Behavior of O-Glycosyl and O-Phosphoryl Proteins in Alkaline Solution

  • John R. Whitaker
  • Robert E. Feeney
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 86)


0-Glycosyl and 0-phosphoryl groups, as well as disulfide bonds, are rapidly removed from proteins in alkaline solution primarily via β-elimination. The reaction is initiated by abstraction of the α-hydrogen of an amino acid residue by hydroxide ion. The carbanion undergoes rearrangement expelling the glyco- or phosphoryl group resulting in formation of a dehydroalanyl (from serine) or β-methyldehy-droalanyl (from threonine) residue. The unsaturated derivatives are reactive with internal protein nucleophilic groups and with external nucleophiles. These addition reactions, some leading to crosslinking, result in changed properties of the protein. Several factors may affect rates of β-elimination and addition. For these studies, we used two unique proteins: phosvitin, a well-characterized protein with 120 0-phosphoryl groups and no cystine or 0-glycosyl groups; and a glycopeptide related to the antifreeze protein from Antarctic fish, a well-characterized protein consisting of (Ala-Ala-Thr)n in which all of the threonyl residues are glycosylated. At the dilute concentrations of proteins used, rates of β-elimination and addition were independent of protein concentration but directly dependent upon the hydroxide ion concentration. With phosvitin, rates of β-elimination and addition were quite dependent on ionic strength and the rate of β-elimination was increased 20-fold in the presence of Ca2+. Activation energies for both β-elimination and addition were near 20 kcal/mole. Implications of these reactions for protein chemistry and protein processing will be discussed.


Alkaline Solution Threonine Residue Antarctic Fish Amino Acid Determination Factor Xiii Deficiency 
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  1. Ahmed, A. I., Osuga, D. T. and Feeney, R. E. (1973). Antifreeze glycoprotein from an Antarctic fish. J. Biol. Chem., 248, 8524.Google Scholar
  2. Asquith, R. S., Booth, A. K. and Skinner, J. D7–796)-7— The formation of basic amino acids on treatment of proteins with alkali. Biochim. Biophys. Acta, 181, 164.Google Scholar
  3. Asquith, R. S. and Carthew, P771972). An investigation of the mechanism of alkaline degradation of cystine in intact protein. Biochim. Biophys. Acta, 278, 8.Google Scholar
  4. Asquith, R. S. and Garcia-Dominguez, J. J. (1968). New amino acids in alkali-treated wool. J. Soc. Dyers Colour., 84, 155.Google Scholar
  5. Asquith, R. S. and Skinner, J. D. (1970). Modification of keratin and related proteins by alkali. Textilveredlung, 5, 406.Google Scholar
  6. Belitz, H. -D. (1965). Egg yolk proteins and their fractionated products. IV. Amino acid sequences in phosvitin. Lebensm.-Untersuch. Forsch., 127, 341.Google Scholar
  7. Bjarnason, J. and Carpenter, K. J. (1970). Mechanisms of heat damage in proteins. 2. Chemical changes in pure proteins. Brit. J. Nutr., 24, 313.Google Scholar
  8. Blackburn, S. 1968 ). “Amino Acid Determination, Method and Techniques.” Marcel Dekker, New York, N.Y.Google Scholar
  9. Bohak, Z. (1964). Nc-(DL-2-Amino-2-carboxyethyl)-L-lysine, a new amino acid formed on alkaline treatment of proteins. J. Biol. Chem., 239, 2878.Google Scholar
  10. Carter, C. E. and Greenstein, J. P. (1946a). A spectrophotometric method for the determination of dehydropeptidase activity. J. Biol. Chem., 165, 725.Google Scholar
  11. Carter, C. E. and Greenstein, J. P. (1946b). Spectrophotometric determination of dehydropeptidase activity in normal and neo-plastic tissues. J. Nat. Cancer Inst., 7, 51.Google Scholar
  12. Corfield, M. C., Wood, C., Robson, A., Williams, M. J. and Woodhouse, J. M. (1967). Formation of lysinoalanine during the treatment of wool with alkali. Biochem. J., 103, 15c.Google Scholar
  13. Dakin, H. D. (1912). The racemization of proteins and their derivatives resulting from tautomeric change. J. Biol. Chem., 13, 357.Google Scholar
  14. DeGroot, A. P. and Slump, P. (1969). Effects of severe alkali treatment of proteins on amino acid composition and nutritive value. J. Nutr., 98, 45.Google Scholar
  15. DeGroot, A. P., Slump, P., Van Beek, L., and Feron, V. J. (1976). Severe alkali treatment of proteins. In “Proteins for Humans:Google Scholar
  16. Evaluation and Factors Affecting Nutritional Value.“ C. E. Bodwell, ed., Avi, Westport, Conn.Google Scholar
  17. Derevitskaya, V. A., Vafina, M. G. and Kochetkov, N. K. (1967). Synthesis and properties of some serine glycosides. Carbohyd. Res., 3, 377.Google Scholar
  18. Downs, F. and Pigman, W. (1976). Determination of 0-glycosidic link- ages to L-serine and L-threonine residues of glycoproteins. Meth. Carbohydr. Chem., 7, 200.Google Scholar
  19. Feeney, R. E. (1974). A biological antifreeze. Am. Scientist, 62, 712.Google Scholar
  20. Feeney, R. E. (1977). Chemical modification of food proteins. In “Improvement of Food Proteins Through Chemical and Enzymatic Modifications,” R. E. Feeney and J. R. Whitaker, eds., Advances in Chemistry Series, Am. Chem. Soc., Washington, D. C.CrossRefGoogle Scholar
  21. Gallop, P. M., Blumenfeld, 0. 0. and Seifter, S. (1972). Structure and metabolism of connective tissue proteins. Ann. Rev. Biochem., 41, 617.Google Scholar
  22. Geschwind, I. I. and Li, C. H. (1964). Effects of alkali-heat treatment on B-melanocyte-stimulating hormone. Arch. Biochem. Biophys., 106, 200.Google Scholar
  23. Gottschalk, A. (1972). “Glycoproteins,” Elsevier Publ. Co., New York, N.Y.Google Scholar
  24. Gross, E. (1977). The chemistry and biology of amino acids in food proteins: lysinoalanine. In “Improvement of Food Proteins Through Chemical and Enzymatic Modifications,” R. E. Feeney and J. R. Whitaker, eds., Advances in Chemistry Series, Am. Chem. Soc., Washington, D. C.Google Scholar
  25. Horn, M. J., Jones, D. B. and Ringel, S. J. (1941). Isolation of a new sulfur-containing amino acid (lanthionine) from sodium carbonated-treated wool. J. Biol. Chem., 133, 141.Google Scholar
  26. Isbell, H. S. (1944). Interpretation of some reactions in the carbohydrate field in terms of consecutive electron displacement. J. Res. Nat. Bur. Stand., Sect. A, 32, 45.Google Scholar
  27. Isbell, H. S., Linek, K. and Hepner, K. E. Jr. (1971). Transformations of sugars in alkaline solutions: II. Primary rates of enolization. Carbohydr. Res., 19, 319.Google Scholar
  28. Lee, H. S., Osuga, D. T., Nashef, A. S., Ahmed, A. I., Whitaker, J. R. and Feeney, R. E. Effect of alkali on glycoproteins: 13-Elimination and nucleophilic addition reactions of substituted threonyl residues. Submitted for publication (1976).Google Scholar
  29. Levene, P. A. and Bass, L. W. (1928). Studies on racemization. VII. The action of alkali on casein. J. Biol. Chem., 78, 145.Google Scholar
  30. Mayo, J. W. and Carlson, D. M. (1970). Effect of alkali and sodium borohydride at alkaline pH on N-acetylchondrosine: reduction vs cleavage. Carbohyd. Res., 15, 300.Google Scholar
  31. Mecham, D. K. and Olcott, H. S. (1949). Phosvitin, the principal phosphoprotein of egg yolk. J. Am. Chem. Soc., 71, 3670.Google Scholar
  32. Mellet, P. (1968). Influence of alkali treatment on native and denatured proteins. Text. Res. J., 38, 977.Google Scholar
  33. Miro, P. and Garcia-Dominguez, J. J. Ç1973T. Action of ammonium and sodium hydroxides on keratin fibers in relation to their morphological structure. J. Soc. Dyers Colour., 89, 137.Google Scholar
  34. Nashef, A. S., Osuga, D., Lee, H. S., Ahmed, A. I., Whitaker, J. R. and Feeney, R. E. Effects of alkali on proteins. Disulfides and their products. Submitted for publication (1976).Google Scholar
  35. Ozeki, T. and Yosizawa, Z. (1971). Glycopeptides isolated from bovine submaxillary mucin. Arch. Biochem. Biophys., 142, 177.Google Scholar
  36. Parisot, A. and Derminot, J. (197)7 Formation of amino acids in wool treated with 0.1 N sodium hydroxide at various temperatures. Bull. Inst. Text. Fr., 24, 603.Google Scholar
  37. Patchornik, A. and Sokolovsky, M. (1964). Nonenzymatic cleavages of peptide chains at the cysteine and serine residues through their conversion into dehydroalanine. I. Hydrolytic and oxidative cleavage of dehydroalanine residues. J. Am. Chem. Soc., 86, 1206.Google Scholar
  38. Pickering, B. T. and Li, C. H. (1964). Adrenocorticotropins XXIX. The action of sodium hydroxide on adrenocorticotropin. Arch. Biochem. Biophys., 104, 119.Google Scholar
  39. Pigman, W. and Moschera, J. (1973). Uses and misuses of bases in studies of glycoproteins. In “Carbohydrates in Solution,” Advan. Chem. Ser., 117, 220.CrossRefGoogle Scholar
  40. Pisano, J. J., Finlayson, J. S., Peyton, M. P. and Nagai, Y. (1971). s-(y-Glutamyl)lysine in fibrin: Lack of crosslink formation in factor XIII deficiency. Proc. Nat. Acad. Sci. USA, 68, 770.Google Scholar
  41. Plantner, J. J. and Carlson, D. M. (1975). Studies of mucin-type glycoproteins. Olefinic amino acids, products of the ß-elimination reaction. Anal. Biochem., 68, 153.Google Scholar
  42. Price, V. E. and Greenstein, J. P. (1947). Dehydropeptidase activity in certain animal and plant tissues. J. Biol. Chem., 171, 477.Google Scholar
  43. Provansal, M. M. P., Cuq, J. -L. A. and Cheftel, J. -C. (1975). Chemical and nutritional modifications of sunflower proteins due to alkaline processing. Formation of amino acid cross-links and isomerization of lysine residues. J. Agric. Food Chem., 23, 938.Google Scholar
  44. Robson, A. and Zaidi, Z. H. (1967). The formation of lysinoalanine during the treatment of silk fibroin with alkali. J. Text. Inst. Trans., 58, 267.Google Scholar
  45. Sen, L. C., Gonzalez Flores, E., Whitaker, J. R. and Feeney, R. E. Reactions of phosphoproteins in alkaline solutions. Submitted for publication (1976).Google Scholar
  46. Simpson, D. L., Hranisavljevic, J. and Davidson, E. A. (1972). β-Elimination and sulfite addition as a means of localization and identification of substituted seryl and threonyl residues in proteins and proteoglycans. Biochemistry, 11, 1849.Google Scholar
  47. Taborsky, G. (1974). Phosphoproteins. Adv. Protein Chem., 28, 1.Google Scholar
  48. Tanaka, K., Bertolini, M. and Pigman, W.-7964). Serine and threonine glycosidic linkages in bovine submaxillary mucin. Biochem. Biophys. Res. Comm., 16, 404.Google Scholar
  49. Tannenbaum, S. R., Ahern, M. and Bates, R. P. (1970). Solubilization of fish protein concentrate. Food Technol. ( Chicago ), 24, 604.Google Scholar
  50. Tanzen, M. L. (1973). Cross-linking of collagen. Science, 180, 561.CrossRefGoogle Scholar
  51. Wakabayashi, K. and Pigman, W. (1974). Synthesis of some glycodipeptides containing hydroxyamino acids, and their stabilities to acids and bases. Carbohyd. Res., 35, 3.Google Scholar
  52. Whiting, A. H. (1971). Isolation of lysinoalanine from the protein-polysaccharide complex of cartilage after alkali treatment. Biochim. Biophys. Acta, 243, 332.Google Scholar
  53. Ziegler, K. (1964). New cross-links in alkali-treated wool. J. Biol. Chem., 239, 2713.Google Scholar
  54. Ziegler, K., Melchert, I. and Lürken, C. (1967). N -(2-amino-2carboxyethyl)-ornithine, a new amino-acid fromsalkali-treated proteins. Nature (London), 214, 404.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1977

Authors and Affiliations

  • John R. Whitaker
    • 1
  • Robert E. Feeney
    • 1
  1. 1.Department of Food Science and TechnologyUniversity of CaliforniaDavisUSA

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