Protein lysinoalanine formation

Nashef et al. (41) also reported that the rate of 6 elimination from cystine was directly dependent on hydroxide ion concentration although the relationship was not linear perhaps because of the complexity of the reaction (Equation 7). Sternberg and Kim (20.) found the rate of lysinoalanine formation in casein to be dependent on hydroxide ion concentration. Touloupais and Vassiliadis (45) also found the rate of lysinoalanine formation in wool to be pH dependent. These workers did not measure the rate of 6 elimination, therefore the rate determining step is not known. These results on proteins appear to be in contradiction to those of Samuel and Silver (46) who reported that hydroxide ion concentration had no effect on the rate of 6 elimination from free phosphoserine between pH 7 and 13.5. Because of the effect... 

Figure 1. Postulated mechanism of racemization and lysinoalanine formation via a common carbanion intermediate. Note that two B-elimination pathways are possible (a) a concerted, one-step process (A) forming the dehydroprotein directly and (b) a two-step process (B) via a carbanion intermediate. The carbanion, which has lost the original asymmetry, can recombine with a proton to regenerate the original amino acid residue which is now racemic. Proton transfer may take place from the environment of the carbanion or from adjacent NH groups, as illustrated. Protein anions and carbanions can also participate in nucleophilic addition and displacement reactions (24, 82, 83).

Since lysinoalanine and at least one D-amino acid are toxic to some animals (35), we wished to distinguish their effects in alkali-treated proteins. Such discrimination is possible, in principle, since we have found that acylating the e-amino group of lysine proteins seems to prevent lysinoalanine formation. Since lysinoalanine formation from lysine requires participation of the e-amino group of lysine side chains, acylation of the amino group with acetic anhydride is expected to prevent lysinoalanine formation under alkaline conditions if the protective effect survives the treatment. This is indeed the case (16). 

Karayiannis, N. (1976). Lysinoalanine Formation in Alkali Treated Proteins and their Biological Effects, Ph.D. Thesis. University of California, Berkeley. 

Sternberg, M. and Kim, C. Y. (1977). Lysinoalanine formation in protein food ingredients. In "Protein Crosslinking Nutritional and Medical Consequences",... 

Alkali-Induced Lysinoalanine Formation in Structurally Different Proteins... 

In previous papers, we have (a) reviewed elimination reactions of disulfide bonds in amino acids, peptides, and proteins under the influence of alkali (5) (b) analyzed factors that may operate during alkali-induced amino acid crosslinking and its prevention (6) (c) demonstrated inhibitory effects of certain amino acids and inorganic anions on lysinoalanine formation during alkali treatment of casein, soy protein, wheat gluten, and wool and on lanthionine formation in wool ( 7, 9) (d) demonstrated that... 

These results, therefore, imply that the extent of lysinoalanine formation may vary from protein to protein. Factors that favor or minimize these reactions need to be studied seprately with each proteins. 

Lysinoalanine formation in casein, lactalbumin, and wheat gluten was measured at 65°C at various pH s for 3 hours. Factors that control the extent of formation of the unnatural amino acid lysinoalanine during food processing and thus the degree of crosslinking in structurally different proteins are discussed. 

Freidman, M. Lysinoalanine formation. Soy Proteins Kinetics and Mechanism, in Food Protein Deterioration Mechanisms and Functionality ACS Symposium Series 1982, 206, 231—273. 

Karayianis, N.I. J.T. MacGregor L.P. Bjelfanes. Lysinoalanine formation in alkali-treated proteins and model peptides. Food Cosmetics Tox. 1979,17, 585—590. 

Savoie, L. G. Parent. Susceptibility of protein fractions to lysinoalanine formation. /. Food Sci. 1983, 48, 1876-1877. 

Tovar (14) performed an experiment designed to evaluate the in vivo effect of D-amino acids in alkali-treated protein without the presence of lysinoalanine. In addition, either lime or caustic soda were used to investigate whether these alkalis had different effects vivo Zein was exposed to O.IN alkali for 4 hours at 85 C. Because lysine is absent from zein, no lysinoalanine formation was observed. Diets were prepared using untreated or alkali-treated zein and were supplemented with casein and free amino acids to meet the nutritional requirements of the... 

Friedman, M. (1982). Lysinoalanine formation in soybean proteins kinetics and mechanisms. In "Mechanism of Food Protein Deterioration", J.P. Cherry Ed., American Chemical Society Symposium Series, Washington, D.C., 206, 231-272. 

In this paper we describe some of the factors that influence lysinoalanine formation and racemization of amino acid residues in food proteins. We also discuss toxicological and nutritional consequences of feeding alkali-treated food proteins, lysinoalanine, and D-amino acids. 

These results and those mentioned above on the pH-dependence of lysinoalanine formation Indicate that for each protein, conditions may exist In which lysinoalanine Is formed as fast as It Is destroyed. Additional studies are needed to verify this hypothesis. 

Mechanistic considerations (Asquith and Otterburn, 1977 Whitaker and Feeney, 1977 Friedman, 1977) suggest that added thiol or sulfite ions can inhibit lysinoalanine formation by at least three distinct mechanisms. The first is by direct competition. The added nucleophile (mercaptide, sulfite, bisulfite, thiocyanate, thiourea, etc.) can trap dehydroalanine residues derived from protein amino acid side chains, forming their respective adducts. In particular, lanthionine side chains (Figure 8)are formed from added cysteine and N-acetyl-cysteine. The second possible mechanism is... 

Tables 6 and 1 compare the effects of several organic and inorganic compounds on the lysinoalanine and lysine contents of alkali-treated wheat gluten and soybean protein. These results show that all these compounds partly inhibit lysinoalanine formation. The extent of inhibition may vary from protein to protein and should be related to both the content and reducibility of the disulfide bonds (Friedman, 1978a Finley et al., 1978 a,b Masri and Friedman, 1982).

Figure 9. Inhibition of lysinoalanine formation by indirect competition. The SH group of a reduced protein combines with the double bond of a dehydro-protein, thus preventing it from reacting with the amino group of lysine to form lysinoalanine.

Figure 13. Preliminary results on the effect of temperature of treatment on D-serine and lysinoalanine formation in soybean protein. Conditions of treatment 1% (w/v) protein in 0.1 N NaOH 3 hours. Note rapid initial rate of production of D-serine which then levels off at about 75 C, where the D/L ratio approaches the maximum theoretical value of 1.0. We thank Dr. D. L. Schwass for his help with the D-serine measurements.

Karaylannis, N. I., MacGregor, J. T. and Bjeldanes, L. F. (1979a). Lysinoalanine formation In alkali-treated proteins and model compounds. Food Cosmet. Toxicol., 17, 585-590. 

As reported by Friedman (1982), the major factor controlling the production of lysinoalanine, once the dehydroalanine precursors are formed, could be the location and availability of "partners for crosslink formation. When they are adjacent or close by, lysinoalanine formation could be facilitated. When treatment allows it, more lysinoalanine could be formed, even involving cross-chain links. Thus, the capacity for forming LAL would vary not only with the treatment applied but with the nature of the protein, involving its secondary or tertiary structure as well as its primary one. 

Friedman, M. (1982) Lysinoalanine formation in soybean protein kinetics and mechanism. In Food Protein Deterioration, Mechanism and Functionality. J.P. Cherry, ed. American Chemical Soc., Wash. p. 231. 

Figure 13.5 Formation of lysinoalanine nucleophilic additions of the e-amino group of the protein-bound lysine to the double bond of DHA residue (a) causes crosslinking of the polypeptide chain (b) lysinoalanine (c) is formed after hydrolysis.

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