- Email: [email protected]
The effect of extraction procedures on sedimentation properties of epidermal proteins
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 66, 167-176 (1957)
The Effect of Extraction Procedures on Sedimentation Properties of Epidermal Proteins D. L. Woemley, C. Carruthers, L. Regent and A. Baumler From the Departments of Biophysics and Biochemistry, Memorial Institute, Butialo, New York Received
April
Roswell
Park
26, 1956
In previous investigations (1, 2) proteins extracted from mammalian epidermis by solvents such as water, urea, and sodium lauryl sulfate were studied by the technique of electrophoresis. The current presentation consists of further characterization of epidermal proteins by means of sedimentation rates obtained by ultracentrifugation analysis. Mercer and Olofsson (3) have investigated a urea extract of the prekeratinous layers of epidermis in order to amplify the knowledge gained from techniques (e.g., x-ray diffraction) applicable to the intact epidermis. More distantly related researches regarding soluble derivatives of wool and feather keratin are those of Mercer and Olofsson (4) and of Woodin (5, 6). An excellent introduction concerning some of the chemical and physical properties of epidermal proteins will be found in the publications of Rudall (7). Since primary neoplasms can readily be produced in mammalian epidermis, it is a highly suitable tissue for making comparative physicochemical studies of the normal and malignant states. This work involves normal tissue proteins and serves as a basis for future comparative research on malignant tissue. PROCEDURE The procedures for the isolation of the proteins from beef snout epidermis with urea were the same as described by Carruthers et al. (1, 2). In addition, urea-extracted beef snout epidermis and epidermis in the fresh state were extracted with 0.05 N NaOH in the cold. The proteins were then isolated as follows: The alkaline extract was diluted threefold with distilled water and then stirred for 2 hr. with a hlagne-stirrer. The pH of the extract was then lowered to 7.0 (glass electrode) by the addition of 1 N HCl. This was followed by 167
168
WOERNLEY,
CARRUTHERS,
REGENT
AND
BAUMLER
centrifugation at 1500 r.p.m. for 20 min. The supernatant fraction was then adjusted to pH 5.5 by the addition of HCI, and the protein obtained at this pH was separated by centrifugation. The supernatant fraction was then adjusted to pH 4.5 by the addition of HCl, and the protein which precipitated was separated by centrifugation. Both of these proteins were purified by dissolving them in slightly alkaline water, centrifuging for 30 min. at 13,309 r.p.m., and then reprecipitating the proteins at the proper pH. This procedure was repeated three times. In this report the principal interest is in the proteins obtained from the first extracts of epidermis with urea or dilute alkali. Other extracts are briefly described since the proteins isolated showed differences in sedimentation properties from those obtained from the primary extracts. The proteins will be designated simply as 4.5, 5.5, and 6.3. These values correspond to the pH at which the proteins were separated from the various extracts by the careful addition of HCl. The pH was measured with a glass electrode (Beckman, model G pH meter). The proteins which were isolated at pH’s 4.5 and 5.5 from urea extracts of epidermis correspond, respectively, to the nonfibrous and fibrous proteins of Rudall (7). The yields obtained of purified proteins were very low. With 0.5, 1.0, 2.0, 3.0, and 6 M urea as extraction media, the percentage yields of purified 4.5 proteins were, respectively, 0.15, 0.13, 0.10, 0.06, and 0.10. From the same extractions the percentage yields of purified 5.5 proteins were respectively 0.13,0.10,0.09,0.04 and 0.32. With 0.05 N NaOH as the extractant, the percentage yields of 5.5 and 4.5 proteins were I.48 and 1.54. It is interesting to note that when 6 M urea was used as extractant, the ratio of fibrous to nonfibrous protein was approximately 3:1, while with smaller concentrations of urea the ratio was 1: 1. Sedimentation rates were obtained at rotational speeds of 59,736 r.p.m. with a model E Spinco ultracentrifuge which was initially tested by a determination of crystallized bovine plasma albumin (control 0123-176, Armour and Company). For preparatory and analytical runs the model A and An-D rotors were respectively employed. Viscosities of the samples of epidermal proteins (which were “cleaned up” by centrifugation at accelerations of 500 X g) were determined with an Ostwald viscometer. For the proteins investigated, there was present a schlieren peak of large area and often another of very small area. The former peak corresponded to a more or less polydisperse system of low sedimentation constant, and the latter to some material of appreciably greater sedimentation constant. To reduce the quantity of the latter material, all epidermal protein samples were centrifuged to the same extent in the preparatory rotor prior to the analytical run. Svedberg’s reduction formula was employed for conversion of measured sedimentation values, sr to s2,,values. For comparative purposes the calculations were made for a constant-run duration. The initial schlieren peaks employed were photographed at the same interval measured from the time the rotor reached its full speed. RESULTS
AND
DISCUSSION
The extrapolated sedimentation constant, SO, (for zero concentration and standard conditions) for bovine albumin in 0.2 N NaCl was de-
FIG. 1. Schlieren patterns for epidermal protein extracted with 6 M urea and which separated out at pH 4.5; solvent, 0.05 M borate buffer pH 8.9; concentration, 1.95 g./lOO ml. solution; time interval between photographs, 8 min.; and time of left-hand pattern, 44 min. after rotor reached full speed.
termined to be 4.54 S. A partial specific volume V equal to 0.730 was employed in the reduction formula. This sedimentation value is about 5 % higher than recently obtained values which fall in the range 4.27-4.32 S. The difference may be due to the effect of the solvent or the nature of the sample. Manufacturer’s products often show differences among batches. The agreement, however, is sufficient for the purposes of this experiment. There are certain inherent difficulties in determining the sedimentation constants for the epidermal proteins so far encountered. In general the extraction procedures are laborious and the extracted proteins are of limited solubility. The most formidable obstacle, however, is that the extracted proteins are more or less polydisperse. This is demonstrated by the fact that the schlieren peaks are unsymmetrical and broaden rapidly in the course of a sedimentation run. At a sample concentration of 2% by volume or lower, since the schlieren peaks for the epidermal proteins broaden out much more rapidly than a monodisperse system, the usuable length of the run with respect t’o measurement is much reduced. See Fig. 1. Another important factor is that while for dilute monodisperse systems the plot of the sedimentation constant against concentration approximates a straight line with an appreciable negative slope, for the polydisperse epidermal proteins in the concentration range 0.2-l % this plot often approximates a straight line with nearly zero slope. Thus in general the following sets of equations do not hold for polydisperse epidermal proteins (8). so
-=1++c
170
WOERNLEY,
CARRUTHERS,
REGENT
AND
BAUMLER
where s is the sedimentation rate at concentration c, so is the rate extrapolated to zero concentration, and A is the specific sedimentation slope ; and A = 0.75 [IV] + 5,
or = [N]
(2)
where m] is the viscosity increment extrapolated to zero volume fraction i.e., c is expressed as,.the volume fraction. The truth of this statement can readily be seenby setting the derivative, ds/dc of Eq. (1) equal to zero, i.e., -80-4 (1 + AC)’ = O
(3)
The only way for this relation to hold at concentrations 0.2-l % is to have A = 0. For the epidermal proteins encountered here, A calculated from N has an appreciable value, in spite of the fact that the sedimentation curves for the epidermal proteins considered here often have zero slope. Thus the primitive theory is not in accord with the experimental evidence. The disagreement may result from the effects of the polydispersity of the protein. As a result, it is not feasible to calculate molecular weights of polydisperse materials from t,he specific sedimentation slopes as proposed by Golder (8). For this purpose the sedimentation value was used in conjunction with the extrapolated viscosity increment. The tendency for the sedimentation constant to be independent of concentration in the concentration range 0.2-l % is shown by the data for the urea-extracted 4.5 protein listed in Table I. All measurements of the position of the schlieren peaks were performed carefully. Although such measurements for polydisperse systems because of the broadness of the peaks cannot be performed with an accuracy equivalent to that obtainable for monodisperse systems of molecular weight greater than 20,000, it is considered from analysis of the data that the tendency mentioned above is, a real one and not one attributed to experimental error. From Table I it can be seen that the first extracts of the epidermal proteins of pH 4.5 have extrapolated sedimentation constants, SO, of about 1.9-2.2 X. This is true for urea as an extractor in the concentration range 0.5-6.0 M and for 0.05 N NaOH (Table II). That is, the extrapolated sedimentation constants for this protein are about the same and are not appreciably dependent on the molarity of the urea-extracting solution in t.he stipulated concentration range. It is also seenthat the sovalue for the first extract of 5.5 protein from
SEDIMENTATION
OF
EPIDERMAL
TABLE Sedimentation
hfolarity
171
PROTEINS
I
Constants of the 4.6 Protein Determined in 0.6 M Borate Bufer, pH 8.9
of urea used for extraction M
Concentration of sample g./100 ml.
szo (Sveds) for main component
0.5 0.5 0.5
0.75 0.60 0.45
1.9 2.1 1.9
1.0 1.0 1.0
1.02 0.82 0.61
1.9 1.9 2.1
2.0 2.0 2.0
0.82 0.65 0.49
2.1 2.1 1.9
3.0 3.0 3.0
0.84 0.68 0.51
2.1 2.1 1.9
6.0 6.0 6.0 6.0
1.95 0.94 0.76 0.57
2.2 2.0 1.9 2.0
6.0 (clot)’ 6.0 6.0
1.45 1.16 0.58
1.7 1.8 1.7
1.32 1.06 0.79
1.1 1.3 1.3
10.0 (from 6.3 protein) b 10.0 10.0
a This clot was obtained upon dialysis of a 6 M urea extract of 94 g. of epidermis. The clot was then treated with 400 ml. of 6 211urea at 20°C. for 22 days to give the 4.5 protein. b This protein was obtained at pH 6.3 from an extraction of 97 g. of epidermis with 400 ml. of 10 M urea for 10 days at 0°C. The 6.3 protein was then treated with 1150 ml. of 6 M urea for 70 days to give the 4.5 protein. 1 M urea would be appreciably lower than the SOvalue for the first ext.ract from 6 M urea (Table III). The epidermis is composed of prekeratinous and keratinous proteins, the latter being insoluble in water and the former, at most, very slightly soluble. Urea, by breaking secondary bonds (e.g., hydrogen bonds), ex-
172
WOERNLEY,
CARRUTHERS,
TABLE
REGENT
AND
BAUMLER
II
Sedinaentafion Constants
of th.e 4.6 and 6.6 Proteins in Borbte Btcffer, pH 8.9
Concentration
Determined sm (Sveds) for main component
Protein
NaOH extractant N
4.5 4.5 4.5
0.05 0.05 0.05
0.845 0.676 0.507
2.0 2.2 2.2
5.5 5.5 5.5
0.05 0.05 0.05
1.060 0.636 0.304
1.9 2.2 2.1
4.5” 4.5 4.5
1.0 1.0 1.0
1.460 1.17 0.88
1.6 1.7 1.7
5.54 5.5 5.5
1.0 1.0 1.0
0.860 0.688 0.516
3.6 3.6 3.4
5.5b 5.5 5.5
0.05 0.05 0.05
0.970 0.776 0.582
2.5 2.4 2.2
g.1100 ml.
a This protein was obtained from a 1.0 N NaOH extract (0” for 2 hr.) of epidermis. The latter had been extracted once with 6 M urea. b This protein was obtained from a 0.05 N NaOH extract (0” for 1.5 hr.) of epidermis. The latter had been extracted twice with 6 111urea.
tracts prekeratinous proteins which separate at pH values of 4.5, 5.5, and 6.3, leaving behind an insoluble keratinous residue. The 4.5 protein is soluble at pH 7.0, and the 5.5 protein is soluble in slightly alkaline solutions. The 6.3 protein is not soluble in water but is soluble in 6 M urea and dilute alkali. For solubilization of the 6.3 protein by urea a considerable amount of time is required. Although in the range 0.5-6 M urea there is no appreciable variation of sedimentation constant wit,h the extractor concentration, there is an important difference: the protein yield is higher for the greater molarities. A characteristic of the 4.5 urea-extracted protein is that it has less of a tendency for forming gellike clots, fibers, or films than the 5.5 protein. Thus the former has been designated as nonfibrous and the latter as fibrous by Rudall (7). Since the polydisperse systems capable of forming fibers tend to lose this ability when the average molecular weight
SEDIMENTATION
OF
EPIDERMAL
TABLE
PROTEINS
173
III
Sedimentation Constants of the 6.6 Protein Determined in 0.06 M Borate Buffer, pH 8.9 Molarity of urea used for extraction dl
Concentration of sample g./lOO
m (Sveds) for main component
ml.
1.0 1.0 1.0
0.70 0.56 0.42
2.2 2.2 1.9
2.0 2.0 2.0
1.00 0.75 0.50
2.2 2.4 2.1
3.0” 3.0 3.0
0.88 0.66 0.50
3.1 3.1 3.2
6.0 6.0
1.35 0.90
2.2 2.5
~2The 5.5 protein obtained from this urea solution was isolated from epidermis extracted with 500 ml. of 3 iii urea a.t 0” for 9 da.ys. The epidermis (61 g.) had been previously extracted with 1000 ml. of 3 M urea at 0” for 7 days.
decreases below a certain value, it is reasonable t,o expect that the so called nonfibrous 4.5 protein would have a lower average molecular weight than the fibrous 5.5 protein. This is substant,iated by data for urea-ext,racted epidermis which will be discussed later. Polydisperse systems are dealt with here, and the line of demarcation between fibrous and nonfibrous is not sharp. It has been noted in Tables I and II that the 4.5 protein has about the same sediment,ation constant when obtained from 0.05 N NaOH or 6 ~13 urea (or less). There are observable differences, however, between the proteins ext,ract,ed by t#hetwo solvents. Those extracted with dilute alkali are definitely less polydisperse than t,hose extract,ed with urea (see Fig. 2). This is evident from electrophoretic as well as ultracentrifugation patterns. In addition, the electrophoret,ic mobilities of this protein obt.ained from two t.ypes of extracts are different. The szOvalues for the 5.5 protein extracted with 6 ill urea are quite concentrat,ion dependent in the concentrat,ion range O-l % (Table III), whereas for t.he 5.5 protein extracted directly with 0.05 N NaOH t.he ~20values appear to be nea.rly independent of concentration (Table II). This is substant,iated by more accurate data given below.
174
WOERNLEY,
CARRUTHERS,
REGENT
AND
BAUMLER
FIG. 2. Schlieren patterns for epidermal protein extracted with 0.05 N NaOH and which separated out at pH 4.5; solvent, 0.05 M borate buffer pH 8.9; concentration, 0.845 g./lOO ml. solution; time interval between photographs, 8 min.; and time of left-hand pattern, 52 min. after rotor reached full speed.
The 4.5 protein obtained by extracting the insoluble 6.3 protein with 10 M urea has an so value of approximately 1.3 S as compared with a first urea extract 4.5 protein from 6 M urea which has an so value of about 2.0 S (Table I). The former extraction requires a much greater length of time than the latter. It is not known whether the former 4.5 protein is a more denatured product or is an altogether different type of 4.5 protein from the latter. The proteins extracted from epidermal residues often exhibit different sedimentation constants from those observed for first extracts even though the pH of least solubility may be the same (Tables I and III). In Table IV are listed viscosity increments, extrapolated to zero volume fraction (i.e., 19= 0), axial ratios, frictional coefficients, so values, and average molecular weights of the 4.5 and 5.5 epidermal proteins which were obtained from primary extracts with urea and dilute alkali. The viscosity increments and so values were determined from measurements, and the other quantities were calculated by well-known procedures (9, 8). A partial specific volume of 0.727 cc/g. was employed in the calculations (10). The reasons for this choice were that the epiTABLE Viscosity
Protein
4.5 4.5 5.5
IV
Increments, Axial
Ratios, Frictional Coe$kients, Sedimentation Constants and Molecular Weights of Epidermal Proteins Viscosity MOllXllkW increment Frictional e=o Extractant Axiil ratio coefiicient so (Sveds) wei!&t 22.0 14.1 1.7 2.0 30,000 6 M urea
0.05 N NaOH 6 M urea
37.1 38.5
19.7 20.2
2.0 2.0
2.1 3.1
39,000 70,006
SEDIMENTd4TION
OF
EPIDERM.4L
PROTEINS
175
dermal proteins investigated are considered to be prekeratinous, and sufficient quantities of these proteins for accurate determinations of partial specific volume were not available. Solvation was neglected in those calculations because its extent is unknown. The so values used in the calculation of the molecular weights correspond approximately to those determined by extrapolating to zero concentration those s20values corresponding to the modes of the initial distribution functions of the polydisperse concentration series. The viscosity increments and the molecular weight for the 4.5 protein from 6 M urea are appreciably less than for the 5.5 protein. This is in agreement with Rudall’s rough classification of the nonfibrous and fibrous epidermal proteins. After completion of the collection of the above data, a synthebic boundary cell was acquired. Some spot checks of the trends discussed above were made. It is believed from this additional work that the synthetic boundary cell is superior to the conventional cell for the study of such polydisperse systems as encountered here because sharper schlieren peaks are initially formed. Thus the necessary measurements can be made with greater accuracy. A concentra6ion series was run on a 5.5 protein which was obtained from a first extract of epidermis with 6 M urea. The solvent for the prot8ein was 0.05 M borate buffer of pH 8.9. The protein was not subjected to a preparatory run as was the case above. For protein concentrations 1.14,0.912,0.684,0.456, and 0.228 g./lOO ml. the s20values were, respectively, 2.03, 2.53, 2.64, 3.34, and 3.44 S. There is thus a definite increase of the 820value with decreasing concentration as evidenced by the more meager data listed in Table III for this protein. This verifies the statement made above that with careful measurements the standard cell can be used to detect trends. A greater accuracy, however, can be attained with the synthetic boundary cell. A concentration series was also run with the synthetic boundary cell for the 4.5 prot,ein which was obtained from a first extract of epidermis with 6 M urea. The solvent was again 0.05 M borate buffer of pH 8.9. The protein was not subjected to a preparatory centrifugation. Since the quantity of 4.5 protein available at this time was small, the series comprised only a small range of concentration. For protein concentrat.ions 0.53, 0.43, and 0.33 g./lOO ml., the ~20values were, respectively, 2.27, 2.12, and 1.93 S. These results (i.e., the szovalues do not increase with decreasing concentrat,ion) are in agreement with t,hose listed in Table I for the same protein.
176
WOERNLEY,
CARRUTHERS,
REGENT
AND
BAUMLER
SUMMARY AND CONCLUSIONS
The sedimentation properties of proteins which were isolated at pH’s 4.5 and 5.5 from extracts of beef snout epidermis with various molarities of urea and 0.05 N NaOH were investigated. It was found that (a) the ~20values for the 4.5 protein of the first extracts were nearly independent of concentration in the range below 1 g./lOO ml.; (b) the so values for the 4.5 protein of the first extracts were nearly independent of molarity of urea employed in the extractions in the range 0.5-6 M urea; (c) the so values for 4.5 protein of the first extracts were approximately the same for the extractors 6 M urea and 0.05 N NaOH although the intrinsic viscosities were different; (d) the szovalues for the 5.5 protein of the first extracts from 6 211urea were definitely concentration dependent; (e) the intrinsic viscosity of the 5.5 protein was greater in 0.05 M borate buffer of pH 8.9 than that of the 4.5 protein in the same buffer; (f) the molecular weight of the 5.5 protein was greater than that of the 4.5 protein when the proteins were extracted with 6 IIf urea; (g) the SOvalue for a 4.5 protein extracted from t,he 6.3 protein with 10 111urea was appreciably lower than that for a first extract of a 4.5 protein with 6 M urea; (h) the epidermal proteins extracted with 6 M urea are more polydisperse than those extracted with 0.05 N NaOH. It was also found that greater accuracy can be attained with a synthetic boundary cell than with the conventional cell in determining t’he sedimentation properties of the more or less polydisperse epidermal proteins. REFERENCES 1. CARRUTHERS, C., WOERNLEY, D. L., BAUMLER, A., AND KRESS, B., J. Invest. Dermatol. 26, 89 (1965). 2. CARRUTHERS, C., WOERNLEY, D. L., BAUMLER, A., AND SHORTS, H., J. Sot. Cosmetic Chemists 6, 324 (1955). 3. MERCER, E. H., AND OLOFSSON, B., J. Polymer Sci. 6,261 (1951). 4. MERCER, E. H., AND OLOFSSON, B., J. Polymer Sci. 6, 671 (1951). 5. WOODIN, A. M., Biochem. J. 67, 99 (1954). 6. WOODIN, A. M., Nature 173, 823 (1954). 7. RUDALL, K. hf., Advances in Protein Chem. 7. 253 (1952). 8. GOLDER, R. H., J. Bm. Chem. Sot. 76, 1739 (1953). 9. SIMHA, R., J. Phys. Chem. 44, 25 (1940). 10. WARD, W. H., Teztile Research J. 32, 405 (1952).
The Effect of Extraction Procedures on Sedimentation Properties of Epidermal Proteins D. L. Woemley, C. Carruthers, L. Regent and A. Baumler From the Departments of Biophysics and Biochemistry, Memorial Institute, Butialo, New York Received
April
Roswell
Park
26, 1956
In previous investigations (1, 2) proteins extracted from mammalian epidermis by solvents such as water, urea, and sodium lauryl sulfate were studied by the technique of electrophoresis. The current presentation consists of further characterization of epidermal proteins by means of sedimentation rates obtained by ultracentrifugation analysis. Mercer and Olofsson (3) have investigated a urea extract of the prekeratinous layers of epidermis in order to amplify the knowledge gained from techniques (e.g., x-ray diffraction) applicable to the intact epidermis. More distantly related researches regarding soluble derivatives of wool and feather keratin are those of Mercer and Olofsson (4) and of Woodin (5, 6). An excellent introduction concerning some of the chemical and physical properties of epidermal proteins will be found in the publications of Rudall (7). Since primary neoplasms can readily be produced in mammalian epidermis, it is a highly suitable tissue for making comparative physicochemical studies of the normal and malignant states. This work involves normal tissue proteins and serves as a basis for future comparative research on malignant tissue. PROCEDURE The procedures for the isolation of the proteins from beef snout epidermis with urea were the same as described by Carruthers et al. (1, 2). In addition, urea-extracted beef snout epidermis and epidermis in the fresh state were extracted with 0.05 N NaOH in the cold. The proteins were then isolated as follows: The alkaline extract was diluted threefold with distilled water and then stirred for 2 hr. with a hlagne-stirrer. The pH of the extract was then lowered to 7.0 (glass electrode) by the addition of 1 N HCl. This was followed by 167
168
WOERNLEY,
CARRUTHERS,
REGENT
AND
BAUMLER
centrifugation at 1500 r.p.m. for 20 min. The supernatant fraction was then adjusted to pH 5.5 by the addition of HCI, and the protein obtained at this pH was separated by centrifugation. The supernatant fraction was then adjusted to pH 4.5 by the addition of HCl, and the protein which precipitated was separated by centrifugation. Both of these proteins were purified by dissolving them in slightly alkaline water, centrifuging for 30 min. at 13,309 r.p.m., and then reprecipitating the proteins at the proper pH. This procedure was repeated three times. In this report the principal interest is in the proteins obtained from the first extracts of epidermis with urea or dilute alkali. Other extracts are briefly described since the proteins isolated showed differences in sedimentation properties from those obtained from the primary extracts. The proteins will be designated simply as 4.5, 5.5, and 6.3. These values correspond to the pH at which the proteins were separated from the various extracts by the careful addition of HCl. The pH was measured with a glass electrode (Beckman, model G pH meter). The proteins which were isolated at pH’s 4.5 and 5.5 from urea extracts of epidermis correspond, respectively, to the nonfibrous and fibrous proteins of Rudall (7). The yields obtained of purified proteins were very low. With 0.5, 1.0, 2.0, 3.0, and 6 M urea as extraction media, the percentage yields of purified 4.5 proteins were, respectively, 0.15, 0.13, 0.10, 0.06, and 0.10. From the same extractions the percentage yields of purified 5.5 proteins were respectively 0.13,0.10,0.09,0.04 and 0.32. With 0.05 N NaOH as the extractant, the percentage yields of 5.5 and 4.5 proteins were I.48 and 1.54. It is interesting to note that when 6 M urea was used as extractant, the ratio of fibrous to nonfibrous protein was approximately 3:1, while with smaller concentrations of urea the ratio was 1: 1. Sedimentation rates were obtained at rotational speeds of 59,736 r.p.m. with a model E Spinco ultracentrifuge which was initially tested by a determination of crystallized bovine plasma albumin (control 0123-176, Armour and Company). For preparatory and analytical runs the model A and An-D rotors were respectively employed. Viscosities of the samples of epidermal proteins (which were “cleaned up” by centrifugation at accelerations of 500 X g) were determined with an Ostwald viscometer. For the proteins investigated, there was present a schlieren peak of large area and often another of very small area. The former peak corresponded to a more or less polydisperse system of low sedimentation constant, and the latter to some material of appreciably greater sedimentation constant. To reduce the quantity of the latter material, all epidermal protein samples were centrifuged to the same extent in the preparatory rotor prior to the analytical run. Svedberg’s reduction formula was employed for conversion of measured sedimentation values, sr to s2,,values. For comparative purposes the calculations were made for a constant-run duration. The initial schlieren peaks employed were photographed at the same interval measured from the time the rotor reached its full speed. RESULTS
AND
DISCUSSION
The extrapolated sedimentation constant, SO, (for zero concentration and standard conditions) for bovine albumin in 0.2 N NaCl was de-
FIG. 1. Schlieren patterns for epidermal protein extracted with 6 M urea and which separated out at pH 4.5; solvent, 0.05 M borate buffer pH 8.9; concentration, 1.95 g./lOO ml. solution; time interval between photographs, 8 min.; and time of left-hand pattern, 44 min. after rotor reached full speed.
termined to be 4.54 S. A partial specific volume V equal to 0.730 was employed in the reduction formula. This sedimentation value is about 5 % higher than recently obtained values which fall in the range 4.27-4.32 S. The difference may be due to the effect of the solvent or the nature of the sample. Manufacturer’s products often show differences among batches. The agreement, however, is sufficient for the purposes of this experiment. There are certain inherent difficulties in determining the sedimentation constants for the epidermal proteins so far encountered. In general the extraction procedures are laborious and the extracted proteins are of limited solubility. The most formidable obstacle, however, is that the extracted proteins are more or less polydisperse. This is demonstrated by the fact that the schlieren peaks are unsymmetrical and broaden rapidly in the course of a sedimentation run. At a sample concentration of 2% by volume or lower, since the schlieren peaks for the epidermal proteins broaden out much more rapidly than a monodisperse system, the usuable length of the run with respect t’o measurement is much reduced. See Fig. 1. Another important factor is that while for dilute monodisperse systems the plot of the sedimentation constant against concentration approximates a straight line with an appreciable negative slope, for the polydisperse epidermal proteins in the concentration range 0.2-l % this plot often approximates a straight line with nearly zero slope. Thus in general the following sets of equations do not hold for polydisperse epidermal proteins (8). so
-=1++c
170
WOERNLEY,
CARRUTHERS,
REGENT
AND
BAUMLER
where s is the sedimentation rate at concentration c, so is the rate extrapolated to zero concentration, and A is the specific sedimentation slope ; and A = 0.75 [IV] + 5,
or = [N]
(2)
where m] is the viscosity increment extrapolated to zero volume fraction i.e., c is expressed as,.the volume fraction. The truth of this statement can readily be seenby setting the derivative, ds/dc of Eq. (1) equal to zero, i.e., -80-4 (1 + AC)’ = O
(3)
The only way for this relation to hold at concentrations 0.2-l % is to have A = 0. For the epidermal proteins encountered here, A calculated from N has an appreciable value, in spite of the fact that the sedimentation curves for the epidermal proteins considered here often have zero slope. Thus the primitive theory is not in accord with the experimental evidence. The disagreement may result from the effects of the polydispersity of the protein. As a result, it is not feasible to calculate molecular weights of polydisperse materials from t,he specific sedimentation slopes as proposed by Golder (8). For this purpose the sedimentation value was used in conjunction with the extrapolated viscosity increment. The tendency for the sedimentation constant to be independent of concentration in the concentration range 0.2-l % is shown by the data for the urea-extracted 4.5 protein listed in Table I. All measurements of the position of the schlieren peaks were performed carefully. Although such measurements for polydisperse systems because of the broadness of the peaks cannot be performed with an accuracy equivalent to that obtainable for monodisperse systems of molecular weight greater than 20,000, it is considered from analysis of the data that the tendency mentioned above is, a real one and not one attributed to experimental error. From Table I it can be seen that the first extracts of the epidermal proteins of pH 4.5 have extrapolated sedimentation constants, SO, of about 1.9-2.2 X. This is true for urea as an extractor in the concentration range 0.5-6.0 M and for 0.05 N NaOH (Table II). That is, the extrapolated sedimentation constants for this protein are about the same and are not appreciably dependent on the molarity of the urea-extracting solution in t.he stipulated concentration range. It is also seenthat the sovalue for the first extract of 5.5 protein from
SEDIMENTATION
OF
EPIDERMAL
TABLE Sedimentation
hfolarity
171
PROTEINS
I
Constants of the 4.6 Protein Determined in 0.6 M Borate Bufer, pH 8.9
of urea used for extraction M
Concentration of sample g./100 ml.
szo (Sveds) for main component
0.5 0.5 0.5
0.75 0.60 0.45
1.9 2.1 1.9
1.0 1.0 1.0
1.02 0.82 0.61
1.9 1.9 2.1
2.0 2.0 2.0
0.82 0.65 0.49
2.1 2.1 1.9
3.0 3.0 3.0
0.84 0.68 0.51
2.1 2.1 1.9
6.0 6.0 6.0 6.0
1.95 0.94 0.76 0.57
2.2 2.0 1.9 2.0
6.0 (clot)’ 6.0 6.0
1.45 1.16 0.58
1.7 1.8 1.7
1.32 1.06 0.79
1.1 1.3 1.3
10.0 (from 6.3 protein) b 10.0 10.0
a This clot was obtained upon dialysis of a 6 M urea extract of 94 g. of epidermis. The clot was then treated with 400 ml. of 6 211urea at 20°C. for 22 days to give the 4.5 protein. b This protein was obtained at pH 6.3 from an extraction of 97 g. of epidermis with 400 ml. of 10 M urea for 10 days at 0°C. The 6.3 protein was then treated with 1150 ml. of 6 M urea for 70 days to give the 4.5 protein. 1 M urea would be appreciably lower than the SOvalue for the first ext.ract from 6 M urea (Table III). The epidermis is composed of prekeratinous and keratinous proteins, the latter being insoluble in water and the former, at most, very slightly soluble. Urea, by breaking secondary bonds (e.g., hydrogen bonds), ex-
172
WOERNLEY,
CARRUTHERS,
TABLE
REGENT
AND
BAUMLER
II
Sedinaentafion Constants
of th.e 4.6 and 6.6 Proteins in Borbte Btcffer, pH 8.9
Concentration
Determined sm (Sveds) for main component
Protein
NaOH extractant N
4.5 4.5 4.5
0.05 0.05 0.05
0.845 0.676 0.507
2.0 2.2 2.2
5.5 5.5 5.5
0.05 0.05 0.05
1.060 0.636 0.304
1.9 2.2 2.1
4.5” 4.5 4.5
1.0 1.0 1.0
1.460 1.17 0.88
1.6 1.7 1.7
5.54 5.5 5.5
1.0 1.0 1.0
0.860 0.688 0.516
3.6 3.6 3.4
5.5b 5.5 5.5
0.05 0.05 0.05
0.970 0.776 0.582
2.5 2.4 2.2
g.1100 ml.
a This protein was obtained from a 1.0 N NaOH extract (0” for 2 hr.) of epidermis. The latter had been extracted once with 6 M urea. b This protein was obtained from a 0.05 N NaOH extract (0” for 1.5 hr.) of epidermis. The latter had been extracted twice with 6 111urea.
tracts prekeratinous proteins which separate at pH values of 4.5, 5.5, and 6.3, leaving behind an insoluble keratinous residue. The 4.5 protein is soluble at pH 7.0, and the 5.5 protein is soluble in slightly alkaline solutions. The 6.3 protein is not soluble in water but is soluble in 6 M urea and dilute alkali. For solubilization of the 6.3 protein by urea a considerable amount of time is required. Although in the range 0.5-6 M urea there is no appreciable variation of sedimentation constant wit,h the extractor concentration, there is an important difference: the protein yield is higher for the greater molarities. A characteristic of the 4.5 urea-extracted protein is that it has less of a tendency for forming gellike clots, fibers, or films than the 5.5 protein. Thus the former has been designated as nonfibrous and the latter as fibrous by Rudall (7). Since the polydisperse systems capable of forming fibers tend to lose this ability when the average molecular weight
SEDIMENTATION
OF
EPIDERMAL
TABLE
PROTEINS
173
III
Sedimentation Constants of the 6.6 Protein Determined in 0.06 M Borate Buffer, pH 8.9 Molarity of urea used for extraction dl
Concentration of sample g./lOO
m (Sveds) for main component
ml.
1.0 1.0 1.0
0.70 0.56 0.42
2.2 2.2 1.9
2.0 2.0 2.0
1.00 0.75 0.50
2.2 2.4 2.1
3.0” 3.0 3.0
0.88 0.66 0.50
3.1 3.1 3.2
6.0 6.0
1.35 0.90
2.2 2.5
~2The 5.5 protein obtained from this urea solution was isolated from epidermis extracted with 500 ml. of 3 iii urea a.t 0” for 9 da.ys. The epidermis (61 g.) had been previously extracted with 1000 ml. of 3 M urea at 0” for 7 days.
decreases below a certain value, it is reasonable t,o expect that the so called nonfibrous 4.5 protein would have a lower average molecular weight than the fibrous 5.5 protein. This is substant,iated by data for urea-ext,racted epidermis which will be discussed later. Polydisperse systems are dealt with here, and the line of demarcation between fibrous and nonfibrous is not sharp. It has been noted in Tables I and II that the 4.5 protein has about the same sediment,ation constant when obtained from 0.05 N NaOH or 6 ~13 urea (or less). There are observable differences, however, between the proteins ext,ract,ed by t#hetwo solvents. Those extracted with dilute alkali are definitely less polydisperse than t,hose extract,ed with urea (see Fig. 2). This is evident from electrophoretic as well as ultracentrifugation patterns. In addition, the electrophoret,ic mobilities of this protein obt.ained from two t.ypes of extracts are different. The szOvalues for the 5.5 protein extracted with 6 ill urea are quite concentrat,ion dependent in the concentrat,ion range O-l % (Table III), whereas for t.he 5.5 protein extracted directly with 0.05 N NaOH t.he ~20values appear to be nea.rly independent of concentration (Table II). This is substant,iated by more accurate data given below.
174
WOERNLEY,
CARRUTHERS,
REGENT
AND
BAUMLER
FIG. 2. Schlieren patterns for epidermal protein extracted with 0.05 N NaOH and which separated out at pH 4.5; solvent, 0.05 M borate buffer pH 8.9; concentration, 0.845 g./lOO ml. solution; time interval between photographs, 8 min.; and time of left-hand pattern, 52 min. after rotor reached full speed.
The 4.5 protein obtained by extracting the insoluble 6.3 protein with 10 M urea has an so value of approximately 1.3 S as compared with a first urea extract 4.5 protein from 6 M urea which has an so value of about 2.0 S (Table I). The former extraction requires a much greater length of time than the latter. It is not known whether the former 4.5 protein is a more denatured product or is an altogether different type of 4.5 protein from the latter. The proteins extracted from epidermal residues often exhibit different sedimentation constants from those observed for first extracts even though the pH of least solubility may be the same (Tables I and III). In Table IV are listed viscosity increments, extrapolated to zero volume fraction (i.e., 19= 0), axial ratios, frictional coefficients, so values, and average molecular weights of the 4.5 and 5.5 epidermal proteins which were obtained from primary extracts with urea and dilute alkali. The viscosity increments and so values were determined from measurements, and the other quantities were calculated by well-known procedures (9, 8). A partial specific volume of 0.727 cc/g. was employed in the calculations (10). The reasons for this choice were that the epiTABLE Viscosity
Protein
4.5 4.5 5.5
IV
Increments, Axial
Ratios, Frictional Coe$kients, Sedimentation Constants and Molecular Weights of Epidermal Proteins Viscosity MOllXllkW increment Frictional e=o Extractant Axiil ratio coefiicient so (Sveds) wei!&t 22.0 14.1 1.7 2.0 30,000 6 M urea
0.05 N NaOH 6 M urea
37.1 38.5
19.7 20.2
2.0 2.0
2.1 3.1
39,000 70,006
SEDIMENTd4TION
OF
EPIDERM.4L
PROTEINS
175
dermal proteins investigated are considered to be prekeratinous, and sufficient quantities of these proteins for accurate determinations of partial specific volume were not available. Solvation was neglected in those calculations because its extent is unknown. The so values used in the calculation of the molecular weights correspond approximately to those determined by extrapolating to zero concentration those s20values corresponding to the modes of the initial distribution functions of the polydisperse concentration series. The viscosity increments and the molecular weight for the 4.5 protein from 6 M urea are appreciably less than for the 5.5 protein. This is in agreement with Rudall’s rough classification of the nonfibrous and fibrous epidermal proteins. After completion of the collection of the above data, a synthebic boundary cell was acquired. Some spot checks of the trends discussed above were made. It is believed from this additional work that the synthetic boundary cell is superior to the conventional cell for the study of such polydisperse systems as encountered here because sharper schlieren peaks are initially formed. Thus the necessary measurements can be made with greater accuracy. A concentra6ion series was run on a 5.5 protein which was obtained from a first extract of epidermis with 6 M urea. The solvent for the prot8ein was 0.05 M borate buffer of pH 8.9. The protein was not subjected to a preparatory run as was the case above. For protein concentrations 1.14,0.912,0.684,0.456, and 0.228 g./lOO ml. the s20values were, respectively, 2.03, 2.53, 2.64, 3.34, and 3.44 S. There is thus a definite increase of the 820value with decreasing concentration as evidenced by the more meager data listed in Table III for this protein. This verifies the statement made above that with careful measurements the standard cell can be used to detect trends. A greater accuracy, however, can be attained with the synthetic boundary cell. A concentration series was also run with the synthetic boundary cell for the 4.5 prot,ein which was obtained from a first extract of epidermis with 6 M urea. The solvent was again 0.05 M borate buffer of pH 8.9. The protein was not subjected to a preparatory centrifugation. Since the quantity of 4.5 protein available at this time was small, the series comprised only a small range of concentration. For protein concentrat.ions 0.53, 0.43, and 0.33 g./lOO ml., the ~20values were, respectively, 2.27, 2.12, and 1.93 S. These results (i.e., the szovalues do not increase with decreasing concentrat,ion) are in agreement with t,hose listed in Table I for the same protein.
176
WOERNLEY,
CARRUTHERS,
REGENT
AND
BAUMLER
SUMMARY AND CONCLUSIONS
The sedimentation properties of proteins which were isolated at pH’s 4.5 and 5.5 from extracts of beef snout epidermis with various molarities of urea and 0.05 N NaOH were investigated. It was found that (a) the ~20values for the 4.5 protein of the first extracts were nearly independent of concentration in the range below 1 g./lOO ml.; (b) the so values for the 4.5 protein of the first extracts were nearly independent of molarity of urea employed in the extractions in the range 0.5-6 M urea; (c) the so values for 4.5 protein of the first extracts were approximately the same for the extractors 6 M urea and 0.05 N NaOH although the intrinsic viscosities were different; (d) the szovalues for the 5.5 protein of the first extracts from 6 211urea were definitely concentration dependent; (e) the intrinsic viscosity of the 5.5 protein was greater in 0.05 M borate buffer of pH 8.9 than that of the 4.5 protein in the same buffer; (f) the molecular weight of the 5.5 protein was greater than that of the 4.5 protein when the proteins were extracted with 6 IIf urea; (g) the SOvalue for a 4.5 protein extracted from t,he 6.3 protein with 10 111urea was appreciably lower than that for a first extract of a 4.5 protein with 6 M urea; (h) the epidermal proteins extracted with 6 M urea are more polydisperse than those extracted with 0.05 N NaOH. It was also found that greater accuracy can be attained with a synthetic boundary cell than with the conventional cell in determining t’he sedimentation properties of the more or less polydisperse epidermal proteins. REFERENCES 1. CARRUTHERS, C., WOERNLEY, D. L., BAUMLER, A., AND KRESS, B., J. Invest. Dermatol. 26, 89 (1965). 2. CARRUTHERS, C., WOERNLEY, D. L., BAUMLER, A., AND SHORTS, H., J. Sot. Cosmetic Chemists 6, 324 (1955). 3. MERCER, E. H., AND OLOFSSON, B., J. Polymer Sci. 6,261 (1951). 4. MERCER, E. H., AND OLOFSSON, B., J. Polymer Sci. 6, 671 (1951). 5. WOODIN, A. M., Biochem. J. 67, 99 (1954). 6. WOODIN, A. M., Nature 173, 823 (1954). 7. RUDALL, K. hf., Advances in Protein Chem. 7. 253 (1952). 8. GOLDER, R. H., J. Bm. Chem. Sot. 76, 1739 (1953). 9. SIMHA, R., J. Phys. Chem. 44, 25 (1940). 10. WARD, W. H., Teztile Research J. 32, 405 (1952).