Casein aspartic acid racemization

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. 

Table 11. Aspartic acid racemization and lysinoalanine (LAL) content of alkali-treated casein and acetylated casein.

The percentage of D-enanticmers relative to the total amount of the amino acid residue can be calculated by the relation (D/DfL) x 100. D-Aspartic acid accounts for 30% of that residue (which is thus 60% racemized) in treated casein, Pranine-D, and wheat gluten. In these three proteins, 22-30% of the phenylalanine (an essential amino acid) is the D-enanticmer, and in wheat gluten, 26% of glutamic acid has been converted to the D-form. 

The rates within each protein were then standardized relative to that of leucine. The order of relative racemization rates is presented in Table II. Relative rates are very similar among the various proteins except for aspartic acid and glutamic acid in wheat gluten. This situation is discussed below. (The relative rate constants estimated for the second region of the casein curves in Figure 2, using the 3-hour and 24-hour points, is k(asp) k(p>he) k(glu) k(ala) k(leu) = 4.0 3.0 2.5 2.5 1.0.)... 

Racemization has also been observed with many peptides and proteins. Casein exhibits racemization at aspartic acid, phenylalanine, glutamic acid, and alanine residues.799 Racemization of serine and histidine residues has been reported for histrelin (a nonapeptide)800 and a decapeptide,801 agonists of luteinizing hormone-releasing hormone (LH-RH). As shown in Fig. 200, the main degradation pathway of decapeptide (an antagonist of LH-RH) above PH 7 was epimerization.802... 

In addition to high pH, increases in the length of treatment and Increases in temperature are factors which promote increased racemization. Friedman, et al. (61) have shown that for casein, half of the amount of aspartic acid that will racemize within 24 hours (at 65 C in O.lN NaOH) will do so within the first hour. Others have shown similar results for proteins such as soy isolate and lactalbumin, with 60% to 80% racemization occurring with the first hour at 100 C (O.lN NaOH)( ). 

Over 500 different a-amino acids have now been synthesized or isolated. About 20 of them form the main components of proteins (see also Chapter 30). a-Amino acids are commerically obtained by fermentation of glucose (arg, asp, gin, glu, his, ile, lys, pro, val, thr) or glycine (ser), or enzymatic attack on aspartic acid (ala) or fumaric acid (asp), by hydrolysis, for example, of casein or sugar beet waste (arg, cys, his, hyp, leu, tyr), by transformation of ornithine (arg) or glutamic acid (gin), or, alternatively, by complete synthesis from aldehydes using the Strecker synthesis (ala, gly, leu, met, phe, thr, trp, val), from acrylonitrile (gly, lys), or from caprolactam (lys). The racemates are obtained by total synthesis, but L-amino acids are produced by all the other processes. The racemates are separated and the D-isomers produced are again racemized. 

Figure 14. The pH dependence of racemization for aspartic acid (0), phenylalanine (a) and glutamic acid ( ) in casein in the pH range 8 to 13. The expression for log shown in the ordinate is derived from Eq. 2. Under our experimental conditions, Eq. 2 can be reduced to this one term when solving for k (Masters and Friedman, 1979). The line is a first-order kinetic plot superimposed on the data points. Horizontal bars denote range of values from several determinations.

---