Protein bound amino acids, racemization

R Liardon, R lost. Racemization of free and protein-bound amino acids in strong mineral acid. Int J Pept Prot Res 18, 500, 1981. 

It may also be surprising how easily this racemization may occur. Friedman and Liardon (126) studied the racemization kinetics for various amino acid residues in alkali-treated soybean proteins. They report that the racemization of serine, when exposed to 0.1M NaOH at 75°C, is nearly complete after just 60 minutes. However, caution must be used when examining apparent racemization rates for protein-bound amino acids. Liardon et al. (127) have also reported that the classic acid hydrolysis, employed to liberate constituent amino acids, causes amino acids to racemize to various degrees. This will necessarily result in D-isomer determinations that are biased high. Widely applicable correction factors are not possible since the racemization behavior of free amino acids is different from that of amino acid residues in proteins (which can be further affected by sequence). Of course, this is not a problem for free amino acid isomer determinations since the acid hydrolysis is unnecessary. Liardon et al. also describe an isotopic labeling/mass spectrometric method for determining true racemization rates unbiased by the acid hydrolysis. For an extensive and excellent review of the nutritional implications of the racemization of amino acids in foods, the reader is directed to a review article written by Man and Bada (128). 

Racemization of Protein-Bound Amino Acids. D/L enantio-meric ratios for seven amino acid residues are given in Table I. Extensive racemization of aspartic acid, phenylala-nine, glutamic acid, and alanine occurred when the four proteins were treated with hydroxide. Valine, leucine, and proline were much less racemized. 

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). 

Amino acid racemization analyses on shell are carried out in two ways sampling all the amino acids extracted from the shell (called total) and sampling only the protein-bound amino acids (referred to as protein). The first method is essentially identical to the processing... 

Species Effects. Another problem with racemization analyses of mollusc shell is that different mollusc species taken from the same deposit can vary considerably in the extent of racemization (20, 21). The variation in alleu/iso ratios again may be attributed to different types of stmctural proteins and to differing rates of diagenetic hydrolysis of these proteins, resulting in variable proportions of free amino acids, small peptides, and protein-bound amino acids (20). 

The calculated activation energy for aspartic acid racemization (20.8) is similar to the value reported by Darge and Thiemann (1971) for racemization of protein-bound aspartic acid. However, this value is about 10 kcal/mole less than the corresponding value reported by Bada (1971) for free aspartic acid determined at pH 7.6. In contrast, activation energies for alanine and phenylalanine residues in casein, determined at pH 12.5 were similar to those found for the same amino acids at pH 7.6. 

LAL is, in fact, 15 to 25 times more nephrotoxic than protein-bound LAL (De Groot et al., 1976). Thus, any decrease in the digestibility of the protein due to alkali treatment could then influence the degree of availability of LAL. The release of biologically active LAL from a treated protein is determined by the proteins accessibility to proteolytic enzymes. As demonstrated by Masters and Friedman (1980), alkaline treatment induces amino acid racemization, with each protein and even each protein fraction reacting differently to the same treatment. Since racemization decreases the digestibility of protein, these differences could explain discrepancies among results reported by different authors. 

Pyridoxal 5 -phosphate dependent enzymes constitute an important class of proteins involved predominately in amino acid metabolism. The PLP-cofactor is capable of catalyzing a variety of reactions at the a-, [3-, and/or y-carbons of amino acid substrates. These reactions include tranamination, racemization, decarboxylation, and aldoyltic cleavage reactions at the a-carbon and elimina-tion/substitution reactions at either the 3-, or y-position of the amino acid substrate (67-74) The chemical properties of the cofactor (67-71) are responsible for the great diversity of reactions catalyzed by PLP, while reaction specificity is ultimately determined by the active site environment imposed by the surrounding apo-protein to which the cofactor is covalendy bound (69). 

Such treatments, however, can induce chemical changes in the secondary as well as primary structure of the protein. These includes racemization of amino acids and crosslinking that leads to the formation of new amino acids (Masters and Friedman, 1980 Friedman et al., 1981). Lysinoalanine (LAL) is one of these amino acids (Friedman, 1982). These chemical changes are of nutritional concern because alkali-treated protein containing protein-bound LAL can produce renal lesions when fed to rats (Woodard et al., 1975). These cytomegalic alterations are characterized by an enlargment of cells of the pars recta epithelium, with disturbances in mitosis and DNA synthesis. 

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