Hydrolysis of proteins performed at high temperatures and for short times with reduced racemization, in order to determine the enantiomers of d- and l-amino acids

Abstract Racemization of free amino acids is considerably lower those bound in peptide. In the same experimental conditions, the rate of racemization of free amino acids is only 20%–80% that of peptide-bound amino acids. When using traditional protein hydrolysis, the racemization was 1.2–1.6 times as high as that obtained at high temperatures (160–180 °C), under conditions ensuring total hydrolysis of the protein. This lower degree of racemization may be explained by the fact that, at high temperatures, the protein hydrolyses more rapidly into free amino acids and the racemization of free amino acids is considerably lower than those bound in polypeptides. When hydrolysis is conducted at lower temperatures for longer times, the amino acids bound in the peptide chain are exposed for a longer time to the effects racemization. As a result, we may say that any factor that speeds up hydrolysis will lower the degree of racemization. Racemization was higher for proteins in milk powder than for pure proteins. This may be explained as a result of catalysis of the racemization by the heavy metals present. After 48 h at 110 °C and in the presence of 4 M barium hydroxide, all amino acids (whether free or bound in peptide) were totally racemized. Therefore, the racemization of tryptophan cannot be determined using barium hydroxide promoted protein hydrolysis. High temperature hydrolysis (at 160 °C for 45–60 min, at 170 °C for 30–45 min and 180 °C for 30 min) is recommended for those who would like to hydrolyse the protein for short times and determine the degree of racemization occurring in the polypeptide chain, but do not wish to use enzyme hydrolysis.