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Studies on the high-sulphur proteins of reduced merino wool
] ournal of Chromatography, 78 (1973) 363-369 © Elsevier Scientific Publishing Company, Amsterdam-Printed in The Netherlands
CHROM.6545
STUDIES ON THE HIGH-SULPHUR PROTEINS OF REDUCED MERINO WOOL THE CHROMATOGRAPHY OF GROUP IlIA ON CELLULOSE PHOSPHATE
L. S. SWART, D. PARRIS
AND
F. J. JOUBERT
National Chemical Research Laboratory, Council for Scientific and I ndustrial Research, Pretoria (Republic of South Africa) (Received December IIth, 1972)
SUMMARY
A method has been developed for the fractionation of a group of high-sulphur proteins from wool by chromatography on cellulose phosphate in 5 M urea at pH z.8. Eight components were obtained and shown to be relatively pure. A scheme for the fractionation of high-sulphur proteins of a-keratins by chromatography on DEAEcellulose followed by chromatography on cellulose phosphate is proposed.
INTRODUCTION
The S-carboxymethyl derivatives of the high-sulphur proteins (SCMKB) of a-keratins have been extensively investigated l - 8 • These studies have shown that the high-sulphur proteins can be divided into groups differing in their charge, molecular size and chromatographic properties. Several fractionation schemes were used to purify the proteins, with varied success. The first pure high-sulphur protein from wool was obtained by SWART et al. 5 • In that study, the high-sulphur proteins were initially separated by column electrophoresis and the electrophoretic fraction III was then fractionated by gel filtration on Sephadex G-roo into two groups, IlIA and IIIB, with molecular weights of 17000 and II 000, respectively. Group IIIB yielded four components when chromatographed on DEAE-cellulose, of which only component IIIBz was found to be homogeneous 9 - 11 • The complete amino acid sequences ofthe three major components, IIIBz, IIIB3 and IIIB4, were deduced and found to be closely homologous 9 - 11 • Chromatography of group IlIA on DEAE-cellulose yielded five poorly resolved components5 , which suggests that group IlIA is composed of micro-heterogeneous proteins. It was evident that DEAE-cellulose chromatography alone would not resolve the components of group lIlA.
L.
S. SWART, D. PARRIS,
F.
J. JOUBERT
This paper describes a simplified method for isolating and purifying group IlIA proteins by chromatography of SCMKB on DEAE-cellulose and cellulose phosphate. EXPERIMENTAL
Preparation of S-carboxymethyl high-sulphur proteins The S-carboxymethylated high-sulphur proteins were prepared from Merino wool by using standard methods 5 • Chromatography on DEAE-cellulose Chromatography was performed on a 3.8 X ISO cm column of DEAE-cellulose (Whatman DE32) at 4° by a method similar to that described by JOUBERT AND BURNS 3 • The protein (20 g) was dissolved in 100 ml of the starting buffer (0.05 M sodium acetate, pH 5.0) and applied to the column. An automatic Beckman Spectrochrom was used and the proteins were eluted at 200 ml/h with a Is-litre linear gradient of 0.05 M to 0.70 M sodium acetate at pH 5.0. The effluent from the column was continuously monitored at 280 nm and 22-ml fractions were collected. Reproducible elution patterns were obtained with this method. Fractions were dialysed free of salt and recovered by lyophilization. Chromatography on cellulose phosphate A modification of the method of GILLESPIE (personal communication) was used for chromatography on cellulose phosphate at low pH. The resin (Whatman PII, 7-4 mequiv./g) was swollen in distilled water and then washed for 30 min with 0.5 M sodium hydroxide, rinsed with water, then washed twice for 30 min with 0.5 M hydrochloric acid with several changes of water between the acid treatments, and finally with distilled water. The resin was extensively de-fined in distilled water immediately before use. The starting buffer contained 0.1 M NaCI and 0.1 M citric acid dissolved in 5 M urea, which had been previously deionized by passage through a Dowex I-X8 column followed by a Dowex 50-X8 column (both columns 5 X 50 cm). The cellulose phosphate was equilibrated with starting buffer and packed in a 3.9 X 50 cm column at 4°. The column was equilibrated until the conductivity of the effluent was the same as that of the starting buffer. The samples were dissolved in the minimum volume , dialysed overnight against the starting buffer and applied to the column. An automatic Beckman Spectrochrom was used and the proteins were eluted at 200 ml/h. The starting buffer was pumped through the column for 4 h before the 8-litre linear gradient was started. The limiting buffer was prepared from the starting buffer by addition of NaCl to a concentration of 0.5 M. The effluent from the column was continuously monitored at 280 nm and 20-ml fractions were collected and kept at 4°. The protein fractions were neutralized with 2 M ammonia solution, dialyzed free of salt and recovered by lyophilization. A mino acid analysis Samples were hydrolysed for periods of 24, 48 and 72 h at IIO o in evacuated glass tubes with constant-boiling hydrochloric acid containing 0.01 M phenol and 0.01 M thiodiglycol. The amino acid compositions were determined on a Beckman
CHROMATOGRAPHY OF HIGH-SULPHUR PROTEINS
r20C amino acid analyzer coupled to a Beckman r25 integrator. The values obtained were extrapolated to zero-time hydrolysis. The presence of tryptophan was detected with the Ehrlich reagent after complete enzymic hydrolysis12 of the proteins. The colour yields were compared visually with those obtained for standard amounts of tryptophan. RESULTS
Chromatography on DEAE-cellulose Large-scale separations of the total high-sulphur protein fraction SCMKB were carried out by chromatography on DEAE-cellulose. A typical elution pattern is shown in Fig. 1. Eighteen fractions were isolated as indicated and accumulated from several separations. E
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Fig. 1. Elution pattern of high-sulphur proteins from DEAE-cellulose. For conditions, see text. Optical path length 2.5 mm.
This pattern is analogous to that obtained by JOUBERT et al. 4 and from their study it is obvious that fractions C6 to C13 (cj., Fig. r) correspond to their electrophoretic fractions E3 to E 5. Electrophoretic fraction III, which is also equivalent to fractions C6 to C13 , was separated into two molecular weight groups, IlIA and IIIB5. From the DEAE-cellulose chromatograms of these groups, it is obvious that group IIIB corresponds to fractions C6 to C9 , and group IlIA corresponds to fractions C10 to C13 with some overlap into the preceeding fractions.
Chromatography on cellulose phosphate In order to obtain overlapping IlIA proteins, fractions C8 and C9 were each chromatographed on cellulose phosphate. The elution patterns obtained are given in Figs. za and zb, respectively. Fraction C8 yielded a component, designated IIIAr, as well as a large amount of material excluded from the column, assumed to be group IIIB proteins. Fraction C9 also gave a large amount of IIIB proteins and in addition yielded two components, IIIAr and IIIAz. Component IIIAr obtained from fractions C8 and C9 was pooled and re-chromatography on cellulose phosphate produced a single peak, proving the materials to be chromatographically identical.
366
L. S. SWART, D. PARRIS, F.
J.
JOUBERT
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Fig. 2. Elution patterns of two chromatographic fractions (cf., Fig. I) from cellulose phosphate. (a) Fraction C8 (7.2 g); (b) fraction C. (6.4 g). For conditions, see text. Optical path length 2.S mm.
Fractions C10 to C13 were pooled and subjected to chromatography on cellulose phosphate and a typical elution pattern is shown in Fig.3a. Group I1IB material was again excluded from the column. Poorly resolved fraction IIIAP and components I1IA4 to IIIAr were isolated 0'3j 0,05""-0'20""
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Fig. 3. Elution patterns-of the components of group IlIA from cellulose phosphate. (a) Fractions C,o to C'3 (3·S g); (b) fraction IIlAP (4 g); (c) component IlIA3 (3 g); (d) component IIlA7 (1.8 g);
(e) component IIlA2 (1.9 g); (f) component IlIA6 (2.r g); (g) component IIlAr (1.4 g); (h) component IlIAS (3 g). The sodium chloride gradient used in each experiment is indicated in each figure; for the other conditions, see text. Optical path length 2.S mm.
r:HROMATOGRAPHY OF HIGH-SULPHUR PROTEINS
as indicated and accumulated from several experiments. The elution volumes of components IIIAz and IlIAr coincided with those obtained for components IIIAz and IlIAr isolated from fractions Cs and C9 (cj., Figs. za and zb). The gradient was altered to 0.05 M to o.zo M N aCl for re-chromatography of fraction IIlAP on cellulose phosphate to give the elution pattern in Fig. 3b. Component IIlA8 was separated completely from the other components and was assumed to be pure. Components IlIA7 to IIIAS were poorly resolved and the separation was not improved by changing the gradient. The small amount of component IIlA4 obtained from fraction IIIAP was added to that obtained previously (cj., Fig. 3a). Components IIIA3, IlIAz and IIlAr were each re-chromatographed on cellulose phosphate using an extended gradient of o.ro M to 0.30 M NaCl. From the chromatographs shown in Figs. 3c, 3e and 3g, respectively, it is evident that the elution volumes are of the order of those expected on the basis of the original experiment (cj., Fig.3a). Components IIlA3 and IlIAr gave single symmetrical peaks while component IIIAz gave a skew peak, showing the presence of IIIAr material. When components IlIA7 and IlIA6, obtained from fraction IIIAP, were rechromatographed on cellulose phosphate, the NaCl concentration of the starting buffer was changed from 0.05 M to 0.08 M, giving the elution patterns shown in Figs. 3d and 3f, respectively. The separation was not much improved and these components cannot be expected to be fully resolved. Components IIIAS and IIIA4 were re-chromatographed on cellulose phosphate with a gradient of o.ro M to o.zo M NaCl, and Fig. 3h shows the pattern obtained for component IlIAS. The pattern obtained for component IIIA4 was similar to that obtained for component IIIAS, but with a larger effluent volume. TABLE I AMINO ACID COMPOSITIONS a (RESIDUES PER MOLE) OF ACID HYDROLYSATES OF COMPONENTS OBTAINED BY CHROMATOGRAPHY ON CELLULOSE PHOSPHATE ._ _ . --------- - - - - - - ---
---- ----------
~--
Amino acid
Component IIlAr
IlIA 2
~--.-------
Trpb Arg SCMCC Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Phe Estimated total
15. 0 33·3 2.0 14-4 II.S S.S IS.1 5·9 3. 0 9. 0 2.0
2.S 1.9 2.S 132
1 15.0 3 2 .9 2.0
15.0 12·5 9·1 20.1 6.0 3.0 9·1 2.1 2·9 2.0 2·9 13 6
IlIA 3
IlIA 4
-----
13·9 3 2 .4 2.0 14. 1 12.1 9·2 19.0 5·7 2·9 S.S 2.1 2·7 1.9 2·9 13 1
IlIAS ----
1 13. 1 3 2 .9 2.0 14·4 12.0 9. S 20·9 6.0 2·9 9. 0 2.0 3. 0 1.4 3. 0 133
1 13. 1 35·9 2.1 14· S 12·3 9·7 19. 0 6.0 2·9 8·9 2.1 2·9 1.5 2·9 135
IlIA 6
IlIA 7
---------
12·9 3 2 .5 2.0 14. 0 12·5 9· S 20.S 6.0 3. 0 9. 0 2.1 3. 0 1.3 3. 0 133
a Values obtained by extrapolation to zero-time hydrolysis. b Identified by Ehrlich reagent. c S-Car boxymethy1cysteine.
11.9 35. 2 2·3 14·7 13·2 10.0 19·5 6.0 3. 1 S·9 2.1 3. 2 1.3 3. 0 135
IlIA 8
----------
11.0 34·9 2.0 14. 0 14· S 10.1 19. 2 5· S 3. 1 S.6 2·3 3. 0 1.5 2.8 134
-
L. S. SWART, D. PARRIS, F. J. JOUBERT
Amino acid compositions The amino acid compositions of the eight components of group IIIA are given in Table 1. These results show that the components have very similar amino acid compositions, the major difference amongst the components being that the arginine content varies from I I to IS residues per mole of protein.
DISCUSSION
The use of chromatography on cellulose phosphate has considerably widened the scope of fractionation of reduced and S-carboxymethylated high-sulphur proteins from a-keratins. This technique, using buffers of pH approximately z.S, separates proteins according to their base content. It therefore combines well with other t echniques, such as DEAE-cellulose chromatography or column electrophoresis, which effect separations mainly according to net charge. Group IIIA of the high-sulphur proteins of wool was previously obtained by a combination of column electrophoresis and gel filtration. On DEAE-cellulose chroma tography, this group yielded a broad peak showing several poorly resolved components 5 . In the present study, chromatography on DEAE-cellulose was used to prepare groups IIIA and IIIB directly from the high-sulphur proteins of wool. By using chromatography on cellulose phosphate, it was possible to isolate eight distinct components of group IIIA. The eight components of group IIIA gave stoichiometric values for most amino acids and have empirical molecular weights of approximately 16,000. Major features of the components are the absence of lysine, histidine and methionine residues. The arginine content of the components decreases stepwise from IIIAr to IIIAS, except for IIIAr and IIIAz and also for IIIA4, IIIA5 and IIIA6, for which constant values were obtained. The isolation of components with identical arginine contents shows that other factors, in addition t o base content, influence their resolution on cellulose phosphate. The integral values obtained for amino acids present in small amounts in the proteins, such as aspartic acid, glycine, alanine, isoleucine , leucine and phenylalanine, but not tyrosine, are evidence of the purity of the proteins. As the amino acid compositions of the components are so closely related, sequence analysis must be the final criterion of their purity. In our study, we have shown that large amounts of protein (3 g) can be rapidly and conveniently separated by chromatography on cellulose phosphate. Minimal shrinkage occurs when a 3.S X 50 cm column is eluted at a flow-rate of zoo mljh and the pressure remains low provided that the cellulose phosphate has been thoroughly de-fined. The choice of the gradient is very important. This fact is illustrated in the chromatograms shown in Figs. 3a and 3b, in which fraction IIIAP is better resolved by a change in the elution gradient, component IIIAS in particular separating as a distinct symmetrical peak. Although the system is sensitive to changes in gradient, excellent repeatability is achieved provided that the experimental conditions are kept constant. Unpublished results of work on mohair proteins have shown that chromatography on cellulose phosphate with sodium chloride gradients starting from zero molarity resolves group IIIB proteins. It has also been shown that proteins equivalent
CHROMATOGRAPHY OF HIGH-SULPHUR PROTEINS to fractions C14 to C18 (cJ., Fig. r) elute with group IIIB components on cellulose phosphate chromatography of total high-sulphur proteins. A rapid and convenient method for the preparation of large amounts of the components of groups IlIA and IIIB of the reduced and S-carboxymethylated highsulphur proteins of a-keratins can now be proposed. This method involves initial separation by chromatography on DEAE-cellulose, followed by chromatography on cellulose phosphate.
ACKNOWLEDGEMENT We thank Mrs. P. G. DRIJFHOUT for performing the amino acid analyses. REFERENCES I J. M. GILLESPIE, A ust. j. Bioi. Sci., 16 (1963) 259· 2 L. S. SWART, F. J. JOUBERT, T. HAYLETT AND P. J. DE JAGER, 3rd Int. Wool Text. Res. Conj., Paris, Institute Textile de France, Paris, I (1965) 493. 3 F. J. JOUBERT AND M. A. C. BURNS, j. S. Afr. Chem. Inst., 20 (1967) 161. 4 F. J. JOUBERT, P. J. DE JAGER AND L. S. SWART, in 'V. G. CREWTHER (Editor), Symposium on Fibrous Proteins, Australia, I967, Butterworths, Australia, 1968, p. 343· 5 L. S. SWART, T. HAYLETT AND F. J. JOUBERT, Text. Res. j., 39 (1969) 912. 6 R. L. DARSKUS, J. M. GILLESPIE AND H. LINDLEY, A ust. j. Bioi. Sci., 25 (1972) 139. 7 H. LINDLEY AND T. C. ELLEMAN, Biochem. j., 128 (1972) 859. 8 E. L. DARSKUS, j. Chromatogr., 69 (1972) 341. 9 T. HAYLETT AND L. S. SWART, Text. Res. j., 39 (1969) 917. 10 T. HAYLETT, L. S. SWART AND D. PARRIS, Biochem. j., 123 (1971) 191. II L. S. SWART AND T. HAYLETT, Biochem. J., 123 (1971) 201. 12 R. L. HILL AND 'V. R. SCHMIDT, j. Bioi. Chem., 237 (1962) 389.
CHROM.6545
STUDIES ON THE HIGH-SULPHUR PROTEINS OF REDUCED MERINO WOOL THE CHROMATOGRAPHY OF GROUP IlIA ON CELLULOSE PHOSPHATE
L. S. SWART, D. PARRIS
AND
F. J. JOUBERT
National Chemical Research Laboratory, Council for Scientific and I ndustrial Research, Pretoria (Republic of South Africa) (Received December IIth, 1972)
SUMMARY
A method has been developed for the fractionation of a group of high-sulphur proteins from wool by chromatography on cellulose phosphate in 5 M urea at pH z.8. Eight components were obtained and shown to be relatively pure. A scheme for the fractionation of high-sulphur proteins of a-keratins by chromatography on DEAEcellulose followed by chromatography on cellulose phosphate is proposed.
INTRODUCTION
The S-carboxymethyl derivatives of the high-sulphur proteins (SCMKB) of a-keratins have been extensively investigated l - 8 • These studies have shown that the high-sulphur proteins can be divided into groups differing in their charge, molecular size and chromatographic properties. Several fractionation schemes were used to purify the proteins, with varied success. The first pure high-sulphur protein from wool was obtained by SWART et al. 5 • In that study, the high-sulphur proteins were initially separated by column electrophoresis and the electrophoretic fraction III was then fractionated by gel filtration on Sephadex G-roo into two groups, IlIA and IIIB, with molecular weights of 17000 and II 000, respectively. Group IIIB yielded four components when chromatographed on DEAE-cellulose, of which only component IIIBz was found to be homogeneous 9 - 11 • The complete amino acid sequences ofthe three major components, IIIBz, IIIB3 and IIIB4, were deduced and found to be closely homologous 9 - 11 • Chromatography of group IlIA on DEAE-cellulose yielded five poorly resolved components5 , which suggests that group IlIA is composed of micro-heterogeneous proteins. It was evident that DEAE-cellulose chromatography alone would not resolve the components of group lIlA.
L.
S. SWART, D. PARRIS,
F.
J. JOUBERT
This paper describes a simplified method for isolating and purifying group IlIA proteins by chromatography of SCMKB on DEAE-cellulose and cellulose phosphate. EXPERIMENTAL
Preparation of S-carboxymethyl high-sulphur proteins The S-carboxymethylated high-sulphur proteins were prepared from Merino wool by using standard methods 5 • Chromatography on DEAE-cellulose Chromatography was performed on a 3.8 X ISO cm column of DEAE-cellulose (Whatman DE32) at 4° by a method similar to that described by JOUBERT AND BURNS 3 • The protein (20 g) was dissolved in 100 ml of the starting buffer (0.05 M sodium acetate, pH 5.0) and applied to the column. An automatic Beckman Spectrochrom was used and the proteins were eluted at 200 ml/h with a Is-litre linear gradient of 0.05 M to 0.70 M sodium acetate at pH 5.0. The effluent from the column was continuously monitored at 280 nm and 22-ml fractions were collected. Reproducible elution patterns were obtained with this method. Fractions were dialysed free of salt and recovered by lyophilization. Chromatography on cellulose phosphate A modification of the method of GILLESPIE (personal communication) was used for chromatography on cellulose phosphate at low pH. The resin (Whatman PII, 7-4 mequiv./g) was swollen in distilled water and then washed for 30 min with 0.5 M sodium hydroxide, rinsed with water, then washed twice for 30 min with 0.5 M hydrochloric acid with several changes of water between the acid treatments, and finally with distilled water. The resin was extensively de-fined in distilled water immediately before use. The starting buffer contained 0.1 M NaCI and 0.1 M citric acid dissolved in 5 M urea, which had been previously deionized by passage through a Dowex I-X8 column followed by a Dowex 50-X8 column (both columns 5 X 50 cm). The cellulose phosphate was equilibrated with starting buffer and packed in a 3.9 X 50 cm column at 4°. The column was equilibrated until the conductivity of the effluent was the same as that of the starting buffer. The samples were dissolved in the minimum volume , dialysed overnight against the starting buffer and applied to the column. An automatic Beckman Spectrochrom was used and the proteins were eluted at 200 ml/h. The starting buffer was pumped through the column for 4 h before the 8-litre linear gradient was started. The limiting buffer was prepared from the starting buffer by addition of NaCl to a concentration of 0.5 M. The effluent from the column was continuously monitored at 280 nm and 20-ml fractions were collected and kept at 4°. The protein fractions were neutralized with 2 M ammonia solution, dialyzed free of salt and recovered by lyophilization. A mino acid analysis Samples were hydrolysed for periods of 24, 48 and 72 h at IIO o in evacuated glass tubes with constant-boiling hydrochloric acid containing 0.01 M phenol and 0.01 M thiodiglycol. The amino acid compositions were determined on a Beckman
CHROMATOGRAPHY OF HIGH-SULPHUR PROTEINS
r20C amino acid analyzer coupled to a Beckman r25 integrator. The values obtained were extrapolated to zero-time hydrolysis. The presence of tryptophan was detected with the Ehrlich reagent after complete enzymic hydrolysis12 of the proteins. The colour yields were compared visually with those obtained for standard amounts of tryptophan. RESULTS
Chromatography on DEAE-cellulose Large-scale separations of the total high-sulphur protein fraction SCMKB were carried out by chromatography on DEAE-cellulose. A typical elution pattern is shown in Fig. 1. Eighteen fractions were isolated as indicated and accumulated from several separations. E
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Fig. 1. Elution pattern of high-sulphur proteins from DEAE-cellulose. For conditions, see text. Optical path length 2.5 mm.
This pattern is analogous to that obtained by JOUBERT et al. 4 and from their study it is obvious that fractions C6 to C13 (cj., Fig. r) correspond to their electrophoretic fractions E3 to E 5. Electrophoretic fraction III, which is also equivalent to fractions C6 to C13 , was separated into two molecular weight groups, IlIA and IIIB5. From the DEAE-cellulose chromatograms of these groups, it is obvious that group IIIB corresponds to fractions C6 to C9 , and group IlIA corresponds to fractions C10 to C13 with some overlap into the preceeding fractions.
Chromatography on cellulose phosphate In order to obtain overlapping IlIA proteins, fractions C8 and C9 were each chromatographed on cellulose phosphate. The elution patterns obtained are given in Figs. za and zb, respectively. Fraction C8 yielded a component, designated IIIAr, as well as a large amount of material excluded from the column, assumed to be group IIIB proteins. Fraction C9 also gave a large amount of IIIB proteins and in addition yielded two components, IIIAr and IIIAz. Component IIIAr obtained from fractions C8 and C9 was pooled and re-chromatography on cellulose phosphate produced a single peak, proving the materials to be chromatographically identical.
366
L. S. SWART, D. PARRIS, F.
J.
JOUBERT
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Fig. 2. Elution patterns of two chromatographic fractions (cf., Fig. I) from cellulose phosphate. (a) Fraction C8 (7.2 g); (b) fraction C. (6.4 g). For conditions, see text. Optical path length 2.S mm.
Fractions C10 to C13 were pooled and subjected to chromatography on cellulose phosphate and a typical elution pattern is shown in Fig.3a. Group I1IB material was again excluded from the column. Poorly resolved fraction IIIAP and components I1IA4 to IIIAr were isolated 0'3j 0,05""-0'20""
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Fig. 3. Elution patterns-of the components of group IlIA from cellulose phosphate. (a) Fractions C,o to C'3 (3·S g); (b) fraction IIlAP (4 g); (c) component IlIA3 (3 g); (d) component IIlA7 (1.8 g);
(e) component IIlA2 (1.9 g); (f) component IlIA6 (2.r g); (g) component IIlAr (1.4 g); (h) component IlIAS (3 g). The sodium chloride gradient used in each experiment is indicated in each figure; for the other conditions, see text. Optical path length 2.S mm.
r:HROMATOGRAPHY OF HIGH-SULPHUR PROTEINS
as indicated and accumulated from several experiments. The elution volumes of components IIIAz and IlIAr coincided with those obtained for components IIIAz and IlIAr isolated from fractions Cs and C9 (cj., Figs. za and zb). The gradient was altered to 0.05 M to o.zo M N aCl for re-chromatography of fraction IIlAP on cellulose phosphate to give the elution pattern in Fig. 3b. Component IIlA8 was separated completely from the other components and was assumed to be pure. Components IlIA7 to IIIAS were poorly resolved and the separation was not improved by changing the gradient. The small amount of component IIlA4 obtained from fraction IIIAP was added to that obtained previously (cj., Fig. 3a). Components IIIA3, IlIAz and IIlAr were each re-chromatographed on cellulose phosphate using an extended gradient of o.ro M to 0.30 M NaCl. From the chromatographs shown in Figs. 3c, 3e and 3g, respectively, it is evident that the elution volumes are of the order of those expected on the basis of the original experiment (cj., Fig.3a). Components IIlA3 and IlIAr gave single symmetrical peaks while component IIIAz gave a skew peak, showing the presence of IIIAr material. When components IlIA7 and IlIA6, obtained from fraction IIIAP, were rechromatographed on cellulose phosphate, the NaCl concentration of the starting buffer was changed from 0.05 M to 0.08 M, giving the elution patterns shown in Figs. 3d and 3f, respectively. The separation was not much improved and these components cannot be expected to be fully resolved. Components IIIAS and IIIA4 were re-chromatographed on cellulose phosphate with a gradient of o.ro M to o.zo M NaCl, and Fig. 3h shows the pattern obtained for component IlIAS. The pattern obtained for component IIIA4 was similar to that obtained for component IIIAS, but with a larger effluent volume. TABLE I AMINO ACID COMPOSITIONS a (RESIDUES PER MOLE) OF ACID HYDROLYSATES OF COMPONENTS OBTAINED BY CHROMATOGRAPHY ON CELLULOSE PHOSPHATE ._ _ . --------- - - - - - - ---
---- ----------
~--
Amino acid
Component IIlAr
IlIA 2
~--.-------
Trpb Arg SCMCC Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Phe Estimated total
15. 0 33·3 2.0 14-4 II.S S.S IS.1 5·9 3. 0 9. 0 2.0
2.S 1.9 2.S 132
1 15.0 3 2 .9 2.0
15.0 12·5 9·1 20.1 6.0 3.0 9·1 2.1 2·9 2.0 2·9 13 6
IlIA 3
IlIA 4
-----
13·9 3 2 .4 2.0 14. 1 12.1 9·2 19.0 5·7 2·9 S.S 2.1 2·7 1.9 2·9 13 1
IlIAS ----
1 13. 1 3 2 .9 2.0 14·4 12.0 9. S 20·9 6.0 2·9 9. 0 2.0 3. 0 1.4 3. 0 133
1 13. 1 35·9 2.1 14· S 12·3 9·7 19. 0 6.0 2·9 8·9 2.1 2·9 1.5 2·9 135
IlIA 6
IlIA 7
---------
12·9 3 2 .5 2.0 14. 0 12·5 9· S 20.S 6.0 3. 0 9. 0 2.1 3. 0 1.3 3. 0 133
a Values obtained by extrapolation to zero-time hydrolysis. b Identified by Ehrlich reagent. c S-Car boxymethy1cysteine.
11.9 35. 2 2·3 14·7 13·2 10.0 19·5 6.0 3. 1 S·9 2.1 3. 2 1.3 3. 0 135
IlIA 8
----------
11.0 34·9 2.0 14. 0 14· S 10.1 19. 2 5· S 3. 1 S.6 2·3 3. 0 1.5 2.8 134
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L. S. SWART, D. PARRIS, F. J. JOUBERT
Amino acid compositions The amino acid compositions of the eight components of group IIIA are given in Table 1. These results show that the components have very similar amino acid compositions, the major difference amongst the components being that the arginine content varies from I I to IS residues per mole of protein.
DISCUSSION
The use of chromatography on cellulose phosphate has considerably widened the scope of fractionation of reduced and S-carboxymethylated high-sulphur proteins from a-keratins. This technique, using buffers of pH approximately z.S, separates proteins according to their base content. It therefore combines well with other t echniques, such as DEAE-cellulose chromatography or column electrophoresis, which effect separations mainly according to net charge. Group IIIA of the high-sulphur proteins of wool was previously obtained by a combination of column electrophoresis and gel filtration. On DEAE-cellulose chroma tography, this group yielded a broad peak showing several poorly resolved components 5 . In the present study, chromatography on DEAE-cellulose was used to prepare groups IIIA and IIIB directly from the high-sulphur proteins of wool. By using chromatography on cellulose phosphate, it was possible to isolate eight distinct components of group IIIA. The eight components of group IIIA gave stoichiometric values for most amino acids and have empirical molecular weights of approximately 16,000. Major features of the components are the absence of lysine, histidine and methionine residues. The arginine content of the components decreases stepwise from IIIAr to IIIAS, except for IIIAr and IIIAz and also for IIIA4, IIIA5 and IIIA6, for which constant values were obtained. The isolation of components with identical arginine contents shows that other factors, in addition t o base content, influence their resolution on cellulose phosphate. The integral values obtained for amino acids present in small amounts in the proteins, such as aspartic acid, glycine, alanine, isoleucine , leucine and phenylalanine, but not tyrosine, are evidence of the purity of the proteins. As the amino acid compositions of the components are so closely related, sequence analysis must be the final criterion of their purity. In our study, we have shown that large amounts of protein (3 g) can be rapidly and conveniently separated by chromatography on cellulose phosphate. Minimal shrinkage occurs when a 3.S X 50 cm column is eluted at a flow-rate of zoo mljh and the pressure remains low provided that the cellulose phosphate has been thoroughly de-fined. The choice of the gradient is very important. This fact is illustrated in the chromatograms shown in Figs. 3a and 3b, in which fraction IIIAP is better resolved by a change in the elution gradient, component IIIAS in particular separating as a distinct symmetrical peak. Although the system is sensitive to changes in gradient, excellent repeatability is achieved provided that the experimental conditions are kept constant. Unpublished results of work on mohair proteins have shown that chromatography on cellulose phosphate with sodium chloride gradients starting from zero molarity resolves group IIIB proteins. It has also been shown that proteins equivalent
CHROMATOGRAPHY OF HIGH-SULPHUR PROTEINS to fractions C14 to C18 (cJ., Fig. r) elute with group IIIB components on cellulose phosphate chromatography of total high-sulphur proteins. A rapid and convenient method for the preparation of large amounts of the components of groups IlIA and IIIB of the reduced and S-carboxymethylated highsulphur proteins of a-keratins can now be proposed. This method involves initial separation by chromatography on DEAE-cellulose, followed by chromatography on cellulose phosphate.
ACKNOWLEDGEMENT We thank Mrs. P. G. DRIJFHOUT for performing the amino acid analyses. REFERENCES I J. M. GILLESPIE, A ust. j. Bioi. Sci., 16 (1963) 259· 2 L. S. SWART, F. J. JOUBERT, T. HAYLETT AND P. J. DE JAGER, 3rd Int. Wool Text. Res. Conj., Paris, Institute Textile de France, Paris, I (1965) 493. 3 F. J. JOUBERT AND M. A. C. BURNS, j. S. Afr. Chem. Inst., 20 (1967) 161. 4 F. J. JOUBERT, P. J. DE JAGER AND L. S. SWART, in 'V. G. CREWTHER (Editor), Symposium on Fibrous Proteins, Australia, I967, Butterworths, Australia, 1968, p. 343· 5 L. S. SWART, T. HAYLETT AND F. J. JOUBERT, Text. Res. j., 39 (1969) 912. 6 R. L. DARSKUS, J. M. GILLESPIE AND H. LINDLEY, A ust. j. Bioi. Sci., 25 (1972) 139. 7 H. LINDLEY AND T. C. ELLEMAN, Biochem. j., 128 (1972) 859. 8 E. L. DARSKUS, j. Chromatogr., 69 (1972) 341. 9 T. HAYLETT AND L. S. SWART, Text. Res. j., 39 (1969) 917. 10 T. HAYLETT, L. S. SWART AND D. PARRIS, Biochem. j., 123 (1971) 191. II L. S. SWART AND T. HAYLETT, Biochem. J., 123 (1971) 201. 12 R. L. HILL AND 'V. R. SCHMIDT, j. Bioi. Chem., 237 (1962) 389.