International Journal of Biochemistry, Biophysics & Molecular Biology

Submit a Manuscript

Publishing with us to make your research visible to the widest possible audience.

Propose a Special Issue

Building a community of authors and readers to discuss the latest research and develop new ideas.

Independent Determination of Cystine in Keratin Proteins

The role of cystine residues in a protein is well recognized, providing the disulfide bonds for the structural integrity of a wide range of proteins. Hence, the determination of cystine in proteins is critical in understanding the structural functionality of proteins. The amino acid analysis (AAA) is a popular method to determine amino acid residue compositions in proteins. In practice, oxidation of or chemical modification to cystine is often performed prior to AAA. However, these pretreatments are indiscriminate towards cystine and cysteine. Hence, it is difficult to distinguish cystine from cysteine in protein AA composition analyses, especially for cystine-rich proteins such as keratin. In this report, we demonstrate that it is possible to determine cystine residues in protein selectively independent from cysteine, using the conventional AAA, without pretreatments. Our experimental results have shown that cystine did not transform into cysteic acid during acid hydrolysis, as has been reported previously. Our results also showed a part of L-cystine transformed to D-cystine. Finally, we applied the same AAA to determine the cystine residue levels in feather and human hair samples successfully and compared those with the results obtained from AAA using the pretreatment by oxidation.

L-/D-cystine, D-cysteine, L-cysteine, Keratin, Amino Acid Analysis

APA Style

John M. Schulze, Ken Tasaki. (2022). Independent Determination of Cystine in Keratin Proteins. International Journal of Biochemistry, Biophysics & Molecular Biology, 7(2), 47-54.

ACS Style

John M. Schulze; Ken Tasaki. Independent Determination of Cystine in Keratin Proteins. Int. J. Biochem. Biophys. Mol. Biol. 2022, 7(2), 47-54. doi: 10.11648/j.ijbbmb.20220702.11

AMA Style

John M. Schulze, Ken Tasaki. Independent Determination of Cystine in Keratin Proteins. Int J Biochem Biophys Mol Biol. 2022;7(2):47-54. doi: 10.11648/j.ijbbmb.20220702.11

Copyright © 2022 Authors retain the copyright of this article.
This article is an open access article distributed under the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Mossuto, M. F. (2013). Disulfide bonding in neurodegenerative misfolding diseases, J. Cell Biology Int. Article ID 318319.
2. Welker, E., Wedemeyer, W. J., & Scheraga, H. A. (2001). A role for intermolecular disulfide bonds in prion diseases? Proc. Natl. Acad. Sci. 98, 4334-4336.
3. Karimi, M., Ignasiak, M. T., Chan, B., Croft, A. K., Radom, L., Schiesser, C. H., Pattison, D. I., & Davies, M. J. (2016). Reactivity of disulfide bonds is markedly Affected by Structure and Environment: Implications for Protein Modification and Stability, Sci. Rep. 6. 38572. 10.1038/srep38572.
4. Davidson, I. (2003). Hydrolysis of Samples for Amino Acid Analysis, In Smith, B. J. (eds) Protein Sequencing Protocols. Methods in Molecular Biology™, vol 211. Humana Press. 119–129.
5. Fountoulakis, M., & Hans-Werner Lahm, H.-W. (1998). Hydrolysis and Amino Acid Composition Analysis of Proteins, J. Chromatography A, 826, 109-134.
6. Wang, Y., Kang, X. J., Ge, W. H., Sun, X. Z., & Peng, J. (2007). Simple, Rapid, and Accurate RPHPLC Method for Determination of Cystine in Human Urine after Derivatization with Dansyl Chloride, Chromatographia 65, 527-532.
7. Inglis, A. S., Liu, T. Y., (1970). The Stability of Cysteine and Cystine during Acid Hydrolysis of Proteins and Peptides, J. Biological Chem. 245, 112-116.
8. Gehrke, C. W.,& Rexroad, P. R. (1987) Quantitative Analysis of Cystine, Methionine, Lysine, and Nine Other Amino Acids by a Single Oxidation-4 Hour Hydrolysis Method, J. Assoc. Off Anal. Chem. 70, 171-174.
9. Tuan, Y. N., & Phillips, R. D., (1997). Optimized Determination of Cystine/Cysteine and Acid-Stable Amino Acids from a Single Hydrolysate of Casein- and Sorghum-Based Diet and Digesta Samples, Agric. Food Chem. 45, 3535–3540.
10. Aitken, A., M., & Learmonth, M. (2002). (Carboxymethylation of Cysteine Using Iodoacetamide/ Iodoacetic Acid. In: Walker, J. M. (eds) The Protein Protocols Handbook. Springer Protocols Handbooks. Humana Press. pp 455-456.
11. Thannhauser, T. W., Sherwood, R. W., & Scheraga, H. A. (1998) Proteins by Amino Acid Analysis: Application to the Characterization of Disulfide-Coupled Folding Intermediates, J. Protein Chem. 17, 37-43.
12. Lee, K. S., & Drescher, D. G. (1979). Derivatization of Cysteine and Cystine for Fluorescence Amino Acid Analysis with the o-Phthaldialdehyde/2-Mercaptoethanol Reagent, J. Biol. Chem. 254 (14), 6248-6251.
13. Ohmori, S., Ikeda, M., Hattori, H., Hagiwara, K., & Iwase, C. A. (1983). Simple and Specific Colorimetric Determination of Cysteine with p-Dimethylaminocinnamaldehyde, J Clin. Chem. Clin. Biochem. 21, 851-857.
14. Barkholt, V., &Jensen, A. L. (1989). Amino Acid Analysis: Determination of Cysteine plus Half-Cystine in Proteins after Hydrochloric acid Hydrolysis with a Disulfide Compound as Additive, Anal. Biochem. 177, 318-22. doi: 10.1016/0003-2697(89)90059-6.
15. Sarwar, G., Botting, H. G., & Peace, R. W. (1988). Complete Amino Acid Analysis in Hydrolysates of Foods and Feces by Liquid Chromatography of Precolumn Phenylisothiocyanate Derivatives, J. Assoc. Off Anal. Chem. 71, 1172-1175.
16. Akinyele, A. F., Okogun, J. I., & Faboya, O. P. (1999). Use of 7-Chloro-4-Nitrobenzo-2-Oxa-1, 3-Diazole for Determining Cysteine and Cystine Incereal and Legume Seeds, J. Agric. Food Chem. 47 (6), 2303-2307. doi: 10.1021/jf9805707.
17. Mokrejs, P., Krejci, O., & Svoboda, P. (2011). Producing Keratin Hydrolysates from Sheep Wool, Oriental J. Chem. 27, 1303-1309.
18. Rouse, J. G., & Van Dyke, M. E. (2010). Review of Keratin-Based Biomaterials for Biomedical Applications, Materials 3, 999-1014.
19. Tanabe, T., Okitsu, N., Tachibana, A., & Yamauchi, K. (2002). Preparation and Characterization of Keratin–Chitosan Composite Film, Biomaterials 23, 817-825.
20. Vasconcelos, A., & Cavaco-Paulo, A. (2013). The Use of Keratin in Biomedical Applications, Current Drug Targets 14, 612-619.
21. Schram, E., & Moore, S. (1954). E. J. Bigwood, Chromatographic Determination of Cystine as Cysteic Acid, Biochem. 57, 34-37.
22. Lollar, R. M. (1955). Theoretical Considerations in the Chemical Modification of Chicken Feather Keratin, in: S. J. Kennedy, A. Schubert, L. I. Weiner (Eds), Utilization of Chicken Feathers as Filling Materials, A conference sponsored by the headquarters quartermaster research and development command, U.S. Army Quartermaster Corps, Natick, Mass. The National Academies Press, Washington, D. C., pp 75-101.
23. O'Donnell, J. (1973). The Complete Amino Acid Sequence of a Feather Keratin from EMU (Droma/US Novae-Holland/AE). Aust. J. BioL. Sci. 26, 415-437.
24. Gregg, K., & Rogers, G. (1986). Feather Keratin: Composition, Structure and Biogenesis, in: J. Bereiter-Hahn, A. G. Matoltsy, K. S. Richards (Eds), Biology of the Integument, Springer-Verlag, Berlin, Heidelberg, pp 666-690.
25. Murphy, M. E., King, J. R., & Taruscio, T. G. (1990). Amino Acid Composition of Feather Barbs and Rachises in Three Species of Pygoscelid Penguins: Nutritional Implication, The Condor 92, 913-921.
26. Zoccola, M., Aluigi, A., Patrucco, A., Vineis, C., Forlini, F., Locatelli, P., Sacchi, M. C., & Tonin, C. (2012). Microwave-Assisted Chemical-Free Hydrolysis of Wool Keratin, Textile Research Journal 82, 2006–2018.
27. Arai, K. M., Takahashi, R., Yokote, Y., Akahane, K. (1983). Amino-Acid Sequence of Feather Keratin from Fowl, Eur. J. Biochem. 132, 501-507.
28. Fujii, T., Takayama, S., Ito, Y. (2013). A Novel Purification Procedure for Keratin-Associated Proteins and Keratin from Human Hair, J. Biol. Macromol. 13 (3), 92-106.
29. Bhavsar, P., Zoccola, M., Patrucco, A., Montarsolo, A., Rovero, G., Tonin, C. (2017). Comparative Study on the Effects of Superheated Water and High Temperature Alkaline Hydrolysis on Wool Keratin. Textile Research Journal 87, 1696–1705.
30. Worth, G., Kelly, R., Krsinic, G., Scott, S., & Roddick-Lanzilotta, A. R. (2015). Topically Applied Keratins for Hair and Skin Care, The Science of Beauty 5, 53-59.
31. Tonin, C., Aluigi, A., Varesano, A., Vineis, C. (2010). Keratin-Based Nanofibres, in: A. Kumar (Ed), Nanofibers, InTech, Rijeka, pp 139-158.
32. Kelly, R. (2016). Keratins in Wound Healing, in: S. Å. Magnus (ed), Wound Healing Biomaterials, Elsevier, Volume 2, pp 353-365.
33. Friedman, M., Krull, L. H., Cavins, A. F. (1970). The Chromatographic Determination of Cystine and Cysteine Residues in Oroteins as S-b-(4-pyridylethyl)cysteine, J. Bio. Chem. 245, 3868-3871.
34. Jacobson, S. J., C. G. Willson, H. Rapoport, Mechanism of Cystine Racemization in Strong Acid, J. Org. Chem. 39 (1974) 1075-1077.
35. Kaiser, K., & Benner, R. (2005). Hydrolysis-Induced Racemization of Amino Acids, Limnol. Oceanogr.: Methods 3, 318–325.
36. Smith, G. G., & De Sol, B. S. (1980). Racemization of Amino Acids in Dipeptides Shows COOH > NH2 for Non-Sterically Hindered Residues, Science 207, 765-767.
37. Darragh, A. J., Garrick, D. J., Moughan, P. J., Wouter, H., & Hendriks, W. H. (1996). Correction for Amino Acid Loss During Acid Hydrolysis of a Purified Protein. Analytical Biochem. 236, 199–207.
38. Robbins, C. R., & Kelly, C. H. (1970). Amino Acid Composition of Human Hair, Textile Research Journal 40, 891-896.
39. Simmonds, D. H. (1958). Automatic Equipment for Determination of Amino Acids Separation on Columns of Ion Exchange Resins, Anal. Chem. 30, 1043-1049.
40. Blum, J. J. (1996). Oxidation of Alanine, Acetate, Glutamate, and Succinate by Digitonin-Permeabilized Leishmania Major Promastigotes, J. Eukaryot. Microbiol. Mar-Apr. 43 (2), 144-150. doi: 10.1111/j.1550-7408.1996.tb04495.x.
41. Recky, J. R. N., Serrano, M. P., Dántola, M. L., & Lorente, C. (2021). Oxidation of Tyrosine: Antioxidant Mechanism of l-DOPA Disclosed, Free Radical Biology and Medicine 165, 360-367.
42. Giulivi, C., Traaseth, N. J., & Davies, K. J. A. (2003). Tyrosine Oxidation Products: Analysis and Biological Relevance, Amino Acids 25 (3-4), 227-32. doi: 10.1007/s00726-003-0013-0.
43. Gregory, B. R., Wilder, O. H. M., & Ostby, P. C. (1956). Studies on the Amino Acid and Vitamin Composition of Feather Meal. Poultry Sci. 35, 234-235.
44. Moran Jr, E. T., Bayley, H. S., & Summers, J. D. (1967). Keratins as Sources of Protein for the Growing Chick 3. The Metabolizable Energy and Amino Acid Composition of Raw and Processed Hog Hair Meal with Emphasis on Cystine Destruction with Autoclaving, Poultry Sci 46, 548-553.
45. Bergès, J., Trouillas, P., & Houée-Levin, C. (2011). Oxidation of Protein Tyrosine or Methionine Residues: From the Amino Acid to the Peptide, J. Phys.: Conf. Ser. 261, 012003. doi: 10.1088/1742-6596/261/1/012003.