Racemization protein stability

Sequence inversion and racemization have been associated with uncatalyzed formation of the cyclic dipeptides and has been shown to greatly complicate the kinetics of formation. Cyclic dipeptide formation, by uncatalyzed processes, is rapid enough to pose an apparent threat to the stability of proteins and a possible rationale for the posttranslational N-acetylation of proteins that have been observed in higher organisms. The rate of DKP formation will also depend on the carbonyl ester protecting groups or the structures of the peptide-resin linkage in the solid-phase mode. Furthermore, cyclization is a concentration-independent reaction and demands the use of dilute solutions. ... 

Proteins, peptides, and other polymeric macromolecules display varying degrees of chemical and physical stability. The degree of stability of these macromolecules influence the way they are manufactured, distributed, and administered. Chemical stability refers to how readily the molecule can undergo chemical reactions that modify specific amino-acid residues, the building blocks of the proteins and peptides. Chemical instability mechanisms of proteins and peptides include hydrolysis, deamidation, racemization, beta-elimination, disulfide exchange, and oxidation. Physical stability refers to how readily the molecule loses its tertiary and/or sec-... 

The correlation between racemization rates in free amino acids and the o values also supports the carbanion-intermediate mechanism of racemization (17). The R-group can act to stabilize the negative charge on the a-carbon so that the carbanion intermediate is more stable. Since the a values also agree with the racemization rates observed in the present study, the same mechanism probably operates with protein-bound amino acids. It is noteworthy, however, that the racemization rate of free aspartic acid is 10-5 relative to those reported here for this amino acid residue in proteins (17-19). (For relevant discussions on the influence of R groups on reactivities of amino acids, peptides, and proteins, see references 21-26). 

Protein instability mechanisms have been reviewed by several investigators.3-13 Chemical reactions such as oxidation, deamidation, proteolysis, racemization, isomerization, disulfide exchange, photolysis, and others will give rise to chemical instability. It is critical that when this happens, the denaturation mechanisms must be identified in order to select appropriate stabilizing excipients. These chemical excipients may be in the form of amino acids, surfactants, polyhydric alcohols, antioxidants, phospholipids, chelating agents, and others. 

Base-catalyzed racemization reactions may occur in any of the amino acids except achiral glycine (Gly) to yield residues in proteins with mixtures of L- and D-configurations. The a-methine hydrogen is removed to form a carbanion intermediate (Fig. 6.26b). The degree of stabilization of this intermediate controls the rate of this reaction. Racemization generally alters the proteins physicochemical properties and biological activity. Also, racemization generates... 

While several laboratories have shown that severe racemiza-tion of proteins can occur during treatment with sodium hydroxide (6,18,22-24,61,62) the effects of other alkalis used in food processing are documented less well. Jenkins, et al. (70) have observed substantial differences in the degree of racemization caused by lime or caustic soda treatment of zein. Lime causes only 50% to 90% of the racemization observed for several amino acyl residues compared to when caustic soda is used. Because a substantial amount of calcium ion remained bound to the protein (approx. 10,000 ppm) compared to l/20th that amount of sodium ion for the caustic soda-treated zein, it is possible that divalent calcium may stabilize the protein making it less susceptible to racemization. Tovar (14) observed increases of 40% to 50% in serine and phenylalanine racemization and a decrease of 30% aspartate racemization for caustic soda-treated fish protein concentrate compared to lime-treated protein (see Table II). These studies indicate that different alkalis have different effects on racemization of proteins specifically, lime may cause less racemization than caustic soda at a similar pH. 

Various side chains affect the extent of racemization in different ways. Thus, the benzyl side chain in phenylalanine contributes to the stabilization of a carbanion and can thereby facilitate proton abstraction from the a-carbon atom. This effect is much more pronounced in phenylglycine (which is not a protein constituent but occurs in microbial peptides) because its chiral carbon atom is benzylic ... 

The a-proton of the azlactone is quite base labile (stabilized carbanion) so that a proton may be lost, but can later add to either side of the azlactone plane with subsequent loss of optical purity. Further, the azlactone may participate in peptide bond forming reactions, so that it disappears during the synthesis, but racemic amino acids are introduced into the product protein. 

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