peptide groups as

\

/

N.H.*-O.H..OC the / H additional water taken up by collagen being coordinated on the molecule of water, functioning as a vehicle for cross linking. Data on the strength of skin pieces cannot be used for a n evaluation of the influence of various types of processing, e.g., tanning, on the strength of the fibers, since the tensile strength measured on a strip of skin really gives the strength of the fiber weave. This aspect of the problem, with due consideration of the effect of various tanning agents on single fibers, was particularly stressed long ago by Jovanovits ( 1 1 3 14), ~ Chernov (22), and Grassmann (50), and recently by Highberger (101). I t is noteworthy that the tensile strength on a collagen basis is greater for vegetable-tanned skin than for chrome-.tanned skin. However, the strength of chrome-tanned .fibers (about 30 kg./mm.*) is about thrice that of vegetable-tanned jibers on the same weight of original collagen (22). In the former instance the macro structure (weave) is the main factor, whereas the strength of the fibers represents the molecular structure of the tanned collagen. The greater strength of the chrome-tanned fiber is in line with the prevailing concept of the degrees of efficiency of the cross linking in the two tannages. \I

3. Binding of Hydrogen and Hydroxyl Ions The maximum acid-binding capacity of collagen (HCl) is reached a t equilibrium pII of about 1 and amounts to 0.9 milliequivalent hydrogen ion per gram collagen, in good agreement with the number of equivalents of basic groups (8). The base-binding capacity is less than half the value expected from considerations of the newest figures of free dicarboxylic acids of collagen. The values are, for collagen, 0.4-0.5 milliequivalent hydroxyl ion per gram protein (8,127,129). Earlier discussions of the acid- and base-binding capacity of collagen have been based on a proportion of 1.5 to 2 basic groups per acidic group (63). The reason for tfhis large difference in the base-binding values of collagen reported (8,127,129), one one hand, and the theoretical values calculated from the content of free dicarboxylic acid, on the other hand, is not clear. The detcrminat,ion of the maximum fixation of alkali by collagen from sodium hydroxide has been carried out in the p H interval 12-13. This determination is subject to large errors. Some errors are inherent in the method and are due to the presence of carbon dioxide and the difficulty of pH determinations a t such high alkalinity. Other errors are due to chemical changes in the collagen at prolonged interaction of alkali such as formation of soluble degradation products and alteration of

PROTEIN-CHEMICAL ASPECTS OF TANNING

363

collagen. Recent figures of the binding capacity of collagen (isoelectric calfskin) for barium hydroxide, a t equilibrium p H values of 12-13, obtained by means of gravimetric analysis of the substrate in equilibrium with the hydroxide solution, are in the range 0 . 5 4 . 6 milliequivalent Ba++ per gram collagen (94). These figures are the final ones, after due correction for sorbed solution in the pressed substrate and the water of hydration of collagen. Another possibility is that collagen requires still higher concentrations of hydroxyl ions than those heretofore employed for attainment of maximum base fixation. The base-binding capacity of gelatin is considerably greater, showing values of 0.7-0.9 milliequivalent hydroxyl ion per gram gelatin (25). The difference of 0 . 3 4 . 4 milliequivalent hydroxyl ion between gelatin and collagen can only in small part be accounted for by the deamidation taking place in the conversion of collagen into gelatin. The possibility that some part of the dicarboxylic groups of collagen is inactivated (for instance by interaction with arginine residues) seems worthy of consideration as a n eventuality if the present low values of the base-binding capacity of collagen prove to be correct. The alkali-binding power of collagen is greatly influenced by the degree of pretreatment of the hide with alkali in the liming process, which leads t o partial deamidation (9,100) and also to rupture of coordinated cross links (hydrogen bonds) and dislocation of part of the saltlike cross links. Also splitting of peptide links, including the -C!O-N= bonds, seems to occur (126)) resulting in a slight increase of hydrogen-ion- and hydroxyl-ion-binding side chains. I n systems for determination of the acid- and base-binding curves of collagen, it is of the utmost importance that the swelling of the protein be repressed by the presence of neutral salts such as NaCl and Na2S04. Otherwise, the different ionic distribution between the solid phase and the external solution will give rise to large errors, as will also the influence of the swelling of the substrate of the sorption of solvent and solute which may result in negative adsorption. This important aspect has been emphasized by Steinhardt and coworkers (189)) references being given t o their publications. The present discussion of the reaction of collagen with hydrogen ions was restricted t o the ideal type of a strong acid forming a practically completely ionized salt and thus avoiding the complications due to fixation of the anion by the protein. With more complicated acids the affinity of the anion must be included and considered, which will lead to greater fixation of hydrogen ions in the higher pH range up to the isoelectric point. This phase of the problem of acid fixation by proteins has been lucidly treated by Steinhardt and collaborators (189,190). I t has for a

364

E. H. GU8TAV80N

long while been realized by investigators of tanning processes that the anion affinity, or complex-forming tendency, is one of the governing reactions in certain tannages (vegetable tannage and fixation of synthetic tannins of sulfo acid type), a fact especially stressed by Otto (165) and by the present author (61). In many instances, e.g., in the irreversible fixation of high molecular lignosulfonic acid (Fig. l), the anion effect dominates over the factor of hydrogen ion concentration in regulating the course of the reaction, as is evident by fixation of anions in the pH range 5-8, i e . , on the alkaline side of the isoelectric point of pelt. 100 . )

c

-

FIQ. 1.-Interaction of hydrochloric acid

(I) and high molecular fraction of lignosul-

90-

fonic acid (11) with standard hide powder aa a function of hydrogen ion concentration of final solutions (78). (The irreversible fixation of lignosulfonic acid obtained indirectly by determination of the hydrochloric acid binding capacity of treated hide powder.)

c 9

5a ao.u. - 70.2 E i6 0 0 0

-

5040-

\

'c)

.-a v-

30-

P

2010

0

1

2

3

4

5

6

I

7

I

8

I

9

I

1

I

0

Final pH value

In the system collagen-aqueous solution of weak acids of high concentration, as e.g. acetic acid, the fixation of the acid in molecular form has been indicated (74). This additional uptake of acid molecules seems to be located in the peptide groups, leading to rupture of hydrogen bonds, evident in the increased reactivity of collagen thus treated toward coordination-active agents. Steinhardt and coworkers (191) have demonstrated the same effect on keratin by a different method. The titration curves of collagen may be analyzed by assigning the original pK values of the particular amino acids to the various amino acid residues (R groups) built into the protein chain. In such allocation of the original pK values to protein groups, certain difficulties have been experienced, for example, in the theory of formaldehyde tannage. It is at present generally recognized that the pK values of an amino acid do

PROTEIN-CHEMICAL ASPECTS OF TANNING

365

not necessarily apply to the amino acid residue built into a peptide chain, a fact especially stressed by Cohn and Edsall (25) and by Greenstein (53). The presence of charged groups on adjacent chains may bring about a large shift of the pK values and the influence of the most common link of protein chains, the peptide group, must also be recognized. Thus, the data of Stiasny and Scotti (195) show for polypeptides the marked influence of the accumulation of peptide groups of the same chain on the strength of the ionic end groups; Greenstein’s researches (53) point in the same direction. One must avoid drawing far-reaching conclusions by application of the method of assigning the pK values of the simple amino acids to the reactive groups of collagen, especially in irreversible systems. That collagen and gelatin, even in their simple reactions, are more complicated than implied by a direct evaluation of reacting groups by the pK values of the amino acids concerned is strikingly proved by comparing the hydrogen ion-binding curves of mammalian collagen, fish collagen, and gelatin in solutions of hydrochloric acid (82). These proteins show identical maximum fixation of hydrochloric acid. However, in the pH range 3-5, gelatin fixes about twice as many hydrogen ions as does native bovine collagen a t the same pH value; fish collagen is intermediate. The difference is probably the result of different degrees of internal inactivation of the polar groups, being a function of the degree of orientation of the chains. At a given pH value the discharge of those free or lightly compensated groups of gelatin would be expected to occur more easily and extensively than that of the strong pair of electrovalent links of native collagen. Since changes apparently take place in the internal compensation of polar groups in the pretreatment of skin for tanning, the degree of depolarization will be important for the behavior of collagen toward electrolytes, including some types of tanning agents. In the formation of soluble protein products by the interaction of acids and alkalis with collagen, neither the mode of attack of these agents nor the nature of the solubilized portion is known. A great deal of painstaking work remains to be done on these and related problems. 4. Swelling of Collagen and the Donnan E$ect

The swelling of hide and its effect upon the inter- and intramicellar space greatly influences the reactivity of hide for large molecules, e.g., vegetable tannins, since the degree of accessibility of the reacting protein groups is a function of the degree of swelling of the hide. The degree of swelling of collagen taking place in solutions of acids and alkalis nearly follows the curves of hydrogen ion and hydroxyl ion binding. By the brilliant application of the principles of the Donnan

366

K . H. GUSTAVSON

equilibrium to the swelling of gelatin and collagen in the classical work of Procter and Wilson (173,211), a basis was laid for quantitative work on protein swelling, even though it was later found that the phenomenon is not so simple as the ideal case considered by the pioneers, among whom the namc of Loeb (139) bclongs because of his outstanding experimental contributions to this problem. A number of problems such as the maximum points of swelling of collagen, the decreased degree of swelling incurred by further increase of the concentration of hydrogen and hydroxyl ions, and the repression of swelling by neutral salts are logically explained by the theory. I n the following sections attention will be called to a few problems to which this simple conception of ionic interaction is not applicable. It may be said that the occasional failure of the quantitative treatment of swelling of collagen according to the Procter-Wilson concept is due t o the complicated reactions taking place in the swelling, not amenable to mathematical treatment. First, the application of the Donnan equation requires complete ionization of the compound formed and a completely reversible system. Accordingly, the anion of the interacting acid should not possess affinity for the protein, and, further, molecular effects, so-called lyotropic effects, should be excluded. Another complication is presented by the changed degree of cohesion of the protein structure a t various degrees of swelling, especially rupture of hydrogen bonds a t high alkalinity, a complication also recognized by the pioneer investigators, which exerts a great effect not yet numerically calculable. The Donnan effect, which undoubtedly plays a prominent part in reactions of collagen, particularly in the preparatory processes, has become a less prominent factor because of the complications mentioned (see, e.g., 126). Furthermore, many problems can be explained equally well by other pliysicochemical treatments. The greatest importance of the Procter-Wilson theory has been its accentuation of physicochemical principles and technique in protein investigations. It constitutes the first attempt to describe the swelling phenomenon on a quantitative basis. It still remains a cornerstone of the theoretical foundations of tanning processes.

5 . Zsoelectric Point of Collagen I n determination of the isoelectric point of collagen, standard hide powder has usually been the substrate. Hide powder is practically pure collagen, obtained from thoroughly limed bovine hide which after deliming is made into a woolly powder. The isoelectric point of hide powder has by several independent methods been localized in the neighborhood of pH 5 , or a value practically coinciding with th a t of gelatin

PROTEIN-CHEMICAL ASPECTS OF TANNING

367

(139). Since the hide material undergoing tanning in practice is always limed, the importance of the isoelectric point of limed (alkali-treated) collagen is obvious. The correlation of the hydrogen ion concentration corresponding to the isoelectric point of this type of collagen with the figures of the contents of acidic and basic groups of collagen and their relative strength was earlier a puzzling problem, since collagen according to the previously accepted values of the amino acid distribution contained a large excess of basic groups. However, in view of the fact that not only the quantity of polar groups but also their strength enter into the equation of the location of the isoelectric point, the possibility that the pK values of the acidic groups are increased, compared to the value of the simple amino acids, by the ionic environment of the protein chains, could not be wholly discounted. It was reported by Briefer (17) and independently by Kraemer and Dexter (117) th at the isoelectric point of gelatin obtained from acid-treated pigskin, which had not previously been limed, corresponded t o p H values in the vicinity of 7. Limed pigskin gave a gelatin with a normal isoelectric point a t p H 4.8. A number of investigators later showed that the location of the isoelectric point of hide collagen is a function of its previous history, particularly the degree of alkali treatment (liming) (33). The final experimental proof of this assertion and an explanation of the displacement of the isoelectric point was given by Beek and Sookne (9) in cataphoretic investigations of collagen of different pretreatment. The isoelectric point of native (unlimed) collagen was found to be a t pH 7. Independently, Highberger (100) obtained figures in the p H range 7.5-8.0. The shift of the isoelectric point some 2 pH units toward the acid side, resulting from thorough liming of collagen, was ascribed to the deamidation taking place by the action of alkali on collagen, whereby the strong carboxyl group is set free (9,104). The new figures of the contents of dicarboxylic acids and amide groups of native collagen (Table 11) offer a rational explanation of the location of the isoelectric point of gelatin and limed collagen in the acid pH range, if due regard is paid to the hydrolysis of acid amide groups, with the formation of free carboxylic groups, taking place in the regular liming process (about half of the amide nitrogen being split by a few days’ treatment of hide in saturated lime solution (104)). Thus, limed collagen will contain about 1 .O mmol free carboxylic groups (1.24-0.24) and 0.87 mmol basic groups, or a surplus of acid groups (0.13 mmol/g. collagen). The new data also satisfactorily account for the localization of the isoelectric point of native (not alkali-treated) collagen in the p H range of 7-8, since the amount of fiee carboxylic groups is 0.77 mmol

368

K. H. QUSTAVBON

(1.24-0.47) and the content of basic groups 0.87 mmol or a surplus of 0.1 mmol basic groups. Highberger and Stecker (104) found that destruction of basic groups (arginine) is only of minor importance in shifting the isoelectric point. Furthermore Kuntzel (126) suggests that in the prolonged alkali treatment of collagen, which occurs in making gelatin, hydrolysis of peptide group links may occur. The effect of the opening up of the -CO-N= on the isoelectric reaction of collagen and gelatin may be an important factor. The location of the isoelectric point of collagen in combination with tanning agents is important for the theory of tanning and will be considered in that connection. 6 . Coordinative Reactions

In the complex reactions of tanning agents with collagen, coordination of the two components is in many instances of a n importance equal t o or greater than that of electrovalent reactions; the coordination is probably mainly localized in the peptide groups. Some of these groups are already internally compensated (hydrogen bonds). However, in view of the fact that limed collagen is generally concerned in fixation of tanning agents, and also since the alkaline pretreatment tends t o weaken or rupture the internal links of the peptide bonds by the swelling of the hide, coordination-active peptide groups should be available in the usual form of limed collagen. Furthermore, the presence of the - CO-N= linkage in every third residue, withdrawing loci for the compensation of adjacent -COgroups of peptide bonds, leaves coordinate loci available even in native collagen. It has been mentioned previously that disorganization of the lattice structure by contraction of the protein chains results in increasing the gap between some of the compensated peptide groups. Thus, the swelling of collagen by hydrotropic agents tends to increase the fixation of reactants of the coordinate type, e.g., vegetable tannins. Simultaneously, the internal cohesion of the structure is impaired, which may have undesirable practical consequences. The opening up of the structure will make the reactive groups more accessible to the tanning agent, and activation of peptide linkages will facilitate multipoint attachment of large molecules with numerous coordination-active groups on peptide groups (71). The coordinate type of reaction is sterically favored since -CO-NHgroups should be more easily accessible than the side chains of collagen to large molecules with regularly interspaced reactive groups. The coordinate type of reaction is indicated to be of governing importance for the fixation of vegetable tannins by collagen, as will be further shown in the section on vegetable tanning.

PROTEIN-CHEMICAL ASPECTS OF TANNING

369

7. Denaturation and Heat Shrinkage Collagen fibers, in the form of skin or tendon, in contact with water of 65-70°C. contract sharply at a given temperature (shrinkage temperature) to about one-third of their original length (34). The shrunken specimens feel gluelike and show rubberlike elasticity. The tensile strength of the fibers is immensely lowered. The original resistance of native collagen to trypsin is destroyed (35). Marked hydrolytic changes, in the form of splitting of peptide groups, do not seem to occur. The elementary composition, including the nitrogen content, is unchanged (50). The maximum acid-binding capacity is not affected (50). The water content remains the same (50). However, the X-ray diffraction pattern of the fiber becomes less well defined and the birefringence is decreased, indicating disorganization of the units constituting the fiber (3). By extension of the shrunken fibers the diffraction pattern is partially restored (3). By intramicellar introduction of certain tanning agents, e.g., aldehydes, almost complete reversal of the shrinkage is attained upon cooling the shrunken fiber structure (Ewald’s reaction, 34), probably due to the presence of aldehyde cross links (154). Similar to the action exerted by heat on moist fiber is the swelling by strong acids, which however only contract the fiber to two-thirds of its original length (121). The swelling incurred in brief treatment is reversed by a subsequent neutralization of the swelled collagen (141). However, upon prolonged interaction with strong acids, and particularly with alkali, the resulting swelling is only partly reversible (121). The contraction of the fiber by lyotropic agents is of an order comparable to the hydrothermal shrinkage. This change is irreversible (121). A close interrelationship of degree of swelling and hydrothermal stability is evident ( 16). The primary reaction of the shrinkage is in all probability a melting of the hydrated crystallites as assumed by Wohlisch (218) and by Meyer (21). Mirsky and Pauling (158) and Kuhn (118) have shown that the change from an oriented structure to a random state of configuration of the protein units is a natural consequence of the tendency of the structure to increase its entropy. The fibrous state is from the thermodynamic point of view artificial and labile, whereas the coiled globular state with a random distribution of the protein chains represents the more probable and stable state of protein configuration. Some investigators like Meyer and collaborators (21,154) consider the denaturation and shrinkage of collagen to be a reversible process. They consider that irreversible alterations occur only in fibers hydrolytically damaged during the denaturation. It seems, however, that the fiber wcave of the hide structure is irreversibly shrunk and permanently altered (120,123). This is indicated by its changed reactivity toward high-molecular compounds (71). However, in spite of the contraction of the chains and the disorganization of the structure, a certain orderly grouping of the units remains (120). Probably the saltlike crosd

370

K. H. GUSTAVSON

links still function as cohesive forces, although weakened by unfavorable steric conditions, maintaining the principal outline of the structural features. The hydrothermal denaturation also affects the hydrogen bonds, which are partly ruptured, resulting in noncompensated pcptide groups with coordinate loci available. This is indicated by the marked increase in the reactivity of collagen toward coordination-active compounds induced by the shrinkage (71,82). The denaturation does not affect the irreversible fixation of ionic agents, e.y., basic chromic salts of low to medium molecular size (two to six atoms of chromium in the molecule). However, the uptake of high molecular agents reacting mainly as coordination compounds, such as certain large chromium complexes and vegetable tannins, is drastically increased as shown by Table 111, in many instances a doubling of the ameunt of fixation being obtained (71).

The instantaneous shrinkage occurring at a certain temperature has its counterpart in similar although less drastic changes induced in the collagen structure by prolonged heating in water of temperature considerably lower than that causing rapid denaturation, for example, in water of about 50°C. This is evident from Grassmann’s data (50), and was more recently noted by Pankhurst (170) in prolonged treatment of pelt in water a t 45°C.(“incipient shrinkage”). Such changes will have practical importance in the pretreatment of hide in tanning. The incipient shrinkage leads to increased affinity of the treated collagen (4045°C.) for coordinate agents, although this is less pronounced than the effect of complete shrinkage (79). Similar changes in the reactivity of collagen to those induced by thermal shrinkage are obtained by the pretreatment of hide collagen with concentrated solutions of lyotropic agents, such as calcium thiocyanate, urea, and 2-3 M solutions of acetic acid (71). The maximum fixation of strong mineral acids such as HC1 is not changed by denaturation, as mentioned earlier. However, the fixation of hydrochloric acid in the p H range 2.5-6 is somewhat increased by denaturation. This is probably due to the easier discharge of the carboxyl groups, present as internally compensated ionic cross links, resulting from the widening of the gap between some of the polar groups, brought about by the steric changes caused by the thermal contraction (82). The fixation of hydroxyl ions is also slightly increased in the pH range 8-11 as a result of denaturation. Noteworthy is the finding of Wohlisch (218), later confirmed by Grassmann’s (50) investigation of single fibers, that the shrinkage temperature is increased when tension is applied to the fiber. This fact will explain why the shrinkage temperature from a compact part of a skin is higher than that of looser sections of the same skin. The fiber bundles of a close-textured weave are exposed to strain from adjacent fibers, whereas the effect of the neighboring fibers on fiber bundles in a loosely interwoven specimen is not apparent. The shrinkage tempera-

PROTEIN-CHEMICAL ASPECTS O F TANNING

371

tures of samples from various parts of the same skin generally do not differ more than 2-3°C. Fish skin collagen shows considerably lower hydrothermal stability than mammalian skin. The shrinkage temperature of fish skins generally is in the range 4@45"C., compared to temperatures of 6O-7O0C. for the mammalian type (71,72). It is noteworthy that gelatin films shrink a t 45°C. (170), which also is the shrinkage temperature of cod skin. It is probable t ha t in the teleost type of collagen saltlike cross links mainly hold the structure together; hydrogen bonding is of secondary importance. This is indicated by a comparison of the reactivity of bovine and fish collagens. Thus, e.g., high-molecular sulfonic acids such a s a-lignosulfonic acid, which is irreversibly fixed by both types of collagen in stoichiometric ratio, according to the available equivalents of basic protein groups, yield with bovine skin a leatherlike product with increased tensile strength compared to native skin, although the hydrothermal stability is not improved. By the corresponding interaction cod skin is converted into a gluelike mass (91). This finding may be interpreted to mean that the ionic pairs of cross links of collagen of fish skin are destroyed in the discharge of the carboxyl ions and by the irreversible attachment of lignosulfonate anions to the basic groups. The electrostatic attraction is eliminated and, since the major part of the peptide groups are not internally compensated, the natural cohesion of the structure is greatly impaired. The corresponding inactivation of the electrovalently compensated cross links of mammalian collagen has only a slight effect on this type of collagen because the main stabilizing links, the hydrogen bonds, are left intact, and also since collagen is not swelled by the sulfo acid. The complete dissolution of fish skin collagen, and also of ichtyocol and elastoidin, two special types of collagens investigated particularly by FaurC-Fremiet and his school (38), in dilute solutions of weak acids, e.g., acetic acid of 0.001 N strength (37), is also of interest in this connection. Mammalian skin collagen is not affected by such solutions, but collagen of tendon, as for example that of the rat's tail, dissolves (134, 159). It is probable that differences in histological structure (the intertwining of fiber bundles in the skin, as contrasted to parallel-grouped structures in the tendon) and in the ensheathing reticulin also play a role in the di&olution. The low degree of interlacing of the structure of fish skin collagen may also contribute to its lability toward acidic solutions.

Native fish skin, such as cod skin, which has been investigated in detail, is easily digested by trypsin (72,75) in contrast to native mammalian skin, which is practically inert toward proteinases (35). This finding indicates that the peptide groups of teleost collagen are largely uncompensated. Since swelling contracts the fibers in the direction of the long axis, it

372

K . H. GUSTAVSON

is evident that the shrinkage temperature (if there is any sense in measuring shrinkage temperatures in such cases) is lowered by acids and alkalis (16). By repressing the swelling of pickled pelt with salt, the dehydration of the structure results in a slightly improved hydrothermal stability (68,123). Complete inactivation of the ionic protein groups by means of a nonswelling acid, resulting in the complete destruction of saltlike cross links, should result in lowered shrinkage temperature (ca. 10-12°C.) (71). The increase obtained by acid saturated collagen from pickle with high salt concentrations evidently shows that some other changestending to increase the stability of the structure-more than outweigh the decrease due to the ionic discharge. Speculatively i t may be conceived that the dehydrating effect of the salt in conjunction with the action of the acid brings the backbone peptide groups closer to each other, increasing the strength of the hydrogen bonds (68). It is interesting that in pickle solutions with increasing concentrations of hydrochloric acid the shrinkage temperature remains constant or slightly increases, compared to neutral pelt, until a concentration of 1 N acid is reached. By further increase of the acid concentration the shrinkage temperature is greatly decreased. Thus, shrinkage takes place at room temperature in acid concentrations greater than 2 N (123). The latter change, which is independent of the neutral salt concentration, is evidently a specific molecular effect, probably due to association and coordination of hydrochloric acid molecules on the peptide links, leading to an irreversible rupture of a part of the stabilizing links at these points. This is an additional example of the dual nature of the action of electrolytes in concentrated solutions upon proteins. Similar effects, decreasing or increasing shrinkage temperatures of collagen according to the concentration of the solute, not involving complicating osmotic swelling phenomenon, are shown by ethanol (123). Mixtures of ethanol and water, containing less than 40 % ethanol decrease the shrinkage temperature of collagen, whereas higher concentrations increase it, the augmentation being about 10°C. in 80% ethanol. Noteworthy is thc behavior of fibers that are heat shrunk in the latter solution. By careful stretching of the fibers it is possible not only to restore them t o their original length but to obtain elongation up to double the original fiber length by applying further tension. The X-ray diagram of the extended denatured fibers shows chains aligned in parallel. This observation of the great extensibility of alcohol-treated collagen (123) is especially interesting in view of the extensibility of collagen fibrils (179). The shrinkage temperature of tanned skin is one of the fundamental criteria of tanning potency (148), although it is important not to evaluate tanning agents merely by this property (101).

8. Neutral Salt Action and Lyotropic Behavior

The effect of neutral salts and lyotropic agents on collagen in the isoelectric state is important practically as well as theoretically. From

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373

a practical standpoint the use of neutral salts, generally sodium chloride, for preservation of the raw hide may be mentioned, as well as the influence of salts on the neutral pelt previous to tanning. The interaction of neutral salts with collagen also involves problems of fundamental importance for the theory of protein reactivity. The specific action of neutral salt ions on soluble proteins, established by Hofmeister (106), applies also to the action of concentrated solutions of neutral salts on collagen. I n this instance the action is mainly a molecular effect. It was shown by Thomas and Foster (203) that, by treating hide powder in solutions of various neutral salts of about 1 M strength, a large percentage of collagen was solubilized by sodium thiocyanate, iodide, and bromide, whereas sodium sulfate acted as a preservative. Sodium chloride had only a slight effect. I n view of the wellknown fact that molecular compounds are formed between amino acids and neutral salts, isolated by Pfeiffer and his school (172), and since the degrees of solubilization of collagen by the various salts on the whole follows the order of the stability of the neutral salt compounds with amino acids, the effect has been considered to be an interaction of the neutral salt with the coordinate valency bonds on the peptide groups (hydrogen bonds) (55). Part of these stabilizing bonds are weakened and ruptured, resulting in creation of new coordinate loci. In reactions between amphoteric ionic structures such as collagen and simple electrolytes, ionic interaction of the type given by simple amino acids and neutral salts has also to be considered, according to the equation (172; cf. 1,135):

+

C O O - C H ~ . N H J + Me+X- e-Me+.COO-CHn.NH8+.X-

This type of reaction or electrostatic interaction probably dominates in dilute solutions. These have no dissolving effect upon collagen. In concentrated solutions of neutral salts, as for instance calcium chloride and thiocyanate, collagen is attacked, being largely dissolved and permanently changed in reactivity and properties. The destructive action of concentrated solutions of lyotropic neutral salts (1-2 M ) seems to be mainly a molecular effect. A proof of the changed reactivity of pretreated collagen, probably involving nonionic groups such as the peptide groups, is supplied by the marked increase of the fixation of high-molecular compounds, e.g., highly aggregated sulfito-sulfato chromiate and vegetable t.annins, compared to native collagen (55,71). The fixation of simple electrolytes, such as acids, alkalis, and chromium salts of low molecular weight, is not affected by the treatment. Table I11 contains some typical data, showing the degree of solubilization of hide powder upon 2 weeks’ treatment under

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K. H . GUSTAVSON

sterile conditions in M solutions of various neutral salts. It also presents data on the fixation of ionic-reacting basic chromium sulfate, highmolecular chromiate, and wattle bark tannins (55). The table also includes results from series of pretreatment5 of neutral pelt (calfskin) with 8 M urea solution and with solutions of 3 M acetic acid (the stock subsequently neutralized and made isoelectric). The effect of hydrothermal denaturation of hide powder (2 minutes a t 70°C.) is also included, since this denaturation also modifies the reactivity of collagen (71). Chrome tanning was carried out in solutions containing 1 equivalent of chromium per liter a t 20°C. for 144 hours. TABLE I11 Influence of Pretreatment of Collagen on Its Reactivity --_ Fixation of tanning agents from Pretreatment

Hide powder Water (blank) M KCNS M CaCL Shrunk at 70°C. Pelt Water (blank) 8 M urea (37°C.) 3 M acetic acid

Collagen dissolved in pretreatment, %

3 24 32

-

0 54 21

66% acid Cr-sulfate, % Cr2W collagen

'Sulfitoehromiate, % CrzOa/ collagen

Wattle bark tannins, % tannin/ collagen

11.2 11.0 11.3 11.7

20.8 31.3 30.6 34.1

52 81

10.3 10.4 11.7

16.5

46

40.3 26.8

80 69

78 84

An excellent illustration of the ionic and molecular effects is presented by acetic acid. I n dilute solution, e.g., 0.1 N a t final p H values of about 3, its action upon collagen is nearly the same as that of a corresponding solution of hydrochloric acid of identical final pH; the effect is mainly due to the hydrogen ion concentration. With increasing hydrogen ion concentration of the solutions, up to pH 1, the swelling of collagen by hydrochloric acid increases until a maximum is reached at pH 2. In contrast to the behavior of strong acids, increased concentration of acetic acid leads to very much greater augmentation of the swelling (74). At the lowest pI-1 values the collagen loses its structural cohesion and goes into solution (74). This peptization is due to the molecular effect of the acetic acid. Since sodium acetate solutions of corresponding concentrations (> 3 M ) do not show any -solubilization effect on collagen and do not alter its coordinate reactivity, it is evident that neither hydrogen

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PROTEIN-CHEMICAL ASPECTS O F TANNING

ions nor acetate anions are responsible for the drastic effect of acetic acid in concentrated, aqueous solutions. The effect is probably caused by the coordination of the monomeric acid molecules on the peptide groups which are thereby partly broken. The result will be diminished internal cohesion of the structure (74). It is noteworthy that strong solutions of mineral acids (above 2 M ) show this molecular type of swelling to a greater degree than the ionic type (123). Phenol reacts in a similar manner (123). The effect of urea on globular proteins, splitting the molecule into several fragments (19), has a counterpart in its behavior toward collagen. The fibers shrink ( 1 16) and the treated product shows markedly increased coordinate reactivity (71). Summing up, the effects of lyctropic neutral salts, organic substances of lyotropic function, weak organic acids in concentrated solution, and heat denaturation of collagen are of the same general type, resulting in a weakening and dislocation of the stabilizing cross links located on the peptide groups (hydrogen bonds). The application of collagen with such activated coordinate loci for the study of the mechanism of tanning processes has been a valuable adjunct as will be shown in the sections on tanning. 111. KERATOLYSIS AND

-4CTION OF

ALKALION

HIDE

The removal of hair and epidermal matter from the hide in order to obtain the leather-forming part, the corium, free from nonleather-forming constituents is accomplished by the liming process, which also brings the unhaired hide, the pelt, into a suitable state for tanning, activating certain protein groups, plumping the hide by water uptake, “opening u p ” fiber bundles, bringing about lateral splitting u p of the individual fibers, saponifying fats, and modifying or removing the accessory proteins, such as interfibrillar matter and reticulin. The hide is treated in a saturated solution of calcium hydroxide, containing a large excess of the alkali and a small amount of a specific depilatory, generally sodium sulfide or hydrosulfide. Within a few hours or days, according to the degree to which the action of the limes is sharpened with depilatory, the hair roots are loosened and unhairjng results. In the fundamental investigations of Merrill (149), the breaking of the disulfide bridge of keratin was established as the main reaction of depilation. This was further confirmed by Marriott (140) and by Windus and Turley (215). The depilatory action of a great number of organic sulfur compounds has been extensively investigated by Turley and Windus in a series of important papers (209,215).

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K . H. QUSTAVSON

According to the modern view, the first reaction of unhairing is the breaking of the disulfide bridge by hydroxyl ions. The action of the depilatory is mainly secondary. Its main function is to prevent the formation of new links which otherwise would stabilize the keratin structure. The breaking of the disulfide cross link in alkaline solution leads to the formation of a thiol and a sulfenic acid group:

\

/

CH.CHz.S.S.CH2.C cystine link

-

&-OR

\

\

/

CH.CHz.SOH sulfenic acid group

+ HS.CHz.CII/

\

thiol group

Then the specific action of the depilatory enters. If only hydroxyl ions are present, the active groups formed by the splitting of the disulfide bridge react furthcr, with formation of new cross links between adjacent protein chains, which leads to increased stability. Among the many reaction mechanisms proposed, experimental evidence has been supplied for the formation of the -CHZSCHZ- link (lanthionine, 27,186). The specific unhairing agent apparently reacts with the sulfenic acid, group, inactivating it and preventing the reformation of cross links. A great many reducing agents possess this property, the most important technically being the alkali sulfides, primarily sodium sulfide (hydrosulfide). Other agents proposed are cyanides (140), thioglycolic acid (157), sulfites (140), and aliphatic amines (147). Among those mentioned the cyanidcs are particularly interesting from a theoretical point of view, since Cuthbcrtson and Phillips (27) recently reported that potassium cyanide in neutral solution converts all cystine of wool to lanthionine. The excellent unhairing action of secondary amines, e.g., dimethylamine, discovered by McLaughlin (147), has not yet been satisfactorily explained (cf. 143). It is noteworthy that the secretion of the cloth moth contains a reductase in alkaline medium (pH 10) which enables the larva to digest wool keratin, as the remarkable histochemical researches of Lindcrstr$mLang and Duspiva (138) show.

The presence of various types of keratins and related proteins in the epidermis of hide further complicates the problem of unhairing. The soft keratins, such as those of the mucous layers of the epidermis, show a lower degree of stability toward reducing agents and alkalis and are also more readily attacked by heat and enzymes than the hard keratins present in hair. This has been explained by the higher frequency of disulfide cross links in the latter. The lower stability of the epidermal keratins has been accounted for by the presence of a lavge part of the sulfur as sulfhydryl groups. However, the cystine content apparently is not the only factor governing the stability. If we take into account Block’s concept of keratin structure, postulating the molecular ratio of basic amino acids as the characteristic property of keratins, some clues to the varying stability of a-proteins may be obtained (177). This is shown by Rudall’s work on the hydrothermal behavior of various epidermal keratins and other a-proteins (177). With decreasing relative proportion of arginine, the stability of these proteins (keratins, myosin and fibrin) is decreased. The guanidyl group may then contribute to the stability of the keratin structure by intermolecular linkage. In view of

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377

Rudall’s important finding (177) th at the mucous layers of the epidermis are readily dissolved in 50 % urea solution, yielding epidermin, the possibility of removal of epidermis by means of strong solutions of lyotropic agents is suggested as a method of unhairing.* The direct chemical action of alkalis on collagen was mentioned in connection with the discussion of the shift of the isoelectric point. It was pointed out that the main reaction apparently is hydrolysis of the amide groups of asparagine and glutamine. By treatment of collagen in lime for 9-10 days, ammonia in an amount equivalent to the hydrolyzed amide groups is formed (104). Partial destruction of the guanidine group of the arginine residue occurs in alkaline solutions, being considerable at high temperatures. However, this reaction does not seem to be appreciable in ordinary liming (104). The extent of the action of alkali on the peptide groups, leading to their cleavage, particularly the effect on the -CO-N= groups, is not known. The hydrolysis of the proline peptide groups should prove especially interesting. From the titration curves of collagen of different degrees of liming no indication of appreciable increase of ionic groups is obtained. However, it must be realized th a t even if the collagen molecule is halved the acid- and base-combining capacity will not show greater increase than 0.1 milliequivalent per gram collagen (1 4). I n conclusion, it may be pointed out that the alkaline swelling is not depressed by the addition of sodium chloride to solutions of sodium hydroxide, as would be expected from considerations of swelling as a Donnan effect. Such an explanation evidently fails in this instance, as aptly pointed out by Kuntzel (126). Sodium sulfate is the only common neutral salt functioning as a n active swelling depressant in alkaline solutions (129). One important function of the liming treatment of hide is the opening up of the fibers, making the structure accessible and permeable to large molecules. Another important aspect is the activation of ionic groups by the breaking up of the saltlike links through hydrolysis of the charged amino groups by the hydroxyl ions, which upon prolonged liming (swelling) involves partly irreversible changes. The increase of carboxylic groups by the deamidation process connotes increased ionic reactivity. Thus, the binding capacity of collagen for electrolytes and tanning agents, both chromium salts and vegetable tannins, is increased. The reactivity of collagens is further favored by the physical changes of the

* Kritainger (117a) reports that by immersion of fresh calfskin in a 10% solution of sodium chloride for 7 days under sterile conditions unhairing takes place. He attributes this to the removal of globulins, but probably the solvent action of the lyotropic agent, sodium chloride, on the epidermis is the main factor of depilation in this instance.

378

K. H. QUSTAVBON

hide in liming and also by the chemical changes in nonionic protein groups, since the swelling of the hide leads to rupture of some of the coordinate bonds between adjacent protein chains (66). The hydrogen bonds are not completely reformed in the subsequent deliming which reduces the swelling. Hence, the reactive protein groups are made more easily accessible.

IV. GENERALASPECTSOF TANNING In the definition of tanning in practical terms, the most striking physical, directly observable changes involved in the process form the criteria. By tanning the easily putrescible hide substance is made resistant to micro-organisms. Further, leather will resist water and moderate temperatures in the moist state and remain soft and flexible upon drying. Through the extended knowledge of the tanning processes acquired during the last decades, it is now possible to define tanning action in a more scientific manner. The first criterion of tanning potency of a substance is its capacity to form an irreversible combination with collagen, resistant to the action of water. Certain reactive protein groups are inactivated. However, the simple incorporation of an irreversibly fixed agent and the reduction of the water-binding capacity of collagen does not effect tanning. The second criterion is the stabilization of the collagen by the tanning agent, improving its resistance to heat and proteinases and preventing the “glueing together” of fibers upon drying (“leathery drying out ”) without detrimentally affecting the mechanical strength of the original hide structure. By the conditioning of the hide, its original resistance to the agents mentioned is lowered, chiefly due to diminished internal cohesion of the weave structure. The function of the tanning agent is to make up for this labilization, incurred in the prior conditioning of the hide, by making it into a more stable structure than it originally was, supplying stable cross links which rivet the chains together. The cross link concept was suggested by Meyer (153). From the practical point of view, it is not always necessary that all criteria of tanning should be realized. Thus, hide treated with agents which do not raise its shrinkage temperature, may nevertheless possess certain specific properties desired, e.g., great tensile strength or pliability. On the other hand, tanning agent making the hide resistant to high temperature may have other disadvantages, such as impairing the tensile strength. It is obvious that tanning agents do not form a distinct class of compounds chemically. On the contrary, substances of very dissimilar

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379

composition and nature possess tanning properties; one need mention only basic chromium salts, vegetable tannins, aldehydes, certain condensed phenols containing sulfonic acid groups, and unsaturated oils. The substances mentioned are the tanning agents of practice; chromium salts and natural vegetable tannins being the principal ones. Since the reaction of chromic salts with collagen (chrome tanning) is best known and offers a great many problems of general interest., the chemistry of chrome tanning will be comprehensively discussed.

V. REACTIONOF CHROMIUM COMPOUNDS WITH COLLAGEN (CHROMETANNING)

Among inorganic substances the chromium compounds, notably the basic chromic salts, hold an unrivaled position as tanning agents. The strong complex-forming capacity of the chromium atom probably is not in itself the deciding factor for the excellent tanning potency. Thus trivalent cobalt is equally efficient as a complex former, but its compounds do not possess tanning potency. Chromium forms polynuclear compounds of an intermediate degree of stability, aqueous solutions forming compounds of the type -Cr-0-Crupon hydrolysis. However, such hydrolytic changes are not restricted to chromium, even the related metals (Fe and Al) show this tendency. Chromium differs insofar as i t forms polynuclear complexes which generally are in hydrolytic equilibrium and reactive. It seems that a combination of these properties is decisive for tanning action. The electronic structure of chromium, with three unpaired electrons entering into resonance with each other in the ionic and covalent chromic compounds, does not permit application of the magnetic method of differentiation of the type of bonds formed between chromic salt and collagen (171). 1. Structure of Basic Chromic Salts of Zmportance in Tanning In practice basic sulfates of chromium containing an equimolar amount of sodium sulfate are commonly employed in tanning. The basic sulfates differ from t h e basic chlorides insofar as the sulfate group possesses a marked tendency to be directly linked to the chromium atom, forming sulfato-chromic complexes (193). The 0 following is an example: (Cr

/

\

\

so,

/

Cr) SO,.

The basic sulfates exist in dilute

solutions, such as those generally employed in tanning, mainly as cationic complexes (77,80,81). Concentrated solutions (greater than 4 N) contain a large percentage of neutral molecules and even negatively charged chromium complexes (77,80,81). On the contrary, the electrochemical composition of basic chlorides is independent of the concentration of the solutions (77,80,81). Chromium salts possess, beside electro-

380

K. H. QUSTAVSON

valent function, the faculty of coordination. The number and nature of the groups in the internal sphere, the coordination sphere, determine the coordinate ability of the central atom. The effect of the nature of the complexly attached acid residues (acido groups) on the coordinate function of chromium is known in a general way (193). The aggregating tendency of the compound is also interrelated with the structure of the internal sphere of the complex. The sulfate group seems to exert optimal action on the coordinate activity of the chromium atom with regard to tanning. Chloride and nitrate groups are weak complex formers, generally being present in the electrovalent state (193). Some strong complex-forming organic acid residues, e.g., the oxalate and tartrate, apparently leave chromium only a slight chance of further coordinate function (193). The tanning potency of basic chromic salts is a function of the coordinate faculty, the degree of aggregation of the salt and its degree of ionization. The size of the complex is important for the polyfunctional activity in the multipoint fixation of the chromium compounds. The properties and behavior of chromium salts are advantageously considered from the point of view of the Werner coordination theory (193). Before proceeding any further with dctails, a general orientation in the probable mode of interaction between a solution of a basic chromic sulfate and collagen may be helpful. A 67% acid chromic sulfate is employed. It may be simply made from the hexaquosulfate by the addition of the required amount of sodium hydroxide to yield a basic sulfate with composition corresponding to the empirical formula Cr2(0H)2(S04)2-Na2S04. This solution is hydrolyzed, and, in the concentration employed for our experiment, 1 equivalent per liter chromium, gives a p H of about 3.0. The neutral collagen combines immediately with the free sulfuric acid. Simultaneously the positively charged sulfatochromium complexes, as e.g. (Cr20S04)++,are attracted to the negatively charged carboxyl groups of collagen, and react with them, the sulfate ions being compensated by the charged amino groups. The chromium complex is extremely firmly attached to collagen; indicating the conversion of the originally electrovalent bond into a type of greater stability (66). The depolarization of the chromium-collagen link is probably the net result of the penetration of the carboxyl group of collagen into the chromium complex, which also completes the reaction by coordination on adjacent coordination-active groups of the protein chains. The final result is the formation of a modified type of an internal complex salt with covalent as well as coordinate multipoint attachment of the chromium complex to groups of adjacent collagen chains, leading to a riveting together of the protein chains by means of the chromium complexes. The formation of chelate compounds explains the high degree of rigidity of the resulting structure and the stability of chrome leather (66,122).

2. Factors Governing the Tanning Efect These are mainly: the type of salt, its basicity (% acidity), type of compound, its concentration, the neutral salt content, the pH value of the system, the presence of complex-forming substances, the temperature of the tanning bath, and the time of interaction. The factors mentioned pertain to the chromium compounds. The nature and the state of collagen enter also heavily in the equation, particularly in regard to the practical issues and the qualities of the leather produced. Such factors are the availability of amphoionic groups, coordination potency of the

PROTEIN-CHEMICAL ASPECTS OF TANNING

381

pelt, and its degree of rigidity (if swelled or flaccid) in the initial stage of the tannage. Many of these factors are evidently interdependent and difficult to treat separately. a. Nature of the Anion. This factor is of primary importance for the tanning function of chromic salts. Nitrates are less satisfactory than chlorides, which in their turn are markedly inferior t o sulfates; the latter as previously mentioned are the most suitable tanning agents. Formato complexes are good tanning agents, especially in mixtures with sulfates (193). Some oxalate complexes possess tanning potency but most of them do not. Of the numerous organo complexes, the acetate and tartrate as well as the oxy acid compounds are devoid of tanning power. The influence of the anion on the tanning potency seems to be a function of its affinity for the central atom, indirectly affecting its coordinate function and secondary reactions (193). b. Basicity of Chromium Salt. The basicity or per cent acidity (expressed in per cent of the equivalents of basic or acid residues present in combination with chromium, calculated on the total amount of equivalents of chromium) (194) is in practice the primary factor. If the basicity is not suitably adjusted, the result of the tanning will be upsatisfactory. By adjusting the percentage of acidity of the chromic salt to the values of 50-66, an average molecular size with two to six atoms of chromium (111,175)is obtained, possessing good diffusibility through the fiber structure a n 4 a suitable degree of affinity for collagen. Sterically, the multipoint interaction is facilitated since further hydrolysis of the fixed chromium salts may occur in the secondary processes and in the subsequent treatments of the leather, yielding in situ still more aggregated complexes. Solutions of this type of salt generally have hydrogen ion concentrations in the range of pH 3-3.5. Hydrogen ion concentrations of this order are favorable for a suitable rate of tanning, since a great part of the carboxyl groups of the amphoionic collagen structure are present as ions. At pH values in the vicinity of 2 or below, only a small part of the carboxyl groups are present in the charged state, slowing up the reaction. On the other hand, a t higher p H values, e.g., 4.5-5,all the carboxyl groups are available in the ionic state. This would result in a very rapid combination. Furthermore, the basic chromic salts formed in this pH range will aggregate. By the rapid uptake of hydrolyzed acid by the neutral pelt, the hydrolysis proceeds. Highly aggregated chromium compounds may be precipitated on the outside of the hide, giving rise to a blocking of the penetration, leaving the interior of the hide in untanned or undertanned state (" case hardening"). c. Concentration of Chromium Salt. As is evident from Fig. 2 the chrome fixation by collagen from solution of basic chlorides increases

382

K . H . GUSTAVSON

steadily with increasing chrome content of the solution (97). This probably reflects the fact that the basic chromic chlorides do not form complexes of greatly loivered affinity for collagen (uncharged or anionic) in concentrated solutions. By interaction of solutions of basic chromic chlorides and sulfates with organic cation exchangers of the sulfonic acid type the same type of chrome fixation curves, as a function of chrome concentration, arc obtained as for corresponding collagen systems (81). The reaction of the basic chromic sulfates with collagen is complicated with increased chrome concentration on account of the formation of uncharged and negatively chargcd chromium complexes (see Fig. 2).

I

0

I

I

50

100

Cr203, q J L

FIG.2.-Fixation of chromium compounds by hide powd; as a function of the chrome content of solutions (66). Time of tanning, 48 hours; composition of chromic salts corresponding to Cr2(0H),X..2NaX. -x-x-, 78 % acid sulfate; ---o-o--, 66 % acid sulfate; -A-A-, 50 ’?& acid sulfate; -0-0-~ 00 % acid chloride.

These show markedly different reactivity toward collagen than the common type of cation chromium complexes present in dilute solutions (80,81). Thus, e.g., the 50% acid chromic sulfate of composition corresponding to the empirical formula Cr4(OH)e(S04)3-2Na2S041 contains only cationic chromium complexes in concentrations up to 1 equivalent per liter chromium. Their amount decreases steadily with increasing chrome concentration, uncharged chromium complexes mainly being formed, and in the most highly concentrated solutions anionic chromium. Thus, a t a concentration of 4 equivalents per liter chromium, the solution contained 60 % cations, 38 % uncharged complexes, and 2% anionic chromium complexes; in solutions containing 8 equivalents per liter chromium, the corresponding figures were 35, 60, and 5 (77,80,81). Since not only cationic chromium complexes are fixed by collagen from concentrated solutions of basic sulfates but also uncharged and negatively charged complexes, the composition of the resulting leather will be very much more complicated than that of collagen tanned in dilute

PROTEIN-CHEMICAL ASPECTS OF TANNINQ

383

solutions (80,81). Further complications arise from the unstable nature of the fixed noncationic complexes and their gradual conversion into cationic ones, for example, by washing the leather, which will result in redistribution of the forces between collagen and the attached chromium compound (68,94). The effect of the uptake of nonionic complexes by collagen is clearly evident in tanning with dilute solutions of basic chromium sulfate, used immediately upon dilution. Although the noncationic complexes are rapidly changed into cationic ones by the dilution (to 60-70% within 4-5 hours), the pelt will combine with the uncharged complexes during a brief duration of tannage, for example within 4 hours. The chrome fixation obtained will be markedly greater than that resulting from the aged solution, which contains only positively charged chromium complexes. Moreover, the composition of the fixed chromium complexes, measured by the ratio of equivalents of sulfate to chromium combined, will give higher % acidity of the stock tanned in freshly diluted solution, due to the fixation of the more acid uncharged complexes (94). Further, in the determination of the shrinkage temperature, on immersing the pressed and lightly rinsed leather in water at about 1°C. below the actual shrinkage temperature (this being localized by preliminary trials), i t will be found that the leather tanned in the aged solution, which contains only cationic chromium complexes, will shrink a t a temperature !5-6”C6 higher than that of the stock tanned in the freshly diluted solution, containing considerable amounts of noncationic complexes (94). A striking illustration of the importance of the mode of determining the shrinkage temperature is supplied by the hydrothermal behavior of the two types of leather. If the determination of the shrinkage temperature is carried out by gradual heating of the stock in water, commencing a t 40°C. and heating a t a rate of 2°C. per minute, the noncationic chromium complexes fixed by collagen have ample time for rearrangement and formation of new attachments to collagen. Both types of leather will show complete resistance toward boiling water whereas, in the instance of direct contact of the specimens with water of the actual shrinkage temperature, the values will be 93 and 99°C. for the leather tanned in the directly diluted and the aged solutions, respectively (94). In actual practice the final shrinkage temperature will be practically identical since in the subsequent washing and processing of the leather the complexes will reach the stable state by structural rearrangement (68). Kiintzel, Kinzer, and Stiasny (128) consider that the main cause of the diminished chrome fixation with increased concentration of chrome is the dehydrating effect of the sodium sulfate on collagen, accumulating in concentrated solutions of “chrome sulfate liquors.” The dehydrated hide should possess less affinity for chromium. They do not consider the constitutional factor of the chromium salt to be of any importance. However, it is difficult to conceive why the large amount of sodium chloride present in concentrated solutions of basic chromic chlorides should not have the same effect (66,97). However, collagen shows a very sharp rise in chrome fixation in tanning with very concentrated solutions of chromic chloride.

384

K. H . OUSTAVSON

The explanation on a constitutional basis receives experimental support by data from the action of neutral sulfates (Na2SO4)on basic sulfates in regard to the chrome fixation by collagen. The formation of noncationic complexes possessing low affinity for collagen runs parallel with the decreasing uptake of chromium from such solution by the cationic exchangers (81).

d. Neutral Salt Effect. The action of neutral salts in the processes of chrome tanning is an important but exceedingly complicated problem. As a rule, the salts mainly concerned are sodium sulfate, a regular constituent of chrome-tanning preparations, and sodium chloride, present in the pickled pelt or added to the tanning bath as a regulator of swelling. The primary role of the neutral salts as agents which depress fiwelling may be illustrated by tanning neutral pelt, or still better pickled pelt, in a solution of a basic chromic sulfate free from sodium sulfate (for example a salt prepared by reducing chromic acid in the presence of sulfuric acid by means of hydrogen peroxide). The pelt will swell and the penetration of the chromic salt through the hide structure will be greatly retarded and tanning practically prevented if highly basic chromic sulfate is used. The cross linking of collagen will be sterically hindered (65). Hence, the primary function of neutral salts in chrome tannin& is the osmotic balancing of the swelling of the hide which takes place in the initial stage of tanning by the preferential fixation of the free acid by collagen. With the concentrations of neutral salts generally used, of the order of a few tenths molar, present in an acid system together with the astringent chromic salt, any direct, specific action of the neutral salt on the protein is not detectable and hardly likely. The main secondary function of the neutral salt will be its effect on the physicochemical properties of the solution, such as alteration of the degree of hydrolysis and, above all, constitutional and electrochemical changes of the chromic salt. The important function of neutral salts in chrome tanning was discovered by Wilson (212-214) some 30 years ago. Fundamental studies of this effect have been carried out by Thomas and coworkers (4,199-201). For details regarding the effect of neutral salts on the hydrogen ion concentration of solutions of chromic salts these papers should be consulted. Only a brief summary of the main principles and results of the neutral salt effect in the various systems can be given. Sodium chloride markedly increases the reactivity of chromic chloride for collagen in dilute solution of chromic chloride, while in concentrated solution the effect is the reverse (59). This action has been explained by the influence of the salt on the composition of the coordinated sphere. By the excess of chloride ions, chlorine groups are being forced into the internal sphere (59). In solutions of very basic chromic chlorides this effect is further

PROTEIN-CHEMICAL ASPECTS O F TANNINQ

385

accentuated by aggregation of the chromic chloride (59). Since in both instances the equivalent weight of the chromium complex will be increased, greater chrome fixation will result. The positive constitutional effect more than counterbalances the negative effect of increased hydrogen ion concentration of the system. I n concentrated solutions of basic chromic chlorides the constitutional change of the complexes has already been attained. Accordingly, it is not further altered by the addition of sodium chloride. I n this instance the main effect of the salt will be to decrease the chrc me fixation by the increased hydrogen ion concentration which it brings about (59). The diminished affinity of basic chromic sulfate for collagen in the presence of sodium chloride seems primarily to be connected with the formation of mixed chloride-sulfate by double decomposition (66). By addition of sodium sulfate to solutions of chromic chlorides, chromic sulfates are formed because of the great complex affinity of sulfate groups. For particulars see Gustavson (56,66). The system basic chromic sulfate-sodium sulfate presents less complication, being a system of a common anion. The addition of small amounts of sodium sulfate to a salt-free solution of basic chromic sulfate tends to increase the chrome fixation by collagen (164). This effect has nothing to do with the neutral sulfate effect on the chromic sulfate. It is probably a purely osmotic effect of the neutral salt on the hide, suppressing its swelling, facilitating the uptake and diffusion of the chromic sulfate. By further addition of sodium sulfate the affinity of the chromic sulfate for collagen is decreased. An over-all decrease is also generally found by the addition of sodium sulfate to the systems of the commonly employed type of chromic sulfate containing sodium sulfate, such as Cr2(OH)2(SO4)2.Na2SO4(212). The retarding effect of sodium sulfate on the chrome fixation cannot be a pH effect, since the hydrogen ion concentration is decreased. The real cause is the change of the chromic sulfate in the direction of uncharged and negatively charged complexes, possessing less affinity for collagen than the cationic ones (66,81). Possibly the formation of addition compounds between the components of the system with lower degree of activity is an additional factor (201). The reaction of chromic sulfates with the cation exchanger as a function of added sodium sulfate (81) shows a similar trend to that of the chrome fixation by collagen. Analysis of solutions by means of the ionic exchange method shows, e.g., that a solution of 66% acid chromic sulfate (1 equivalent per liter chromium) contains practically all the chrome in cationic form. Upon the addition of sodium sulfate, making the solution 1 M in NapSO,, it contains 65% cationic, 6% anionic, and 29% uncharged complexes. Since concentrated solutions of basic chromic sulfate contain mainly noncatiouic complexes, the retarding effect of sodium sulfate upon the chrome fixation by collagen should in this case hardly be perceptible, since by the addition of neutral

386

K. H. QUSTAVSON

sulfate further shift of the equilibrium of the various forms should not be expected. Thia is also the case (201). An important property in the investigation of tanning processcs, easily measured and stated, is the hydrothermal stability of the leather as a function of the neutral salt content of the tanning bath, measured by the shrinkage temperature. Generally decreased chrome fixation and formation of more acid chromium compounds in the skin, associated with the neutral salt effect, will tend to decrease the shrinkage temperature. That is exemplified by the system chromic sulfate-neutral salts. Sodium sulfate affects the hydrothermal stability only slightly, although it lowers chrome fixation more than does sodium chloride, whereas sodium chloride greatly decreases the hydrothermal stability (66). This may be ascribed to the fact that chromium chlorides do not yield leather of such high shrinkage temperature as that tanned with chromic sulfates. The formation of chloro-chromium complcxes in leather tanned with solutions of basic chromic sulfates containing large amounts of sodium chloride explains the low degree of stability of this type of leather (66). Concerning the systems chromic chlorides-neutral salts, it may be said that addition of neutral sulfates as well as of neutral chlorides improves the hydrothermal stability of the treated collagen, the stabilization being particularly marked in the first instance. The explanation of the action of neutral sulfate is the formation of basic chromic sulfate, which cross links the protein chains more effectively than chromic chloride (66,130).

The neutral salt effect in chrome tanning, presenting intricate theoretical problems, is also of great importance to practical tanning. It forms a versatile tool to regulate the degree of swelling of pelt in the initial chrome fixation which largely determines the character of the final leather. e. Hydrogen Ion Concentration. The hydrogen ion concentration of the tanning system, employing neutral pelt, is a factor interrelated with the composition of the chromic salt, primarily its basicity. The extremes are: simple complexes and high hydrogen ion concentration of solutions of the normal salts and aggregated larger complexes with lower hydrogen ion concentration present in the basic salts. Thus, increased baficity of the chromic salt will make itself felt in two respects, both favorable for the tanning effect: ( I ) increased size of the complex, facilitating multipoint attachment to the protein, and ( 2 ) lowered hydrogen ion concentration of the tanning bath, which means a greater number of carboxyl groups of collagen in the ionic form. A competition for these ionic groups of collagen is set up between hydrogen ions and cationic chromium complexes. In the chrome tanning agents usually employed a certain upper limit to the pH values of the solutions is set by the fact that the incorporation of too many hydroxyl groups will lead to such a high degree of aggregation that the ensuing compound will be too bulky and insoluble. Undoubtedly the hydrogen ion concentration is a very important factor, in combination with the factors discussed earlier. However, a

PROTEIN-CHEMICAL ASPECTS OF TANNING

387

too one-sided accentuation of one single factor may be precarious as shown by recent tendencies (146). According t o the hydrolytic concept (32), as modified by McLaughlin (146), the acid-binding function of collagen is the governing factor in chrome tanning. This hypothesis is in conflict with scveral fundamental facts of physical chemistry and our knowledge of chrome tanning. Among the many experimental findings contradicting the hydrolytic hypothesis (88) the following may be mentioned: Since the acid fixation by collagen reaches equilibrium in 24-48 hours and the chrome and acid fixations are interrelated, the chrome fixation should come to a standstill within the time given (87,88). This is not the case. Furthermore, the chrome fixation by various proteins is not a direct function of their acid-binding capacity; the ratio of fixed chromium to fixed acid in equivalents shows values ranging from 1.0 for silk fibroin t o 10.3 for blood fibrin (88). If tanning occurs in a n unchangeable system as assumed by the hydrolytir concept, the chrome fixation should be independent of the temperature of tanning, since the fixation of strong acids is not affected by the temperature (87). In reality, the chrome fixation is greatly increased by augmented temperature (56,151,164). Collagen pickled with acid and in equilibrium a t a given p H value, e.g., 2.5, should not take up chrome from a solution of basic chromic sulfate of a higher p H value, e.g., p H 3.5, according to the hydrolytic concept. However, this is not true. The practice of chrome-tanning pickled stock of lower p H value than that of the tanning bath is in fact in direct contradiction t o the adsorption hypothesis. Finally, the excellent tanning artion of certain complex salts in solutions with p H values on the alkaline side of the isoelectric point of collagen invalidates this concept (57). Also the findings of the neutriil salt effect reported conflict with this hypothesis, since no direct relationship exists between the hydrogen ion concentration of the solution of chromic salt in the presence of neutral salts and the fixation of chromium by collagen (88). Still, the hydrolytic concept must be given the credit of having called needed attention to this important detail of the chrome-tanning mechanism, particularly Elod’s scholarly contributions (32) demonstrating the importance of changes in the chromic salt fixed by the protein during the tanning and in the subsequent processes.

The rate of chrome fixation is increased by increase of temperature (151). A number of factors are involved in the temperature function of the system. The equilihria in hydrolytic systems are complicated by constitutional changes of the solutes which are greatly temperaturedependent in regard to the degree of hydrolysis (pH), degree of aggregation of the basic chromium compounds, their constitution, and electrochemical behavior. Furthermore, the protein component, being an amphoionic structure, is highly temperature-dependent, the ratio of charged to uncharged protein groups decreasing with increasing temperature (87). f. Influence of Previous History of Collagen. The influence of the hydrogen ion concentration of the environment upon the degree of reactivity is a general phenomenon, discussed in connection with the various factors. The effect of various pretreatments of collagen, leading to certain irreversible changes of the protein, upon the chrome fixation

388

K. H. OUSTAVSON

will be briefly considered, since it supplies certain information regarding the mechanism of the reaction (66). The acid-binding capacity of collagen is not changed by its pretreatment in strong solutions of lyotropic neutral salts, as, e . g . , 1-2 M calcium chloride and thiocyanate, nor by previous prolonged immersion in solutions of hydrotropic agents, such as urea in concentrated solution, weak organic acids, or 2-3 M acetic acid; furthermore hydrothermal denaturation does not alter this acidbinding capacity. The fixation of chromium by collagen from solutions of simple cationic chromic sulfates and chlorides generally used in tanning is not influenced by these pretreatments, with the exception of the acetic acid pretreatment, which leads to increased chrome uptake, probably as a result of the liberation of acidic groups of collagen by means of the deamidation occurring in prolonged treatment with acid solutions. Accordingly, the conclusion seems justified that forces of coordinate nature do not govern the primary fixation of simple chromic salts. However, the fixation of highly aggregated salts, e . g . , those with acidities less than 50%, is a function of the pretreatment, considerably increased chrome fixation being noted by pretreated collagen. This increase may be of the order of 75-100% of the original fixation for the highly aggregated sulfito-sulfato chromiate, containing mainly uncharged and negatively charged chromium complexes (Table 111). The coordinate effect is also evident in the fixation of compounds of the type of tetraoxalatodiol chromiate (57), and generally in reactions involving coordination of the tanning agent on collagen (84). By prolonged pretreatment of native collagen in solutions of alkali in the range p H 12-13, the coordinate reactivity of collagen as well as its ionic reactivity by means of the carboxyl groups is increased (66,71). The uptake of the simpler types of chromium compounds which are not affected by changes in the coordinate property of collagen is increased by the alkali pretreatment, indicating ionization of carboxyl groups. 3. Nature of the Chrome-Collagen Compound

Even such small amounts of combined chromium as 0.5-1.5 g. per 100 g. collagen impart a high degree of stabilization. With chromium contents of or greater than 2-2.5 g. per 100 g. collagen, the leather will as a rule withstand the action of boiling water. I n earlier sections it was pointed out that a tentative explanation of the reaction mechanism of basic chromic salts with collagen is given by the concept of the formation of a modified type of internal complex salts (chelate compounds, involving groups of different protein chains). According to this concept, the initial reaction is an ionic interaction of cationic chromium complexes,

PROTEIN-CHEMICAL ASPECTS OF TANNING

389

such as (Cr20SOd),2n+, with the charged carboxyl groups of collagen, the sulfate ions being compensated by the NHs ions. The carboxyl groups, having a great tendency to complex formation and for a direct attachment to chromium, penetrate into the coordination sphere, forming a covalent-coordinate bond. Since several chromium atoms are present in large chromium complexes, and in view of the secondary aggregation of the fixed chrome complexes by further hydrolysis, possibilities may be a t hand for a multipoint interaction of one chainlike chromium complex with several carboxyl ions of the collagen lattice, resulting in the linking of adjacent protein chains by strong bonds by means of the chrome bridge. Shuttleworth's conductivity data for gelatin solutions containing chromic salt point to the inactivation of carboxyl ions a s the main reaction (182). This type of cross linking an olated chromium complex, containing several

/

oc \ HC /

NH

O )

X--'C:..-OH,

/

*\

HO, 'OH (CH,),COO-'CT-------NH,(CH,)~CH /

'.

/

HO.

'b

'.'OH

/

H2O-.C?-X

/

\

,% ,

\ HN

\

FIG. 3.

chromium atoms, by means of two or more carboxyl groups on adjacent protein chains, accounts sat,isfactorily for the chemical behavior of chromed collagen, particularly the increased reactivity of the basic protein groups resulting from the chrome tannage ((31). I n view of the new data (Table 11) of the free carboxyl groups, previous objections based on spatial and stoichiometric considerations are largely removed. The numerous hydroxyl groups may further act as supplementary links since the oxygen of this group possesses a residual negative charge (183). Moreover, the chromium atom possesses great coordination power. Coordinate centers are numerous in the protein chains in the form of basic groups and particularly as hydroxyl and peptide groups, containing both carbonyl and imino groups with coordination-active 0- and H- groups. A possible type of combination is illustrated by Fig. 3. The postulation of the formation of secondary stabilizing links between the chromium complex and these groups is chemically sound and may represent the most probable course of the reaction, although direct evidence for this concept has not yet been adduced. Indications th a t chrome fixation by collagen is intermicellar, only affecting the surface ionic groups of micelles,

390

K . H . GUSTAVSON

have been supplied by Kiintzel (122). The hardening of chrome leather upon drying and certain aspects of its reactivity are in accord with this view. The most direct indication is of a qualitative sort (130). The bluegreen solution of basic chromic sulfate, diffusing into a gelatin gel, imparts a strongly violet color to the layer penetrated. Chromium compounds containing carboxyl groups directly attached to chromium, e.g., chromium acetate, show the very same color. The direct interaction of carboxyl groups of gelatin with the chromium atom is thus indicated to take place. It was early recognized (54,62) that a prototype of the chrome-collagen compound was the structure of the internal complex salts which copper and chromium form with simple amino acids. These compounds have been extensively investigated by Ley (136), who together with Pfeiffer (172) first conceived them as internal complex salts. In such structures the central atom, chromium in this casc, completes its coordination number by primary (covalent) interaction with the carboxyl groups and coordination with the amino groups of the same molecule, forming a stable system. The diol-chromium glycinate (137) is the classical example of these interesting compounds and especially useful for the present problem:

In chrome tanning various groups of adjacent protein chains are probably incorporated into the coordination sphere of the aggregated chromium complex, resulting in the riveting together of different chains on the same complex. The nonionic nature and the high degree of stability of the formed chromium-collagen compound is thus explained. Kiintzei’s spectrophotometric investigations of chromium glycinates and the complexes formed between gelatin or degraded collagen and chromium nitrate show very similar type of curves (124,130). This similarity constitutes the best available indication of the presence of structures of the type of internal complex salt as the reaction product of collagen and gelatin with basic chromium salts. By inactivation of carboxyl groups of collagen by methylation, the chrome fixation decreases about 70%. I t is interesting to note that the chrome taken up by collagen lacking in ionic carboxyl groups does not sfabilize the structure, although the same amount of chromium in untreated collagen markedly increases its shrinkage temperature and

PROTEIN-CHEMICAL ASPECTS O F TANNING

39 1

results in tanning. Deamination of methylated collagen only causes a further slight decrease in chrome fixation (13a). These interesting findings are in harmony with results obtained in a study of the fixation of chromium by collagen, with its carboxyl groups completely discharged (pH 1.0) from solutions of basic chromic sulfates (68). Acid-saturated collagen fixes only small amounts of chromium, which do not exert tanning effects, as measured by shrinkage temperature and tryptic resistance of tlhe stock. These investigations also show that ionized carboxyl groups play a major part in the fixation of chromium and for the tanning effect. These findings further suggest that the chromium complexes do not combine with amino groups independently of carboxyl groups. The interaction of chromium complexes with electrovalent groups of collagen should be expected to shift the isoelectric point of the original protein. By removing the protein-bound acid of hide powder, tanned with basic chromic sulfate, by means of pyridine (60), and then carefully freeing it from the latter, the isoelectric point of the chromed collagen was located in the p H range 6-7 (212) by the dyestuff fixation technique (206). The isoelectric point of the original hide powder was 5.5. The dioxalatochromiate tannage shifted the isoelectric point toward the acid side, p H 4 . 0 4 . 5 being obtained (212). Thus, the fixation of cationic chromium complexes by collagen leads to inactivation of acid protein groups, whereas the anionic oxalato compound is indicated to combine with basic protein groups. In recent investigations by Theis (196), examining suspensions of tanned hide powder in a cataphoretic cell using buffer solutions, values in the opposite directions to those previously mentioned were obtained. According to Theis’ findings, the cationic chromium complexes should mainly inactivate basic protein groups. He found collagen tanned with chromic sulfate isoelectric a t p H 4.50. Determinations of the isoelectric point of chrome-sulfate-tanned hide powders, using borate buffers, which do not interefere with the composition of the fixed chromium complex, by cataphoresis, gave values of pH 6.5-7.0* The oxalato-chromed hide powder did not migrate a t pH 4.5.* By the use of complex-forming buffers, containing phosphate and acetate, the composition of the cationic chromium complex was radically changed, leading to dislocation of bonds. The sulfate-tanned hide powder then was isoelectric a t p H values below 5. The importance of employing buffers without complex affinity for chromium is strikingly demonstrated. By the electrokinetic technique of Neale (160), the isoelectric point of the pelt tanned with cationic chromium sulfate was * Determinations carried out at the Swedish Institute of Textile Research, Gothenburg (unpublished) by Mr. B. Olofsson whose cooperation is gratefully acknowledged.

392

K. H. GUBTAVBON

found to be in the vicinity of p H 7 ; that of oxalato stock a t p H 4.* These values were obtained in the absence of buffers in 0.02 M potassium chloride solutions. Theis’ findings are probably vitiated by the secondary action of the unsuitable buffers employed (phosphate and acetate). It is interesting to note that by light drying or acetone dehydration of chrome leather its isoelectric point is displaced toward the acid side (94), possibly due to gradual inactivation of basic protein groups.

4. Some Important Properties of Chrome Leather of Theoretical Interest The most significant property of chrome leather is its high degree of hydrothermal stability as mentioned previously. A well-tanned chrome leather will withstand a few minutes’ boiling in water. I n a recent method the determination of shrinkage temperature of leather is carried out in a glycerol-water mixture in order to assess shrinkage temperatures above 100°C. (148). Objections may be raised against the use of such mixtures since the environment is altered. The use of cod skin, immersed in water, for the determination of the degree of stabilization of the collagen lattice by chromium salts is free from this objection. Cod skin with a shrinkage temperature of 45°C. leaves a range of 55°C:. to the boiling point whereas bovine skin only gives a margin of about 30°C. A fully tanned chrome leather is not digested by trypsin, as first noted by Thomas and Seymour-Jones (208). At that time nothing definite was known about the relation of cross linking and chemical structure, on one hand, and the stability of proteins toward proteinases, on the other hand. Since trypsin generally is considered to hydrolyze peptide groups of proteins, it was natural to conclude that chrome fixation is localized to and inactivates peptide groups. However, i t was later found that native collagen with its internal cohesion intact is resistant toward trypsin and further that structures stabilized by means of covalent cross links, e.g., keratin, are not attacked. Accordingly, the inertness of chrome leather toward trypsin only shows that the protein structure is effectively stabilized by the new cross links (64). By chrome tanning of collagen, its reactivity for acid dyestuffs of the sulfonic acid type is greatly increased as is also the irreversible fixation of vegetable tannins (61). Since in the pH range (3-5) involved in the latter reaction collagen contains a large part of its ionic groups internally compensated and thus not available for interaction with the reactants mentioned, their fixation will be governed by the degree of ionization * Determinations carried out by Professor 5. M. Neale (College of Technology, Manchester, England), whose kind cooperation is gratefully acknowledged.

PROTEIN-CHEMICAL ASPECTS OF TANNING

393

of these groups according to the equilibrium pH of the system; and only that part of the total binding capacity of collagen can be utilized by the anionreacting agents. The chrome-tanned collagen, on the other hand, contains all or the greatest part of its basic groups in the free, reactive state, independent of the p H value of the system, as the result of the breaking up of the internal salt structure of collagen by the ionic interaction of the carboxyl ions with chromium complexes. I n order to obtain the same degree of fixation of vegetable tannins by collagen as by chromed collagen, extensive periods of tanning (several years) (169) and high hydrogen ion concentration of the tanning systems are required; whereas the maximum fixation of vegetable tannins by chromed collagen is reached within a few days’ time by applying vegetable tannins in solution of p H 4-5 (61). This activation of the vegetable tannin fixation is of practical importance and extensively applied in the manufacture of combination-tanned leather, although it was not until recently that this important function of the chrome-tanning process was fully realized. It is a n excellent example of the import.ance of the availability of reactive protein groups. The acido groups of the chromium complexes fixed by collagen may be completely or partly displaced by other groups, which may result in changed hydrothermal stability. In reactions in solutions certain anion series are shown, e.g., oxalate, acetate, formate, sulfate, chloride, and nitrate, with the oxalate possessing the greatest and the nitrate group the lowest degree of affinity for chromium (193). I n the interaction of complex-forming salts with chrome leather this order may be modified by the mass action effect. In the treatment of collagen tanned with basic sulfates, the protein-bound sulfuric acid being previously removed, only slight amounts of sulfate are present in ionic form in contrast to the high concentration of salt used for the treatment. The mass action effect of the solute will dominate over the complexforming tendency. This will explain why sulfato groups of chromium-sulfate-tanned collagen are nearly completely displaced by treating the stock for a few days in a 1-2 M solution of sodium chloride containing a group of low degree of complex formation (65,66). The original leather, standing the boiling test, will upon such a treatment show large shrinkage in boiling water. On the other hand, the treatment of a sulfate-tanned leather of a shrinkage temperature of, e.g., 93°C. for a few hours in a solution of 1 M sodium sulfate will mostly result in a leather resistant to boiling (66). In order to explain the stabilizing effect of sodium sulfate on the sulfato-chromecollagen eompound, it has been suggested that by the penetration of sulfate groups into the internal sphere several sulfate groups may become associated with one chromium atom in the polynuclear complex. This particular chromium atom with an excess of sulfate groups acquires negative charge (131). Accordingly the fixed complex will function as a n amphoionic structure; t h e negatively charged chromium atom serving as a locus for additional stabilization of the structure by its electrostatic interaction with positively charged protein groups. The concept of Latimer and Porter (133) regarding the residual charge of a particular atom, e.g., N in NHz or NHa+,applied by Smythe and Schmidt (183) to ferric proteins is also of interest in thia connection (68).

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€I QUBTAVSON .

These examples ehow the importance of the constitution of the chromium complex fixed by collagen for its hydrothermal stability and for other physical properties. Evidently the valency partition within the chromium complex, especially the nature of the acido groups, determines the efficiency of the chromium atom as a center of coordination, a function of fundamental importance for the stability of the chrome-collagen compound. The state of ionic protein groups and steric conditions of the protein chains are the main factors of the protein component.

VI. VEQETABLE-TANNINQ PROCESS 1. General Properties of Vegetable Tannins

The tannins, polyphenols of complicated structure, may conveniently be classified as: (1) Hydrolyzable tannins, which are esters of hexoses and phenolcarbonic acids, the simplest type being tannic acid. ( 2 ) Condensed tannins or the catechin type, the latter being the prototype for this important category. Hydrolyzable tannins containing ellagic acid are sometimes considered as a third group. Tannins produce on the tongue the sensation of puckering. Substances causing this effect are called astringent in proportion to the degree of the effect. A numerical classification of the various tannins and vegetable-tanning materials according to their degrees of astringency is not possible, since a number of factors are involved. The astringency appears to be a function of the molecular size of the tannins, the proportion of tannins to total solubles (degree of purity), the hydrogen ion concentration of the solution, the electrical charge of the particles, nature of anions and neutral salt present, temperature, and tannin content of the solution. Finally, the state of the protein substance is an important factor. It has been assumed that the affinity of the tannins for collagen, at comparable experimental conditions, is a measure of the astringency. I t has also been suggested that the type of protein groups involved in the fixation of the tannins by collagen may affect the astringency (70,83). Quebracho and tannic acid are classified as astringent tannins, whereas gambier and myrobalans, possessing lower degree of affinity for collagen, belong to the nonastringent group. Table I V contains data for some important tannins, regarding their source, type, degree of purity, pK value (5a), and molecular weight (30), measured by the depression of the freezing point of electrodialyzed solutions (1-2.5% total solubles). The molecular weight of the tannins purified by dialysis is in the vicinity of 1000-4000 (30,110) but also higher values are indicated; the

395

PROTEIN-CHEMICAL ASPECTS OF TANNING

molecular weight is influenced by the hydrogen ion concentration, high acidity tending to aggregation. The problem of the charge of the tannins has been variously interpreted. Some investigators have found them to be uncharged (18); others consider them to be charged (202). Since the polyphenols are very weak acids (pK > 6 ) , the ionization of the phenol groups should be marked only at rather high pH values and also their reactivity as electrolytes depressed a t low pH values. The tannins containing stronger acidic groups, e.g., carboxyl as in valonia (185), cerTABLE IV Some Properties of Vegetable Tanning Material8 Material

Source

Galls on leaves of species of Quercus and Rhu8 Hydrolyzable Fruits of Terminnlic Hydrolyzable Myrobalans chebula Wood of Caetanea Hydrolyzable Chestnut vesca Leaves of Uncaria Condensed Gambier species Condensed Mimosa (wattle Bark of Acacia species bark) Wood of Quebracho Condensed Quebracho colorado Wood of Quebracho Condensed .Sulfited colorado quebracho

Molecular weight

Purity

Tannic acid

89 62

5.c 4.5

3 100-3400 1900

76

5.c

1550

55

4.5

520

79

> 6.0

1600- 1700

89

> 6.0

2420

89

> 6.0

760

tainly react ionically with collagen in the pH range (3-5) usually encountered in vegetable tannage. Recent investigations of purified solutions of tannins by electrophoresis show a part of the wattle and quebracho tannins as well as the valonia tannins to be charged (29). However, the high-molecular fractions did not migrate (29). Reactions between vegetable tannins and collagen generally seem to involve both electrovalent and coordinate forces, the latter type being the dominating one in some tannins. Investigators of the mechanism of vegetable tannage have surprisingly enough emphasized either the one or the other function. However, the final answer will probably have to recognize both types of reaction, with preference for a particular reaction in certain instances. That will not necessarily mean that one type of tannin is attached by electrovalent reactions to the basic protein groups and another type of tannin coordinated on peptide groups. It rather

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seems that the very same large molecule may interact with collagen by means of both types of valencies (multipoint fixation). Mechanical deposition of the tanning material in hide probably accounts for a large part of leather formation. 2. Factors Governing the Reaction

a . Hydrogen Ion Concentration. The hydrogen ion concentration of the system is probably the foremost factor in vegetable tanning. Our present knowledge of this factor is due mainly to the pioneering investigations of Thomas and his school (205). This effect is very intricate. First, it involves the influence of the hydrogen ion concentration on the tannins, in regard to their charge, degree of aggregation, and secondary chemical reactions (at high pH values). Second, the effect of the pH of the system upon the collagen substrate is important, and no doubt is the deciding factor with respect to the degree of swelling, the accea-

0

2

4

6 8 Final pH

1

0

FIG.4.-Average curves of the irreversible fixation of vegetable tanning by hide powder as a function of the concentration of the tannin solutions at attained equi, 2 weeks’ tanning; - - -, 24 hours’ tanning. librium (212).

-

-

sibility of reactive groups, and the internal resistance of the hide structure to the diffusion of the tannins. In this tannage the state of the hide fiber weave enters heavily. Fig. 4 shows the average curves of the fixation of six commercial tannins by hide powder run in series for 24 hours and 2 weeks, as a function of the final hydrogen ion concentration of the systems (212). The sharp minimum of fixation in the pH range of the isoelectrio point of collagen is especially noteworthy, as is the greatly increased tannin fixation which occurs on lowering the pH value. On the alkaline side of the isoelectric point a marked fixation also takes place. However, a sharp drop is clearly evident a t pH values greater than 8. These fixation curves illustrate the irreversible fixation of tannins, i.e., the part

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397

which forms a compound with collagen resistant to water of p H values corresponding to the equilibrium p H values of the tanned stock. The total sorption of substances by collagen is far less pH-dependent than the irreversible tannin fixation. The curve of the irreversible tannin fixation after 2 weeks’ tannage is also more evened out in the whole pH range than the corresponding curve from the short period of tanning. The formation of coordinate compounds between tannins and collagen is indicated to be the main reaction in the pH-independent process. Simple prototyes of such molecular compounds between amino acids and anhydrides, on one hand, and phenols, on the other hand, have been isolated by Pfeiffer (172). The importance of such coordinated systems for the vegetable-tanning process has especially been emphasized by Freudenberg (44) and Stiasny (193). The coordinate reactivity of collagen should be pH-independent over the whole pH range if swelling and rupture of hydrogen bonds is excluded; this is shown in fixation of tannins in solutions of pH values lower than the pK values of the phenolic groups, since intact hydroxyl groups are necessary for coordination. The influence of the hydrogen ion concentration on the molecular weight of the tannins is a complicating factor, more or less prominent according to the type of tannins. b. Protein Groups Involved. The tannins contain numerous phenolic groups interspaced on the chainlike structure and also, more sparingly represented, ionic groups which interact with favorably located protein groups of adjacent chains, resulting in a multipoint attachment of the tannins. According to the nature of the tanning agent, especially the relative proportions of electrovalent and coordinate groups, and the experimental conditions, primarily hydrogen ion and tannin concentrations and time of interaction with a given collagen substrate, the preponderance of one of the two main binding types will be determined. Our present knowledge of the protein groups concerned in this tannage will be briefly reviewed. Regarding the basic groups, the first direct demonstration of their participation in the fixation was adduced by Thomas and collaborators (204), who showed that deamination of collagen (removal of c-amino groups of lysine) decreases its capacity for irreversible fixation of tannins. Further, by complete inactivation of the basic protein groups by means of irreversibly fixed sulfo acids, e.q., pentanaphthalenetetramethylenedisulfonic acid, a more or less marked decrease of the tannin fixative capacity of collagen will result; this constitutes further evidence for the importance of the basic groups (70). In a comparative study of tannin fixation by untreated hide powder and hide powder with inactivated basic groups, the following findings are of interest (70). The former will react with all available groups, ionic and nonionic, and the latter mainly with the nonionic groups (peptide

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K. H. GUSTAVSON

groups). The difference of the fixation obtained in the two series represents the fraction of tannins fixed by the basic groups. With increasing hydrogen ion concentration the participation of the basic groups is more prominent. The hydrothermal stability, imparted to the fibers by the action of the tannins, is found to be associated mainly with the fraction of tannins attached to the basic groups. The equilibrium of the reaction with the basic groups is rapidly attained. It was further indicated that the uptake of fannins by collagen upon prolonged tanning is mainly accounted for by the fraction reacting with peptide groups. Further, a t increased concentrations of tannins as well as of hydrogen ions, the peptide-bound fraction of tannins is not affected; the increased reactivity is due mainly to the ionic protein groups (70). Further evidence for the participation of the basic groups in vegetable tannin fixation may be cited. Vegetable tannins displace rather completely the fixed highpolymeric phosphate in hexametaphosphate-treated hide (94). Displacement of tannins is further shown by certain sulfo acids, which have a selective affinity for the basic groups (85,92). Even 0.1 N solutions of mineral acids may displace some fixed tannins (92). The isoelectric point of hide powder tanned with wattle bark or quebracho tannins is displaced 1 pH unit toward the acid side, or from pH 5.5 to 4.0-4.5, indicating inactivation of basic protein groups (212).

Regarding the nonionic protein groups, primarily the peptide groups as loci for coordinate valency forces, a great array of facts may be cited as indication or evidence for their important function. First, the different behavior of vegetable tannins and simple sulfo acids toward water-soluble urea-formaldehyde condensation products, introduced by Grassmann and coworkers (51), merits attention. The Grassmann reagent, consisting of -NHCO-NHCH2*NH*CO*NH- units, forms heavy precipitates with weakly acid solutions of vegetable tannins, being the most sensitive tannin reagent known. On the contrary, condensed sulfo acids, such as the condensed naphthalenesulfonic compound earlier mentioned, which do not carry coordination-active groups, are not reacted upon or precipitated by the Grassmann reagent, which seems t o be specific for agents reacting with the main bonds of the urea-formaldehyde, the peptide groups. This fact together with certain considerations of the acidbinding capacity of vegetable-tanned collagen led Grassmann and coworkers (51) to the conclusion that the basic protein groups are not involved in the reaction of tannins with collagen. The data from the vegetable tanning of collagen with blocked basic groups also show the presence of nonionic protein groups with affinity for tannins (70). Further indications have been supplied by the study of the influence of certain pretreatments on the amount of irreversibly

PROTEIN-CHEMICAL A S P E C T S O F T A N N I N G

399

fixed tannins. Pretreatment of hide powder in solutions of lyotropic salts of 1-2 M strength does not alter the acid-binding capacity of collagen, but largely increases the fixation of tannins; the increase generally is of the order of 50-100% (55). The same effect, but t o a still larger degree, is obtained by pretreatments in 3 M acetic acid and in 6-8 M urea (71,79). Finally, by hydrothermal shrinkage of collagen the very same trend is shown. As mentioned in the section on chrome tanning, the fixation of simple ionogens is not affected by the pretreatments mentioned, which forms conclusive evidence for the participation of nonionic groups in the irreversible attachment of tannins to collagen (71). c. Degree of Stabilization. Further illustration of this point has been obtained by investigation of the stability of the irreversibly fixed vegetable tannins in leather toward repeated treatment in 6-8 M urea (83). The action of urea on soluble proteins is generally considered to be specifically directed toward the peptide groups, although in view of the dipolar nature of urea, involving resonance with a dipolar ionic form, reactions with ionic groups may also occur to some minor extent. I t is interesting to note that practically all or the greater part of the tannins of the nonastringent class are removed by one week’s treatment of the leather in 8 M urea. The detanned leather shows shrinkage temperatures below that of the original pelt, probably as a result of the denaturation of the detanned collagen by urea. Collagen in combination with astringent tannins loses about half the total amount of combined tannin. However, the extracted leather is not pelty and gluelike as in the abovementioned case but has retained its leathery properties; the shrinkage temperature is lowered only a few degrees. The order of the degree of removal of various tannins by urea solution is practically identical with the series arranged according to the ratio of tannins combined with peptide groups to those attached to basic groups, commencing with myrobalan and ending up with wattle bark and quebracho tannins. The shrinkage temperature of collagen in combination with sorbed matter, including nontans and reversibly fixed tannins, is markedly increased by the removal of the sorbed matter; increases up to 6°C. have been recorded (168). This is not due to the p H effect on the leather, since even leather tanned a t pH 4-6 behaves similarly (85). Since i t is indicated t h at the initial stage of vegetable tanning involves primarily the basic protein groups, and the later stage coordination reactions (70), it is interesting t o note t h a t the optimum of hydrothermal stability is obtained at rather low percentages of combined tannin (short duration of tannage) (168,197). Further increase of the degree of tannage leads to a slight lowering of the shrinkage temperature. The great decrease of the stability induced by sorbed matter is probably due to a breaking up of hydrogen bonds through the association of sorbed matter on the peptide groups without cross-link formation. Vegetable tannage imparts to collagen

400

K. €I GIUSTAVSON .

increased resistancetoward trypsin (12,75). Of great interest is the drastic removal of fixed tannins by aqueous acetone, reported by Merrill and coworkers (150), and also the fact that tannins dissolved in ethanol are not fixed by collagen (20), which evidently shows that water must be involved in this tannage. By saturation of hide with tannins dissolved in water-miscible organic solvents, e.g., acetone, pressing out the excess of the solution, and subsequent immersion of the impregnated stock in water, tanning takes place, yielding vegetable-tanned leather (176).

3. General Comments on the Theory of Vegetable Tannage

I n their suggestive paper on “Collagen Structure and the Vegetable Tanning Process,” Braybrooks, McCandlish, and Atkin (16) justly remark that very few of the tanning theories take into consideration the very important factor of the molecular structure of the hide itself, and further the fact that the vegetable tanning agents are colloidal. The starting point in their discussion is the accessibility of the amino groups to the tannins. Modifying their concept by including the coordinate groups, which according to data to be discussed later are the groups chiefly affected by swelling, the accessibility factor means a n important advance in our conception of the vegetable tanning process. As pointed out by these authors, the vital factor is the swelling of the micellar structure of the hide in order to make it accessible to the tannins. The parallelism between the curves showing the effect of the p H value of the systems on the vegetable tannin fixation, on one hand, and on the degree of swelling of the hide, on the other hand, is striking. Braybrooks, McCandlish, and Atkin advance as a final proof of their assertion the fact that hydrogen ion concentration does not affect the tannin fixation after the hide structure has been “struck through ” (penetrated) by tannins and thus somewhat fixed by the light tannage. It is shown that the initial swelling is the governing factor in tannin fixation. The influence of the osmotic condition of the hide substrate on its affinity for vegetable tannins will explain the important practical finding of the English school ( 5 ) regarding the controlling importance of the content and type of neutral salts in the tannin solutions and the importance of the ratio of free and total acid of the solution for the vegetable tanning process. The primary role of the p H factor in vegetable tannage on the swelling of collagen was first recognized and experimentally indicated by Vogl (219). In order to demonstrate the fallacy of the Procter-Wilson extension of the Donnan effect (211) to vegetable tanning, Vogl carried out the following, very simple but convincing experiments. Portions of hide powder in equilibrium with aqueous solutions of p H 5 and 3, the latter accordingly considerably swelled, and the former not swelled a t all, were lightly treated with formaldehyde in order to fix the degree of

PROTEIN-CHEMICAL ASPECTS OF TANNING

40 1

swelling. The hide powder specimens were then tanned in solutions of various tannins at p H 3 and 5. The amounts of irreversibly fixed tannins obtained were the same in tanning a t p H 3 and 5 for the specimens of the same degree of initial swelling. This proves the contention of Vogl that the main effect of the hydrogen ion concentration in vegetable tanning is directed toward the protein, regulating its degree of swelling. The data may also be interpreted in terms of the activation of the protein groups involved in the tannage (ionic as well as coordinate valencies), as a function of the hydrogen ion concentration of the system. The importance of the degree of accessibility of the basic groups to tannins is also stressed by Page (167,168). It may be remarked that, from consideration of the amphoionic nature of collagen and the internal compensation of oppositely charged groups, it is self-evident th a t the activity of the basic groups is a function of the hydrogen ion concentration. Since the maximum degree of swelling as well as the corresponding point of tannin fixation coincide with the p H value a t which the complete discharge of carboxyl ions and maximum activation of charged basic groups is accomplished, the concept of accessibility does not necessarily need to be restricted to the osmotic swelling, although i t is likely th a t the micellar changes resulting from the disorganization, due to osmotic forces, in themselves are most important. Page cites some data of Beek (7), who in experiments on the acid equivalent of fully tanned vegetable leather found that only about half of the basic groups had combined with tannins. The maximum degree of inactivation of the basic groups was reached in the initial stage of tannage, a t rather low values of fixed tannin, further tannin combination not affecting the free basic groups. This finding is in complete agreement with the results of the tannin fixation by collagen with inactivated basic groups discussed earlier. Page mggests that the apparent inaccessibility of half of the basic groups to the tannins is due to the inaccessibility of certain basic groups to the high-molecular tannins, deduced from Huggins’ (109) model of collagen structure. Page’s hypothesis is in harmony with findings on the accessibility factor in the reaction of high-molecular sulfo acids to be discussed in the following section. The pH independence of vegetable tannin fixation by chrome-tanned hide (61) discussed earlier has also some bearing upon the problem of accessibility of reactive groups. Since optimal conditions for reactivity of collagen are created by the chrome tannage, the maximum fixation of tannins is easily obtained without the aid of hydrogen ions. Furthermore, topochemical complications due to diminished rate of diffusion of the tannins through the gel-like hide structure are eliminated since swelling is avoided.

402

K. H. GUSTAVSON

I n this connection, it is interesting to note that in tanning hide powder with wattle tannin, which is only slightly acid- and salt-sensitive, at pH 2 and 5 in the presence of 4 volume per cent salt, practically the same degree of vegetable tannin fixation is obtained (94). Since complete discharge of ionic carboxyl groups of collagen and consequently liberation of charged basic groups are obtained at pH 2, without altering the accessibility of coordinate loci on the peptidc groups, which requires disorganization of protein chains by swelling, the findings indicate the primary role of the coordinate type of reaction in vegetable tannage. Further, it proves the insufficiency of the concept of the activation of the basic groups and their accessibility as the regulating factor in the fixation of tannins by collagen.

VII. REACTION OF CONDENSED SULFOACIDS(SYNTANS) WITH COLLAGEN The synthetic tanning agents called syntans are generally condensation products of aromatic hydroxy compounds and formaldehyde, made soluble by introduction of the sulfonic acid group(s). The irreversible fixation of strong sulfo acids by hide protein is regulated by the stoichiometric acid-binding capacity of collagen, the degree of affinity of the sulfo acid anion for the basic protein groups, and the stability of the bonds formed. The reaction of the large sulfo acid molecule containing several reacting groups is primarily governed by the anion affinity, a problem discussed in connection with the reaction of acids with collagen. The effect of the molecular size of the sulfo acid on its reaction with collagen and the nature of the compound formed is illustrated by naphthalenesulfonic acid and its condensation products. @-Naphthalenesulfonic acid, which does not swell hide (pH 2), is partly irreversibly fixed by collagen. The breaking of ionic cross links of collagen by the fixation of the acid leads to decreased hydrothermal stability; the shrinkage temperature being lowered 10-12°C. By condensing three or four naphthalene units by means of formaldehyde and introducing two terminal sulfonic acid groups, the resulting compound will be irreversibly fixed to a large extent. These compounds will convert hide into a leatherlike product. The hydrothermal stability is not decreased. Compounds of still higher degree of condensation yield leather with shrinkage temperatures a few degrees higher than that of pelt. Both the sulfonic acid groups of the irreversibly fixed compounds interact with the protein. Since no coordination-active groups are present in this type of C O W pound, the behavior of these compounds toward collagen illustrates the importance of the molecular size of the tanning agent for stabilization of the proteins. More complicated types of compounds, indicated to react entirely by means of electrovalent forces, are present in the high-

PROTEIN-CHEMICAL ASPECTS O F TANNING

403

molecular fraction of lignosulfonic acid (76,78). These compounds, with molecular weights of 5,000-10,000 and with an average equivalent weight of about 500, are to a great extent irreversibly fixed by collagen. The equivalent weight of the fixed sulfo acid is also about 500 (96). I t has been proved that the lignosulfonic acid molecule, containing from ten to twenty sulfonic acid groups, is irreversibly attached to collagen by means of all available sulfonic acid groups (96). Further, the reversibly sorbed part of the lignosulfonic acid only reacts by means of two to four sulfonic acid groups of the ten to twenty groups present. By the multipoint attachment of the large molecule on collagen by means of numerous sulfo acid groups a highly resistant compound is formed. The previous discussion has concerned the mechanism of the reaction of collagen with such sulfo acids as predominantly react electrovalently. In the tanning agents of sulfo acid type, however, the presence of coordination-active substituents seems to be necessary (phenolic groups). In the remarkable work of Wolesensky (216)) reported in the early twenties, condensed polyphenols not containing sulfo groups were studied. Resorcinol and pyrogallol were condensed by means of formaldehyde to water-soluble products. Particularly the former compounds possessed excellent tanning properties, yielding in neutral solution a full and stable leather. The fixation was quite independent of the hydrogen ion concentration of the system within a wide pH range, which is typical for the coordinate type of protein reactions. However, the accumulation of phenolic groups on a n extended molecule does not lead to a sufficient degree of water solubility of the product, as shown by Wolesensky’s experiments. The correct balancing of the size of the molecules, or rather their size distribution, seems to be the fundamental problem, controlling water solubility and reactivity as well as the degree of irreversible fixation with multipoint linking of the various groups. Polydisperse systems are advantageous, and the proportion of ionic groups (generally sulfo) and coordinate groups (generally hydroxyl) is important. See also the papers of Croad (26) and Kuntzel and Schwank (132). The use of simple condensed sulfo acids for inactivation of the basic protein groups and the influence of this pretreatment of collagen on the subsequent retannage with vegetable tannins have been mentioned in the section on vegetable tannage. The behavior of this type of sulfo acids toward vegetable-tanned collagen throws further light on the mechanism of the two processes. In comparing the fixation of a strong mineral acid (HC1), a condensed naphthalenedisulfonic acid (two to three naphthalene units), and the high-molecular fraction of lignosulfonic acid by dried vegetable-tanned collagen subsequently hydrated a t optimum p H values of the fixation, the following interesting findings were obtained (96).

404

K . H. QUSTAVSON

The fixation of hydrochloric acid by the thoroughly hydrated, vegetabletanned hide powder (tanned by means of wattle, quebracho, and myrobalan tannins) was 85-95 % of the maximum binding capacity of untreated hide powder (collagen). The uptake of the naphthalenesulfonic acid by the vegetable-tanned collagen was 5 0 4 0 % of the figure for untreated collagen and finally the high-molecular lignosulfonic acid reacted with vegetable-tanned hide powder only to 3040 % of the maximum potency obtained by native collagen. Thus, e.g., the wattle-tanned hide powder with a combining capacity of 0.89 milliequivalent hydrochloric acid per gram collagen fixed only 0.31 milliequivalent lignosulfonic acid. The lignosulfonic-acid-treated, wattle-pretanned collagen fixed further 0.52 milliequivalent hydrochloric acid from 0.1 N solution. Hence, free acid-binding groups (carboxyl and amino) are present in vegetable-tanned hide powder in equilibrium with lignosulfonic acid of pH 1.5, the optimum for its fixation. I n the irreversible fixation of the high-molecular fraction of lignosulfonic acid by native collagen and by vegetable-tanned collagen, all sulfo groups react with the protein. Since i t has been demonstrated by various methods (76,78,95) that the reaction of lignosulfonic acid with collagen does not involve coordinate reactions, in the initial fixation a t least, the very drastic decrease of the irreversible fixation of lignosulfonic acid by vegetable-tanned collagen cannot be caused by the inactivation of coordinate loci of collagen by the vegetable tannins fixed. The probable explanation is to be found in the special nature of the lignosulfonic compound, its molecular size, and the presence of numerous strong acid groups in the molecule. Although the incorporation of large vegetable tannin molecules in the collagen lattice and the valency interaction of the same with the reactive protein groups do not materially hinder the reaction of small ions, such as hydrogen and chloride ions, with the ionic protein groups, some sort of steric hindrance may interfere with the reaction of collagen with the very large anion of lignosulfonic acid, which for its stabilization in the protein lattice requires a multipoint attachment. The blocking of the large anion will in its turn affect the fixation of the hydrogen ions, decreasing the same, since the reactions of hydrogen ions and anions of the sulfo acid are interdependent; the maintenance of electroneutrality of the system is essential. The sum total is lowered degree of fixation. The accessibility factor is also involved in the fixation of highly basic chromic salts by vegetable-pretanned hide. In retanning vegctable-tanned collagen by means of extremely basic chromic salts and high-molecular chromium compounds generally, the chrome fixation is only 10-20% of the corresponding fixation by native collagen. Dried vegetable-tanned hide powder has practically no affinity for chrome (94).

PROTEIN-CHEMICAL ASPECTS OF TANNING

405

VIII. TANNING POWER OF ALDEHYDES 1. Tanning Action of Various Aldehydes

Among the aldehydes, formaldehyde holds a unique position as a tanning agent. Since the chemistry of the reaction of proteins with formaldehyde (F.) has been comprehensively treated in Volume I1 of this series (43),only some special problems characteristic of the tanning process will be discussed. A survey of the general behavior of various types of aldehydes toward collagen will be included. Applying the usual criteria of tanning potency, i.e., the degree of hydrothermal stability, inertness to swelling agents and proteinases, and the “leathery drying” of the fibers of the treated skin, it will be found that, among the simple saturated aliphatic aldehydes, the tanning power of the first member of the series, formaldehyde; is outstanding, when the effects of the various aldehydes are compared in solutions of equal parts of water and acetone. Acetaldehyde has a feeble action and the higher homologs are devoid of tanning power. Among the more common unsaturated aldehydes, acrolein and crotonaldehyde show a fair degree of tanning potency (67). By introduction of ethyl and propyl groups in the 2 and 3 positions in acrolein, this property is lost (94). The dialdehydes are an interesting class from the point of view of tanning theory, since the presence of two aldehyde groups on a small molecule would be expected to facilitate the cross linking of adjacent protein chains, as first pointed out by Seligsberger (181). The simplest dialdehyde, glyoxal, is a fair tanning agent, particularly in certain organic solvents (89). Surprisingly enough, the methyl derivative, pyruvic aldehyde, shows excellent tanning properties; in some solvents it is fully comparable with that of formaldehyde (90). Both these aldehydes possess a very marked tendency to condensation and polymerization. However, the tanning effect is indicated to be mainly associated with the monomers. The aromatic aldehydes form a chapter in themselves (46,67). Benzaldehyde and related compounds with the CHO group built into the aromatic structure will combine with collagen, when used in organic solvents or water mixtures of the same (67). However, the effect of their combination with collagen is not a stabilization of the structure. On the contrary, they exert marked hydrotropic action or labilization of the protein lattice, similar to the action of many related organic compounds, e.g., simple phenols. The valency action of these aldehydes evidently is directed toward the hydrogen bond loci; rupturing part of these cross links. The tanning power of aldehydes of diphenols is inter-

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esting. However, since the methoxy derivatives behave similarly to the simple aldehydes, being devoid of tanning power, the tanning action shown by dihydroxy aldehydes is probably due to the presence of two phenolic groups, as indicated by Gerngross’ work (46). Aromatic aldehydes containing the CHO group in an aliphatic side chain usually possess weak tanning power (67). At the present state of our knowledge, i t is not possible to describe the tanning action of aldehydes on a common basis. Probably the activation of the aldehyde group by adjacent groups is the underlying cause of the apparently elusive behavior of the aldehydes toward proteins. 2. Tanning with Formaldehyde

a. General Aspects of the Reaction. By the fixation of F. by collagen the strength of the acidic groups is increased (13,99), probably as a result of the inactivation of the basic gr0up.s by F. and breaking up of the internal compensation of the electrovalent groups. Hence, the alkali binding is increased (48) and further, as a direct result of the inactivation of the. basic groups, the capacity of acid fixation decreased (45,192). The isoelectric point of gelatin and collagen is displaced about 1 pH unit to the acid side by F. tannage (47,212). b. Participation of Lysine Groups. Since the development of a quantitative method of determining fixed F. (15,102), an array of data obtained by numerous investigators indicate the main reaction of importance for the tanning function of F. to be located a t the c-amino group of the lysine residue. It is also generally considered that the tanning effect is a result of the formation of cross links by means of F. (46,153). The maximal fixation of F. a t the highest pH value in the range mentioned amounts to 0.4-0.5 millimole F. per gram collagen (15,102). Hence, it is nearly equivalent to the content of lysine residues although this may merely be a coincidence. The formation of -CH*cross links between two adjacent amino groups on different chains should only require half the amount of fixed F. found; this point has especially been stressed by Nitschmann and Hadorn (161) and by French and Edsall (43). It has also been pointed out (67) that the chance that two amino groups of different peptide chains should approach each other close enough for the formation of a methylene bridge should be rather slight (cf. 181). Hence, it was suggested that only a minute part of the bound F. is bridge-forming, the main part reacting without interlocking the collagen chains, with the formation of simple -NH*CHvOH structures. The drastic change of the internal cohesion of polystyrene polymers effected by the introduction of covalent bridges (187) (one bridge per 33,000 residues)

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in the form of divinylbenzene in the polystyrene linear polymers was mentioned as an analogous reaction. * A very important advance in our concept of F. tannage is marked by the researches of Nitschmann and Hadorn (161), who concluded from their comprehensive investigation of the reaction of F. with casein, that the F. cross linking takes place by the formation of -CH2bridges between an e-amino group and the imino group of the peptide link of adjacent chains. The concept of Nitschmann and Hadorn does away with the steric objection to the hypothesis of methylene bridge formation between two amino groups of differentchains, since the assumption of the proximity of amino and peptide groups on adjacent protein chains seems reasonable. Further, it also recognizes the bonding strength of the compounds formed, as remarked by French and Edsall (43), and agrees quantitatively with the ratio of one F. bound to one NHs group of F.-treated collagen of maximum F. content in its zone of maximum stability (pH 7-8). c. Reaction of Arginine Groups. The role of the guanidyl group of the arginine residue for the fixation of F. from solutions of final p H values greater than 8, indicated by the investigations of Highberger and Salcedo (103), is particularly interesting in view of the high pK value of this residue in the simple amino acid (pK about 13), which would require p H values about 3 or 4 units greater than the value given for reaction with the discharged guanidyl group, if the pK of the guanidyl group of arginine as amino acid applies to this group built into peptide chains. The very same discrepancy is noticeable between the p H of the reactivity of the lysine amino group (pH 5-8) and the p K of the simple amino acid (pK 9). It seems likely that the influence of the ionic environment of the protein groups as well as the effect,of F. may change the pK value of these amino acids considerably (69,86). The shift of the equilibrium NH3+-+ N H z groups, due t o inactivation of NHz groups by F. should not be ignored (73). The F. fixed by the arginine residue does not stabilize the protein structure, since it has been shown that deaminated collagen reacting with F. a t pH 11-13, although fixing 0.4t o 0.5 millimole F. per gram collagen, retains the shrinkage temperature of the original deaminated pelt and is more extensively swelled by strong acids and considerably less resistant to trypsin than the untreated deaminized stock (73). In view of the possibility of cross linking by F. of the guanidine and lysine groups occurring in simple prototypes (42), the lack of stabilization strictly proves only the absence of cross links between arginine residues. Recent

* Cross links between pairs of amino groups by means of condensed F. are too labile to withstand washing (private communication from Dr. H. Fraenkel-Conrat).

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criticism of this view (101), based upon the difference of the shrinkage temperature of collagen treated a t pH 12 and F. collagen tanned at pH 12, is unwarranted because of lack of information, and further has no bearing upon the findings reported which are based on the behavior of deaminated collagen. Yet it must be pointed out that the interrelationship of shrinkage by heat and by swelling has been overlooked (101). In comparing pelt and F. pelt of such high alkalinity as pH 12 (the point of maximum alkaline swelling), the swelling of the blank (pelt) will yield values of shrinkage temperature far too low, whereas this effect is practically eliminated by F. tannage. The value of the shrinkage increase will be too large a t p H 12. If the experiments are carried out in 5 % solutions of swelling-depressant sodium sulfate, it will be found that the increased hydrothermal stability obtained by tanning a t p H 12 is practically equal t o that obtained in tanning a t p H 8, compared to the blanks (pelt) (93). This also rules out bridge formation between lysine and arginine groups by means of condensed F. (93). I n both instances of tanning the stabilizing reaction is apparently located in the e-amino groups involved in F. fixation a t the low pH range (73), which, of course, also is a part of the total F. fixation in the high pH range. Partial removal of guanidyl groups does not decrease the hydrothermal stability of collagen F. tanned a t optimal pH for these groups, pointing to the nonparticipation of the guanidyl group in the stabilization of collagen by F. bridge formation (69). I n practical tanning the pH range 5-8 is generally preferred (212). Precipitated chalk is an excellent pH regulator (pH 7.5-8). Since this range forms the zone of minimum swelling of collagen and the affinity of collagen for F. is sufficiently great a t these p H values, reactive protein groups being present, theory and practice agree. A marked lowering of the pH value would mean insufficient F. fixation and too low tanning action, while increasing the p H value of the system too far, would promote swelling, rupture of hydrogen bond cross links, lead t o disorganization of the structure, and accordingly create unfavorable steric conditions for cross linking. The interaction of F. with the basic groups is also shown in retanning chrome leather with F. It has been proved (73) that F. displaces the protein-bound acid of chrome-tanned collagen in equilibrium with a weakly acid solution of F. This is an additional proof of the discharge of NH8+ ions by F. in the acid range. If lightly chrome-tanned leather, preferably that tanned by means of basic chromic chlorides, is used, with shrinkage temperature in the range 85-90°C., the F. treatment results in a boiling-resistant leather (shrinkage temperature greater than 100°C.)

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in a p H milieu of 3 4 . A marked displacement of protein-bound mineral acid by F. is noted (73). Of practical importance also is the uie of F. in the retannage of vegetable-tanned leather, which also is hydrothermically stabilized, indicating the presence of free basic groups in vegetable-tanned leather (70,197). Some interaction of F. with the vegetable tannins fixed in the leather may also occur (70,73). d. Participation ojPeptide and Other Groups. The participation of the peptide groups in F. binding is an interesting part of the problem. A labile fixation of this type seems to occur over the whole p H range, being a pH-independent reaction. This type of reaction may be prominent in solutions of high F. content. However, it does not appear to contribute measurably to the stabilization of the lattice. This is shown in tanning pelt and deaminated pelt at low p H values, e.g., p H 2, adjusted by hydrochloric acid and eliminating the swelling by using 5 % sodium chloride solution containing high concentrations of F. (5-10%). The shrinkage temperature of the pelt will upon prolonged treatment (about 2 weeks) increase 5-10°C., whereas the shrinkage temperature of deaminated pelt remains unchanged (73). This also excludes cross linking of collagen through amide groups but leaves open a possible linking of amide and amino groups. However, in both instances the peptide groups are equally accessible to F. The deaminated pelt will bind F. probably on the peptide and amide groups. The fixation of F. by collagen in the pH range 1-3 is an exceedingly slow reaction, probably connected with the gradual discharge of NH*+ groups, according to: -NHs++ H+, high F. concentration and prolonged interaction being -NH2 required. The cross linking will gradually lead t o increased stability of the lattice (73). The kinetics of the F. binding does not fit a reaction with the amide group. This type of reaction (40,41,217)recently discovered in F. combination with amide-rich proteins (casein and vegetable proteins) cannot be prominent with limed collagen, which only contains small amounts of such groups. However, the greater F. fixation of native collagen (not alkali-treated) compared t o alkali-treated collagen (limed) from weakly acid solutions is, as Highberger (101) points out, logically explained by the F. fixation by means of the amide groups of the nat'ive collagen. The discharge of ammonium ions of collagen in tanning with F. in concentrated solutions a t low p H values is demonstrated by the diminished fixation of sulfuric acid from 0.1 N solutions containing 4 volume per cent sodium sulfate with increasing F. content (73). It is further indicated by the behavior of collagen with its basic

+

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K . H . OUSTAVSON

groups completely inactivated by polymethylenenaphthalenedisulfonic acid that F. fixed by the nonbasic groups of collagen (amide and peptide) does not stabilize the protein lattice. Condensed naphthalenesulfonic acid is specific for the acid-binding groups and does not interfere with the affinity of the peptide linkages. The F. fixed by collagen with its basic groups completely blocked, probably attached mainly to the peptide groups, does not increase the shrinkage temperature (67). The cross linking of proteins by F. was suggested by Meyer (153). Experimental evidence for the condensation type of cross-linking protein chains by F. was first supplied by Nitschmann and coworkers (lGl), who showed that in the reaction of gaseous F. with casein water is liberated. Fraenkel-Conrat and collaborators (40,41) have furnished further proof. The recent researches of Fraenkel-Conrat and Olcott (42) have largely extended our knowledge of the mechanism of F. tannage. By model experiments, employing simple amides and amino compounds, these authors have demonstrated that a t room temperature and over the range bridges are formed in the interaction of F. with of pH 3 to 7.5,-CH*a primary amide and an amine or amino acid. Such F. condensation products could be isolated and characterized. The reaction between amino acids and amides is favored by alkaline medium, whereas the condensation of amines with amides is facilitated in weakly acid solution. Secondary amides (-COaNHR) do not react with F. to give cross linking. This finding evidently invalidates the hypothesis postulating cross linking of protein by F. through condensation of two adjacent imino groups of the peptide linkages. The improbability of such a linking has been indicated by other experiments, mentioned in the foregoing. The primary reaction of F. with proteins probably is the formation of methyl01 amines. Simple amines condense with guanidines and F., but amides and guanidine do not react. The results of experiments with proteins and macromolecular model compounds, such as polyglutamine, rich in amide groups, were in line with the findings of the simpler models. The authors consider that the F. tanning of proteins of average composition, i.e., containing equal numbers of amino and amide groups, is largely due to the secondary cross linking of amino and amide groups by means of condensed F. (methylene bridges). This reaction probably occurs in F. tanning of native collagen in slightly acid solution, as previously mentioned (101). However, in view of the low content of amide nitrogen of limed collagen, usually employed in practical tanning, this hypothesis does not appear to be applicable to the F. tannage of pelt and gelatin. Summing up, it may be said that the only firmly established fact is that the hydrothermal stabilization and tanning of collagen by F. is bound up with the c-amino groups of lysine residues.

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41 1

e. Znjluence of Solvent. Since F. in aqueous solution mainly exists as the monohydrate methylene glycol, CH,(OH)1 (210),whereas the carbonyl form predominates in organic solvents (210),it is noteworthy that F. dissolved in the common alcohols, acetone, dioxane, and benzene cxcrts as good a tanning action as it does in aqueous solution (67,178). Any influence of the dielectric constant of the solvent on F. fixation is not cvident (67). Hence, colloidal polymethylene glycols cannot be the effective tanning ngent (67). This speculation is also invalidated by the particular p H function of the F. tannage; the maximum F. fixation being located in the range of slight alkalinity under which conditions the polymerized glycol is rapidly converted into the monomer. In connection with the polymeric forms of F., attention will be called t o a detail of practice. In tanning with F. in neutral or slightly acid solutions great variations in the efficiency of the tannage are a t times experienced, probably because of the failure to take due precautions t o allow for sufficient ageing of the diluted formalin or adjusting its p H value for effective depolymerization. f. Ewald Reaction. Ewald (34)found that heat-shrunk F.-treated tendon spontaneously re-extends t o almost its original length upon cooling. This reaction is specific for F.-treated collagen fibers and has even been proposed as a tes: for both F. and collagen. Evidently, the re-elongation of the shrunk fiber on cooling is intimately connected with the changed micellar tension of the collagen lattice due t o its combination with F. (cross links) (123). It is interesting to note that native elastoidin fibers also show reversible hydrothermal contraction (36). Since this special collagen contains sulfur, the presence of sulfur bridges may explain the unique behavior of elastoidin. Kuntzel (123)applies the Meyer-Ferri (155)concept of the mechanism of the elastic behavior of tendon t o the reversed contraction of F. collagen. According to Meyer and Fcrri, two opposing tensional systems of opposite temperature coefficients arc a t work in elastic structures such a s tendons. In the melting and contraction of the fiber the lcngthwise tension is increased. The cross-sectional tension sets in upon cooling of the fiber, bringing about reorientation of the structural units and accordingly elongation of the fiber (123).

IX. QUINONETANNAGE The remarkable tanning power of p-benzoquinone was discovered

40 years ago by Meunier and Seywetz (152)in the course of their inveeti-

gation of the use of oxidized phenols as photographic developers and hardening agents for gelatin film. Quinone tannage was later comprehensively investigated by Thomas and Kelly (207). Its effect on subsequent vegetable tannage was also investigated. In the fundamental researches of Hilpert and Brauns (105)in 1925,the principles of the reaction of quinone with collagen were established by means of preparation of simple prototypes involving reaction be tween quinones and various amino compounds and by the study of the actual tanning process. The more recent investigation of Stecker and Highberger (188)is in excellent agreement with the older work (105). The established facts of quinone tannage are as follows. Benzoquinone tans well in alcoholic solution (212). In that, as in many other respects, it shows similarity t o F. The monomer is evidently the active tanning agent in this case. From aqueous solutions of pH values less

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than 7 collagen fixes the monomer, increasing acidity markedly lowering the degree of fixation. In slightly alkaline solutions polymerized guinones partake in the reactlion. Upon prolonged tanning, formation and fixation of such polymerization products also occur in solutions of pH less than 7. In the reaction of quinone with collagen in solutions of pH greater than 8 the polymerized products are mainly involved (188). The study of model compounds by Hilpert and Brauns (105) indicates that the old conception of the reaction (152), assuming the CO groups to be the active groups of tanning, is incorrect. Instead, the formation of compounds of the type O=

q;:

seems most likely. This con-

cept is also supported by the fact that tetrachloroquinone is devoid of tanning power (198). Quinone tannage can accordingly be formulated as: (1) A rapid reaction of the monomer with the amino groups of collagen, predominant in neutral and slightly acid media. (2) A relatively slow reaction of polymerized quinone in alkaline solutions, probably by attachment of the high molecular products to the peptide groups of collagen. This reaction also seems to occur as a secondary fixation to (1). I t is doubtful if this type of interaction results in tanning. A mechanical impregnation with preformed products and by means of substances polymerized in situ has also been made probable (188). The shrinkage temperature of collagen is raised about 25°C. by quinone tanning under optimal conditions (at pH 6). Hence, quinone is superior to F. in this respect. A recent investigation of Highberger (101) regarding the tensile strength of single fiber bundles of collagen (kangaroo tail tendon) is of interest in this connection. Tanning of the tendon by means of F. or quinone markedly decreases the tensile strength of dry fibers. These observations cause the author to question the general applicability of the cross-linking concept of the tanning mechanism to the mechanical stability of fibrous proteins, since the effect predicted on the basis of cross linking appears to be at variance with the findings. However, it seems possible that two different types of forces are involved in the hydrothermal and mechanical stabilization of collagen; the former being due to intra- and intermicellar cross linking and the latter also to interfibrillar forces (93). Some physiological aspects of quinone tanning may be mentioned. The possible role of quinonelike substances in the hardening and formation of larval cuticles has received a great deal of attention. Pryor (174) first recognized the hardening of insect cuticles &B a tanning reaction, probably due to the oxidation products of an o-dihydroxyphenol which combine with the water-soluble proteins to form a dark brown, insoluble, tanned protein. The recent researches of Fraenkel and Rudall (39) also s ~ p p l yexperimental indications of the formation of stiff nondeformable structures of insects by the stabilization of proteins b y means of quinonelike substances formed i n vivo by deanination of tyrosine. Glyoxal haa also been claimed as a hardening

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413

agent for the nondarkening proteinous parts of insects (177). Finally, the increased solubility of certain naphthoquinone derivatives in solutions of serum albumin, mentioned by Edsall (31),is of interest in this connection. The tanning action of inorganic poly acids, e.g., tungstic and metaphosphoric, zirconium compounds, aluminum and ferric salts, as well as unsaturated oils, has not been included in this survey; the reason is not only consideration of space but primarily the lack of fundamental theoretical knowledge of these tanning systems, and secondly their limited application.

X. GENERALCOMMENTS I n the processes of tanning and leather formation complexities in addition to the intricacies of heterogeneous systems of high molecular proteins are introduced by the two phase nature of the systems. The insoluble lattice-structured protein differs in reactivity fundamentally from the soluble proteins, since factors of little or no importance for the latter are often of governing importance for the behavior of the fibrous protein. The inherent properties of a biological product, such as the weave structure of the fiber bundles, the organization and macro structure of the fibers and fibrils, and the micro and molecular structure of the micelles and protein chains, show variations according to the origin of the hide, the location of the sample tested, and the previous history of the hide prepared for tanning, including the nature of the pretreatments. Beside being a function of purely chemical factors, the interaction of tanning agents with hide protein is intimately bound up with the physical state of the substrate, particularly the rate of diffusion of the tanning agents, frequently in polydisperse solution, into the interior of the hide (macroscopic) and into the interior of the fibrils. Finally, the tanning agent may preferentially react on the surface groups of the micelles, intermicellarly, or penetrate into the micelles, reacting with the individual protein chains (intramicellarly). The attainment of equilibrium is a vexing problem in this type of reaction; and this difficulty is one of the greatest obstacles in the investigations of this field. Topochemical reactions are of major importance, and the conditions for reaction may accordingly be altered during the process. The main reaction and ultimate nature of tanning is incorporation of substances possessing affinity for various protein groups; the tanning agent being immobilized in the protein lattice. The protein enters into a more or less stable combination with the tanning agent which in its turn leads t o stabilization of the protein structure. The most effective way of obtaining this appears to be the function of the tanning agent as an artificial bridge between reactive protein groups of adjacent chains. The possibility of some kind of depolarization of the active valency centers of the protein by means of the tanning agent, the type of long range effect,

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may also be involved, as is the simple inactivation of hydrophilic protein groups. It is likely that the strength of the valency forces between the fixed tanning agent and collagen does not in itself govern the efficiency of the stabilization. The spatial factor exerts a marked influence on the reactions, as, for instance, the size and shape of the introduced molecule and the distance between reactive protein groups and between those and the reactive loci of the tanning agent. Knowledge is lacking regarding the elementary background to the problem of the spatial relationship of the reactants, the sequence of amino acid residues, and their spatial environment. Furthermore, some tanning agents probably interact with collagen mainly intermicellarly, not affecting the interior, ultimate units, but only the surface-active groups of micelles and fibrils which offer quite different spatial environment and valency distribution than the more closely packed protein chains. The whole subject of fine structure of proteins of the collagen group, and the problem of the mechanism of tannage, definitely invites profound investigations by application of new tools and refined techniques. It is obvious that our conceptions of the nature of tanning processes are based upon and reflect the status of the fundamental chemistry of the interesting groups of substances concerned in these complicated reactions. The problem of tannage is not confined to industrial applications. It appears to be of general importance for the elucidation of the nature of metal-protein compounds, and other complexes of proteins and smaller molecules, with important biochemical functions. ADDENDUM ADDED I N PROOF

Bowes and Kenten (220) recently presented detailed data on the combination of modified collagens with various tanning agents; some results of this investigation were mentioned in the text (preliminary statement of €?owes (13a)). Of particular interest is their finding that no chromium is fixed by methylated collagen (with its carboxyl groups completely blocked) from dilute solutions of chromium sulfate of pH 24. The original shrinkage temperature of the methylated collagen is not changed. From these findings and the behavior of deaminated collagen, the authors concluded that the combination of chromium salts and collagen involves coordination of both the carboxyl and amino groups with the same chromium complex. Their data confirm earlier findings of the nonreactivity of collagen with its carboxyl groups in the nonionized state towards cationic chromium complexes (68). Since complete methylation of collagen requires repeated treatment by the methylating agent (methyl sulfate), the resulting preparation of collagen may be radically modified in many reHpects, as indicated by its behavior towards other tanning agents. Hence, precaution is needed in drawing conclusions. In two papers on the mechanism of the hydrothermal shrinkage of collagen (tendon) (221,222), Weir opened a new approach to the investigation of the nature of tanning processes. The thermodynamic treatment of tanning processes appears to

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be (very) promising. He (221) measured the coefficient of the cubical expansion of hative tendon and tanned tendon in water of increasing temperature up to the point of complete shrinkage. The data are interpreted a8 indicating that shrinkage does not occur a t a characteristic temperature but is a rate process, involving a reaction of the first order. This is in agreement with earlier findings on skin collagen (50,79,170) and also with Rudall's observation of shrinkage curves of sigmoid form in the contraction of epidermin within a wide temperature range (177). Weir reported the heat of shrinkage of untreated tendon collagen to be 141 kcal./ mol., the entropy 349 cal./mol. deg., and the free energy 24.7 kcal./mol. at 60"C., with standard deviations of 15, 43 and 0.6 units, respectively. Weir (222) further attempted to divulge the type of binding between various tanning agents and collagen through determination of heat, entropy, and free energy changes in the shrinkage of tendon collagen, pretreated with various substances, including tanning agents. By applying the theory of absolute reaction rates to the shrinkage of tanned and otherwise pretreated collagen, he concluded that tannages with the salts of aluminum, iron and zirconium as well as with F., seem to reduce entropy more than heat, thereby increasing the free energy. The tannage with chromium salts is unique inasmuch as it increases not only the free energy but also the entropy of activation. The heat of activation, identified with the degree of disorganization occurring in the activating process, is drastically increased, and this trend is in agreement with the concept of chrome tanning as an internal stabilization of the collagen lattice by crosslinking the protein chains through chromium complexes. One per cent CraO, fixed by collagen is sufficient to produce the maximum degree of stabilization. Weir concluded that only a fraction of the acidic and basic protein groups possess the required spatial orientation to react with the chromium complex to form crosslinks. The amount of chromium combined with collagen in excess of 1 % probably combines with collagen in the same manner as the other tanning agents mentioned, decreasing the entropy of activation. Hence, this additional chrome will add nothing to the orientation of the protein chains and their stabilization. Incorporation of large amounts of chromium will mean some loss of orientation of the protein lattice. Weir's concept of the mechanism of denaturation of collagen, the type of stabilizing bonds, and the nature of the chrome tanning process are on the whole in harmony with the views on these problems outlined in the present chapter.

REFERENCES 1. Anslow, W. K., and King, H. (1927). Biochem. J . 21, 1168. 2. Astbury, W. T. (1933). Trans. Faraday SOC.29, 193. 3. Astbury, W. T. (1940). J . Intern. SOC.Leather Trades' Chemists 24, 69. 4. Baldwin, M. E. (1919). J . A m . Leather Chemists' Assoc. 14, 10. 5. Balfe, M. P. (1948). J . Intern. SOC.Leather Trades' Chemists 82, 39. 5s. Balfe. M. P. (1948). Progress in Leather Science. 111. 610. London. 9. (1944). . J . Amy Chem. SOC.66, 1927; (1942) 64,'729. 6. Bear, 7. Beek, J., Jr. (1930). I d . Eng. Chem. 22, 1373. 8. Beek, J., Jr. (1938). J . Research Natl. Bur. Standards 21, 117; J . A m . Leather Chemists' Assoc. 83, 621. 9. Beek, J., Jr., and Sookne, A. M. (1939). J . Research Natl. Bur. Standards 23, 271. 10. Bergmann, M. (1935). J . Biol. Chem. 110, 471. 11. Bergmann, M., and Niemann, C. (1936). J . Biol. Chem. 116, 77; (1937). 118, 301.

k.

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