Disulfide bonds in wool were reduced with tri-n-butylphosphine in 50% n-propanol at pH 7.6. Native and generated sulfhydryl groups were then alkylated in the same medium with 2-vinylpyridine. Cystine and the original of cysteine residues were thus changed quantitatively to residues of S-β-(2-pyridylethyl)-L-cysteine (2-PEC). Occurrence of this new amino acid in hydrolyzates of the modified wool was confirmed by independent synthesis. 2-PEC is stable to acid hydrolysis and is eluted from the basic physiological column of an amino acid analyzer as a discrete peak before arginine. Quantitative modification of sulfhydryl groups in reduced keratins to 2-PEC permits quantitative determination of cysteine or of cystine via cysteine by two independent techniques-ion-exchange chromatography and ultraviolet spectroscopy. The new procedure is suitable for routine analyses.
Cavins, J.F. and Friedman, M., Automatic Integration and Computation of Amino Acid Analyses, Cereal Chem.45, 172-176 (1968).
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Cavins, J.F. and Friedman, M., An Internal Standard for Amino Acid Analyses: S-β-(4-Pyridylethyl)-L-Cysteine, Analyt. Biochem., 35, 489-493 (1970).
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Cavins, J.F. and Friedman, M., Specific Modification of Protein Sulfhydryl Groups with α,β-Unsaturated Compounds, J. Biol. Chem.243, 3357-3360 (1968).
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Crewther, W.G. , Dowling, L.M., Inglis, A.S., and Maclaren , J.A., The Formation of Various Cross-linkages in Wool and Their Effect on the Supercontraction Properties of the Fibers, Textile Res. J.37, 736-745 (1967).
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Crewther, W.G. , Fraser, R.D.B., Lennox, F.G., and Lindley, H., The Chemistry of Keratins, Adv. Protein Chem.20, 191-346 (1965).
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Fletcher, J.C. and Robson, A., A Method for the Determination of Cystine Plus Cysteine in Proteins, Biochem. J.84, 439-444 (1962).
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Fletcher, J.C. , Robson, A., and Todd, S., The Sulfur Balance of Wool, Biochem. J.87, 560-567 (1963).
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Friedman, M. , Krull, L.H., and Cavins, J.R., The Chromatographic Determination of Cystine and Cysteine Residues in Proteins as S-β-(4-Pyridylethyl) Cysteine, J. Biol. Chem., 245, 3868-3871 (1970 ).
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Friedman, M., Haddon, W.F., and Rohwedder, W.K., Presented at 159th ACS National Meeting, Houston, Texas , Feb. 22-27, 1970.
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Friedman, M. and Sigel, C.W., A Kinetic Study of the Ninhydrin Reaction , Biochemistry5, 478-485 (1966).
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Friedman, M. and Krull, L.H., A Novel Spectrophotometric Procedure for Half-Cystine Residues in Proteins, Biochem. Biophys. Res. Commun.37, 630-633 (1969).
12.
Friedman, M. , Cavins, J.F., and Wall, J.S.Relative Nucleophilic Reactivities of Amino Groups and Mercaptide Ions in Addition Reactions with α,β-Unsaturated Compounds, J. Amer. Chem. Soc.87, 3672-3682 (1965).
13.
Kohler, G.O. and Palter, R., Studies on Methods for Amino Acid Analysis of Wheat Products, Cereal Chem.44, 512-520 (1967).
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Leach, S.J. , Meschers, A., and Springell, P.H., Micro andSubmicro Methods for the Estimation of Thiol and Disulfide Groups in Insoluble Proteins Using Radioactive Mercurials, Anal. Biochem.15, 18-30 (1966).
15.
Leach, S.J., in "A Laboratory Manual of Analytical Methods of Protein Chemistry ," Alexander, P. and Lundgren, H. P., Eds., Oxford, Pergamon Press, 1966, Vol. 4, Chap. 1.
16.
Ward, W.H., in "A Laboratory Manual of Analytical Methods of Protein Chemistry ," Alexander, P. and Lundgren, H. P., Eds., Oxford, Pergamon Press, 1966, Vol. 4, Chap. 5.
17.
Lewis, B., Robson A., and Tiler, E.M., An Investigation of the Sulfur Compounds in Acid Hydrolyzates of Wool, J. Textile Inst.51, T653-T664 ( 1960).
18.
Maclaren, J.A. , Kilpatrick, D.J., and Kirkpatrick , A., Reduced Wool Fibers, Their Preparation and Alkylation, Australian J. Biol. Sci.21, 805-813 (1968).
19.
Maclaren, J.A. and Sweetman, B.J., The Preparation of Reduced and S-Alkylated Wool Keratins Using Tri-n-butylphosphine, Australian J. Chem.19, 2355-2360 (1966).
20.
Maclaren, J.A. , Leach, S.J., and Swan, J.M., A Study of Some Problems in Protein Chemistry Using New (Nonhydrolytic) Methods for the Determination of Thiol and Disulfide , J. Textile Inst.51, T665-T677 ( 1960).
21.
Menkart, J. , Wolfram, L.J., and Mao, I., Caucasian Hair , Negro Hair, and Wool: Similarities and Differences, J. Soc. Cosmetic Chemists17, 769-787 (1966).
22.
Moore, S., On the Determination of Cystine as Cysteic Acid, J. Biol. Chem.238, 235-237 (1963).
23.
Inglis, A.S. and Lennox, F.G., Studies in Wool Yellowing. Part IV. Changes in Amino Acid Composition Due to Irradiation, Textile Res. J.33, 431-435 (1963).
24.
Spackman, D.H. , Stein, W.H., and Moore, S., Automatic Recording Apparatus for Use in the Chromatography of Amino Acids, Anal. Chem., 30, 1190-1206 (1958).
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Spackman, D.H. , Accelerated System for the Automatic Analysis of Amino Acids, Federation Proc.22, 244 (1963).
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Sweetman, B.J. and Maclaren, J.A., The Reduction of Wool Keratins by Tertiary Phosphines, Australian J. Chem.19, 2347-2354 (1966).
27.
White, L.M. and Secor, G.E., Micro-Kjeldahl Determination of Nitrogen in Gramicidin and Tryptophan. Comparison of Gunning-Arnold-Dyer and Friedrich Methods, Ind. Eng. Chem. Anal. Ed., 18, 457 (1946).
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Wibaux, G., Mazingue, G., and Van Overbeke, M. , Le Bilan De Soufre De La Laine, Bull. Inst. Textile France86, 41-52 (1960).
29.
Association of Official Agricultural Chemists , "Official Methods of Analysis," 10th Ed., Sec. 38.012-39.014, pp. 744-745, Washington, D. C., 1965.
