Lysinoalanine formation

Lysinoalanine formation is not restricted to alkaline conditions—it can also be formed by prolonged heat treatment. Any factor favoring lower pH and less drastic heat treatment will reduce the formation of lysinoalanine. Hurrell (1984) found that dried whole milk and UHT milk contained no lysinoalanine and that evaporated and sterilized milk contained 1,000 ppm. More severe treatment with alkali can decompose arginine into ornithine and urea. Ornithine can combine with dehydroalanine in a reaction similar to the one giving lysinoalanine and, in this case, omithinoalanine is formed. 

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).

Discriminating Between the Effects of Racemization and Lysinoalanine Formation, A mechanism of racemization and lysinoalanine formation is illustrated in Figure 1. 

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). 

Although acetylation appears to minimize or prevent lysinoalanine formation, our findings indicate that acylation does not significantly change the extent of racemization after 3 hr at 65°C (Table IV). These results show that it is possible in principle to discriminate between the alkali-induced effects of racemization and lysinoalanine formation. Additional studies in progress are designed to further delineate this principle. 

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... 

The postulated mechanism of lysinoalanine formation (Figure 5) is at least a two-step process. First, hydroxide ion-catalyzed elimination reactions of serine, threonine, and cystine (and to... 

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... 

Since SH groups react more readily with double bonds than do NH2 groups (43,44,49,54), addition of thiols should trap the residue of dehydroalanine as described below. These competitive reactions should minimize lysinoalanine formation. These expectations were realized. 

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