Studies on a parent gelatin from ichthyocol

Studies on a Parent Gelatin from Ichthyocol’ Paul M. Gallop2* 2e From

the Department

of Biology,

Massachusetts Massachusetts Received

April

Institute

of Technology,

Cambridge,

29, 1954

Past studies on gelatins have probably always involved polydisperse preparations. Scatchard, Oncley, Williams, and Brown (18) proposed a model in which molecular units of gelatin (parent gelatin) are linked end to end by weak, heat-labile bonds into a linear aggregate. These investigators studied acid-degraded gelatin preparations in which few, if any, linear aggregates of parent gelatin molecules remained. They assumed a Montroll-Simha distribution to characterize the polydispersity and were able to extrapolate to a hypothetical parent gelatin unit, approximated by an ellipsoid 17 by 800 A., weight 110,000 avograms. Salo (17) subjected acetic acid solutions of the ichthyocol from carp swim bladders to mild heating and observed a decline of intrinsic viscosity with time to a limiting value which was consistent, with a molecule of dimensions in the range described by Scatchard et al. The present paper describes the preparation and characterization of a monodisperse gelatin obtained by mild heating in citrate buffer of a solution of acid-extracted ichthyocol, described in a preceding paper (6) and similar to the procollagens of Orekhovitch Tustanovskii, Orekhovitch, and Plotnikova (13) and of Orekhovitch(l3a). Since this mono1 Supported in part by a a grant-in-aid for the study of connective tissue structure under the supervision of Richard S. Bear, from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council. 2 Experimental material presented herein is drawn in part from a thesis submitted to t,he Graduate School of the Massachuset,ts Institute of Technology, September, 1953, in partial fulfilment of requirements of the Ph.D. degree. 2% Present address: Dept. of Medicine, Long Island Jewish Hospital, New Hyde Park, N. Y. 501

502

PSUL M. GALLOP

disperse gelatin preparation is obtained under conditions in which only weak, heat-labile bonds are broken, the term parent gelatin proposed by Scatchard et al. is retained for convenience. PREPARATION

OF THE PARENT

GELATIN

Wet material, obtained from the phosphate dialysis of the pH 4.3 citrate extract of the carp swim bladders as described in detail in the previous paper, is dissolved in a pH 3.7 citrate buffer, 0.1 M with respect to citric acid and 0.05 M in sodium citrate. These solutions are cleared by centrifugation at about 50,009 X y in a Spinco preparative ultracentrifuge at 5°C. The intrinsic viscosity of these solutions is about 13 and the specific optical rotation near -350”. If these solutions are brought to 3O”C., the viscosity and the optical rotation decrease. The optical activity falls rapidly and reaches a limiting value of about -110” in approximately 2 hr., while the viscosity continues to fall linearly with the logarithm of the time, reaching a limiting value of 0.34.5 in about 3 days. In several runs the intrinsic viscosity was near 5 when the optical rotation first reached its lower value, which approximates the rotations reported for gelatin sols by many workers (10, 16, 21, 23).3 The reaction is carried out at 40°C. for the preparat,ion of the parent gelatin, since the changes occur in a matter of minutes at this temperature. Routinely the solutions are kept at this temperature for 1 hr. The gelatin solutions, which will now gel at lower temperatures, are centrifuged 1 hr. at 50,000 X g at 30°C. and dialyzed against the pH 3.7 buffer at room temperature overnight. The solutions are heated to 40°C. and allowed to come down to the temperature of the experiment. The parent gelatin concentration is determined by a calorimetric biuret procedure similar to that of Gornall, Bardawill, and David (7). CHARACTERIZATION

OF THE PARENT

GELATIN

Investigations in the Ultracentrifuge A Spinco model E analytical ultracentrifuge, equipped with a PhilpotSvenson cylindrical lens optical system, was used. Each run took about 2.5 hr., and the temperature of the rotor was measured at intervals by the method of Waugh and Yphantis (24). Runs were done in a temperature range of 21-26”C., and in the course of each individual run the temperature rose about one degree every 2 hr. The temperature used in the calculations was the average temperature between the first and last sedimentation photographs. Sedimentation constants were calculated in the usual manner and corrected to a solvent having the properties of water at 20°C. Sedimentation diagrams for several concentrations of the parent gelatin are shown in Fig. 1. The main peak is symmetrical and is characteristic 3 C. Cohen, unpublished

results, 1953.

PARENT

GELATIN

FROM

ICHTHYOCOL

FIQ. 1. Several sedimentation runs at 60,006 r.p.m. on ichthyocol solutions (at pH 3.7 in a 0.15 M citrate buffer).

503

parent geIatin

of a peak arising from molecules of one characteristic sedimentation constant. Commercial gelatins show a skewed peak typical of a distribution of sedimentation constants. A very small second peak, possibly similar to that described by Bresler, Finogenov, and Frenkel (2) for procollagen, seemsto vary with ionic strength, but this was not investigated and has been ignored in the calculations. Several runs at different concentrations were made in order to extrapolate to infinite dilution. The sedimentation constant has a strong concentration dependence (Fig. 2), increasing in value with decreasing concentration as is characteristic of asymmetric molecules. The extrapolation to infinite dilution (Fig. 3) was made by plotting sgoversus s;o X c (5). The best line was determined by the method of least squares and the extrapolated value for the sedimentation constant is 3.31 svedbergs. Di$usion and Partial SpeciJic Volume A Pearson electrophoresis-diffusion apparatus, equipped with a Longsworth scanning system, was empioyed. The cell was of 11-ml. capacity and of the standard type supplied by Pyrocell. Hydrostatic compensa-

504

PAUL

M.

GALLOP

tion was used, and no attempt was made to sharpen boundaries. The runs were done at 29.4 and 29.8”C. in order to prevent gelation, since each run takes 2-3 days. The diffusion constants were calculated by the method of area and maximum ordinate, and were corrected to a solvent having the proper3400-r

FIG. 2. The concentration dependence of the corrected sedimentation constants of ichthyocol parent gelatin solutions (at pH 3.7 in a 0.15 M citrate buffer).

FIG. 3. Extrapolation to infinite dilution of the corrected sedimentation constants of ichthyocol parent gelatin solutions (at pH 3.7 in a 0.15 M citrate buffer).

PARENT

GELATIN

FROM

ICHTHYOCOL

505

FIG. 4. Several diffusion diagrams of ichthyocol parent gelatin solutions (at pH 3.7 in a 0.15 M citrate buffer). Runs were done at 29.4 and 293°C. in order to prevent gelation.

ties of water at 20°C. Figure 4 is a reproduction of several of the diagrams from runs at three different concentrations. The extrapolation to infinite dilution is made by plotting Dozeversus concentration (Fig. 5) and is theoretically justified by the OnsagerFuoss equation (12). The best line obtained by the method of least squares yields an extrapolated diffusion constant equal to 3.91 X 10-T sq. cm./sec. Measurements of the partial specific volume were made in 22- and 24ml. pycnometers, which were weighed to 0.1 mg. The procedure is similar to that described by Svedberg and Pedersen (22). The measurements were done at 31°C. and the value obtained for the partial specific volume was 0.705 f 0.005. Molecular Weight and Frictional Ratio The molecular weight, obtained by using the Svedberg equation with the extrapolated sedimentation and diffusion constants, is 69,700 f 4200, and the frictional ratio f/f0 is calculated as 2.025. This ratio yields, for a prolate ellipsoid, an axial ratio of 20 for zero hydration, and 15 for 30 %

506

PAUL M. GALLOP

FIG. 5. Extrapolation ichthyocol

parent

gelatin

to infinite solutions

dilution (at pH

of the corrected diffusion constants 3.7 in a 0.15 M citrate buffer).

of

hydration, when the procedure of Oncley (11) and the equation of Perrin (14) are employed.

The viscosities were measured in Ostwald-Fenske capillary viscometers having flow times for water at 20°C. of about 270 sec. No study wasmade on velocity gradient dependence. The intrinsic viscosity is obtained by plotting TJJC versus concentration and extrapolating to infinite dilut,ions, as is shown in Fig. 6. The three runs shown were done at 39.3, 29.8, and 20.3”C., in this order. At the lowest temperature gelation can occur, and after several hours an increase, first in the slope and then the intercept, is apparent. At temperatures where there is negligible gelation the intrinsic viscosity is 0.34 in the pH 3.7 citrate buffer. The viscosity increment, which is 100 times the ratio of the intrinsic viscosity and the partial specific volume, was found to be 48.2. Correction for hydration was made by dividing the viscosity increment by (1 + w/p p), where w is the hydration in grams of solvent per gram dry weight of protein, p is the solvent density, and v the partial specific volume. Axial ratios on the basis of an ellipsoid can be calculated by using the corrected viscosity increment and the Simha equation (20, 3). For zero and 30% hydration, the axial ratios are, respectively, 23 and 18. On the

I’:iRENT

GELATIN

FROM

507

ICHTHPOCOL

basis of a random coil the effective length R (the root-mean-square distance between chain ends) can be calculated from the relation of Flory and Fox (4), Eq. (l), by employing the molecular weight, 111, intrinsic viscosity, [T], and the value of 2.1 X 1031 for @, a universal constant. R was thus found to be about 225 A.

[Tj]= cp$

(1)

Light Scattering Measurements were made in the apparatus described previously (6). The refractive-index increment, (n - no)/c, measured in a Brice-Speiser differential refractometer, was 0.192 for the 4368-A. mercury line at 25°C. Difficulty was encountered in cleaning the heated ichthyocol solutions, since the solutions become slightly hazy on heating to gelatin. However, centrifugation at 50,000 X g at 30°C. for about 6 hr., with careful handling and filling of the scattering cells, sufficed to clear the solutions optically. No change in protein concentration could be detected chemically during the clearing process, although very slight precipitates were seen at the bottoms of the centrifuge tubes. The clean gelatin solutions studied at 30°C. had dissymmetries of 1.0, within experimental error, and accordingly a standard plot of

0.1

0.2

0.3

0.4

0.5

0.6

CONCEwrRLTlOW i+-

6. Plots of the reduced parent gelatin solutions measured 3.7 in a 0.15 M citrate buffer). FIG.

viscosity at 39.3,

cc

versus concentration for ichthyocol 29.8, and 20.3”C., respectively (at pH

508

PAUL

M.

GALLOP

zm-

ICHTHYOCOL

‘PARENT

!+b’)

?.mCITRATE

.ooi 0

.OOiO

“I. BUFFER

GELATIN’ CONC.

p”

.0030

?,.T

.oo*o

CONCENTRATION 9 -cc

FIG. 7. Light-scattering run on ichthyocol parent gelatin solutions at 30°C. (at pH 3.7 in a 0.15 M citrate buffer). The extrapolated value, [Hc/T,+,], corresponds to the reciprocal of the weight-average molecular weight.

Hq’r

versus concentration was made. Here H is a constant equal to [(n - no)/$, c the concentration in grams/ml. and. 7 the turbidity. The intercept of this plot (Fig. 7) is the reciprocal of the weight-average molecular weight. The molecular weight as thus determined was 68,000 =t 7,000, the large error resulting from a 5 % experimental error and a 5% error in calibration. (32*3n~/3X4)

Scheraga-Mandelkern

Treatment of Data

Instead of the classical treatment of sedimentation-diffusion and viscosity data, an alternative treatment described by Scheraga and Mandelkern (19) may be used. Substitution of the extrapolated sedimentation and diffusion constants into their equations led in the present caseto a value of 2.79 X lo6 for their quantity 0. If the molecule of parent gelatin were a completely flexible coil, /3 should be about 2.5 X 106, although this result could also be given by a prolate ellipsoid of a certain axial ratio. The value of p cannot exceed 2.15 X lo6 with ablate ellipsoids. The “effective hydrodynamic volume” of the gelatin particles, corresponding to the p given, is 0.509 X 10-ls cc., as compared to the classical anhydrous volume of 0.816 X 10-ls cc. In order to obtain agreement between these two volumes, negative hydrations must be assumed. This situation is taken by the authors as indicating that the

PARENT

GELATIN

FROM

ICHTHYOCOL

509

molecules are probably free draining and that the classical treatment is erroneous in taking a solid ellipsoidal model. It is possible to use either the classical treatment or the ScheragaMandelkern treatment, which serve equally well for interpreting experimental results, although neither is completely satisfactory. In the treatment of Scheraga and Mandelkern, the concept of the hydrodynamically effective volume is vague and no satisfactory physical model is presented. However, one important conclusion can be drawn from the large discrepancies in the volumes calculated by the two methods; the molecule of parent gelatin must have a relatively open structure under the experimental conditions described. Optical Activity As mentioned previously, the gelatin solutions above 25°C. at pH 3.7 have a specific rotation of - 110 f 20”, as compared to -350 f 30” for the unheated ichthyocol solutions. On cooling below 2O”C., the optical activity of the solutions tends toward its initial value, approaching about -29Oo.3 In this respect, it is interesting to note that in films of gelatin prepared at high temperatures so that the rotation remains low, the wide-angle diffractions are amorphous. However, ‘with films prepared at low temperat,ures the optical activity of the films increases and the wide-angle collagen pattern returns (8, 9, 16). DISCUSSION

The results are summarized in Table I. According to the ultracentrifugal investigations, the gelatin studied has a monodispersepeak and can be considered as a parent gelatin, since the preparation is carried out under conditions where only weak heat-labile bonds can be broken. The molecule has a weight of 69,700 f 4,200 (sedimentation-diffusion),. 68,000 f 7,000 (light scattering), with an axial ratio of about 20 from the viscosity increment and the frictional ratio, and can be represented as a prolate ellipsoid about 20 X 400 A. If the molecule is assumedto be randomly coiled the effective length is about’225 A. The transformation of protofibrillar particles to a monodispersepopulation of parent gelatin molecules has been accomplished with an extracted fraction which represent’sonly a small portion of the collagenous material of a native connective tissue (6). Nevertheless, this conversion is virtually complete in vitro for a system originally capable of yielding fibrillar structures similar to those of native collagens generally.

510

PAUL

M.

GALLOP

TABLE

Summary _.of Important

1. &g 2. ?2,0 3. P 4. M 5. f/f0 6. a/b 7. 191 8. Y 9. a/b 10. v 11. p 12. ve

13. R

I

Physical-Chemical Data on Ichthyocol Parent Gelatin Solutions in 0.16 M Citrate, pH 3.7

Extrapolated sedimentation constant Extrapolated diffusion constant Partial specific volume Molecular weight Frictional ratio Axial ratio, from ratio Intrinsic viscosity Viscosity increment

frictional

Axial ratio, from viscosity increment Anhydrous molecular volume Scheraga-Mandelkern factor Hydrodynamically effective volume Effective length, random coil treatment

3.31 X lO+(cm./dyne 3.91

X 10-r

sec.)

sq. cm./sec.

0.705 f 0.005 69,700 68,000

f f

2.025 20 (zero 15 (30%

4200 (sed.-diff.) 7000 (light scattering) hydration) hydration)

0.34 48.2 (zero

hydration) 33.7 (30% hydration) 23 (zero hydration) 18 (30yo hydration) 0.816 X lo-‘9 cc. 2.79 x 106 0.509 x 10-19 cc. 225 A. (from

intrinsic

viscosity)

The question arises as to whether there may not be other parent gelatins. Gelatin particles of weight ranging between 15,000 and 250,000 have, for example, been isolated by Pouradier, Roman, and Venet (15). Observations of this kind do not, however, immediately remove the possibility of special significance for the molecules of 70,000 weight. Smaller particles may be products of chemical degradation. Larger ones may result from preservat,ion of heat-stable cross linkages established in the native state between collagen units of weight 70,000. An argument for more than casual significance of the units of about 70,000 weight can be derived from consideration of the structure of collagen fibrils. Bear (1) has suggestedmolecular units of collagen 12 A. thick and 640 A. long (in the dry state), dimensions which were derived from x-ray diffraction and electron optics; density considerations indicate these units would have weight near 65,000 avograms. It has not yet been demonstrated that collagen units of this description can be dispersed from native fibrils; rather acid extracts are found to contain predominantly protofibrillar aggregates (6), presumed to be linear strings of such units. It seemsprobable, however, that the parent

PARENT

GELATIN

511

FROM ICHTHYOCOL

COLLAGEN FISRIL

PARENT GELATIN MOLECULES M - 70.000 [ql = 0.3 - 0.5 [a],: -,lO’* 20’

FIG. gelatin.

8. Mechanism

proposed

for

the

conversion

of collagen

fibrils

to parent

gelatin molecules herein described correspond to somewhat altered (randomly crumpled) collagen units. Heat treatment has caused the latter to lose certain features typical of their original condition: the ability to reconstitute typical banded fibrils, and their initially high intrinsic viscosity and optical rotation. These changes indicate a general decrease in asymmetry of shape and loss in specificity of configuration. A diagrammatic indication of these relationships is shown in Fig. 8. ACKNOWLEDGMENTS The author wishes to express thanks to Dr. Richard S. Bear for interest and encouragement in the investigations reported in this and the preceding paper. Special indebtedness is acknowledged also to Dr. David F. Waugh, for the use of ultracentrifugation and diffusion apparatus, as well as for many helpful suggestions. Informative discussions with Drs. Irwin W. Sizer and Myles Maxfield, as well as with Dr. Paul Doty of Harvard, contributed much to this study.

SUMMARY

Mild heating of citrate-extracted ichthyocol solutions results in a breakdown of long, thin collagen protofibrils into a monodisperse parent gelatin. Viscosity, sedimentation-diffusion, and light-scattering studies indicate a molecular weight of about 70,000, dimensions 20 X 400 A. A mechanism is proposed for the conversion of collagen to parent gelatin.

512

PAUL

M.

GALLOP

REFERENCES 1. BEAR, R. S., Advances in Protein Chem. 7, 69 (1952). 2. BRESLER, S. E., FINOGENOV, P. A., AND FRENKEL, S. Y., S.S.S.R. 72, 555 (1950). 3. EDSALL, J. T., in “Proteins, Amino Acids, and Peptides” 4. 5. 6. 7.

Doklady Akad. Nauk

(E. J. Edsall, eds.), Chapts. 19 and 21. Rheinhold Publ. Corp., New FLORY, P. J., AND Fox, T. G., JR., J. Am. Chem. Sot. 73, 1904 FRITH, E. M., AND TUCKETT, R. F., “Linear Polymers,” Chapt. Green, and Co., London, 1951. GALLOP, P. M., Arch. Biochem. and Biophys. 64,486 (1955). GORNALL, G. A., BARDAWILL, C. J., AND DAVID, M. M. J. Biol.

Cohn and J. T. York, 1943. (1951). 7. Longmans,

Chem. 177,751 (1949). 8. KATZ, J. R., DERKSEN, J. C., AND BON, W. F., Rec. trav. chim. 60, 725 (1931). 9. KATZ, J. R., Rec. trav. chim. 61, 835 (1932). 10. KRAEMER, E., AND FANSELOW, J. R., J. Phys. Chem. 29,1169 (1925). 11. ONCLEY, J. L., Ann. N. Y. Acad. Sci. 41,121 (1941). 12. ONSAGER, L. AND FUOSS, R. M., J. Phys. Chem. 36,2659 (1932). 13. OREKHOVITCH, B. N., TUSTANOVSEII, A. A., OREKHOVITCH, K. D., AND PLOTNIHOVA, N. E., Biokhimiya 13, 55 (1948); Doklady Akad. Nauk S.S.S.R. 60, 837 (1948). 13a. OREKHOVITCH, B. N., Intern. Congr. Biochem. Communs. 2nd Congr. Paris, 1962; Acad. Sci. U.S.S.R., Moskow, 106 (1952). 14. PERRIN, F. J., J. phys. radium 7.1 (1936). 15. POURADIER, J., ROMAN, J., AND VENET, A. M., J. ehim. phys. 47,ll (1950). 16. ROBINSON, C., “Nature and Structure of Collagen” (J. T. Randall, ed.). Academic Press, New York,

1953.

17. SALO, T. P., J. Am. Chem. Sot. 71,2276 (1949). 18. SCATCHARD, G., ONCLEY, J. L., WILLIAMS, J. W., AND BROWN, A., sot. 66, 1890 (1944). 19. SCHERAGA, H. A., AND MANDELKERN, L. J. Am. Chem. Sot. 76, 20. SIMHA, R., J. Phys. Chem. 44.25 (1940). 21. SMITH, C. R., J. Am. Chem. Sot. 41,135 (1919). 22. SVEDBERG, T., AND PEDERSEN, K. O., “The Ultracentrifuge.”

J. Am. Chem. 179 (1953).

Press, Oxford, 1940. 23. THAUREAUX, 24. WAUGH, D.

J., Bull. eoc. chim. biol. 27, 327 (1945). F., AND YPHANTIS, D. A., Rev. Sci. In&. 23, 609 (1952).

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