Total hydrolysis of proteins with strongly reduced racemization of amino acids

Abstract

A new method has been devised for the complete hydrolysis of proteins with an extremely low level of racemization of amino acids. Proteins are incubated in 10 M HCl at a low temperature to obtain partial hydrolysis. They are then incubated with pronase and finally with leucine aminopeptidase and peptidyl-d-amino-acid hydrolase from Loligo vulgaris. The proposed method ensures the total hydrolysis of either purified proteins contained in a crude homogenate of animal or vegetable tissue. In both cases, the racemization of amino acids (expressed as rate of d form / d + l form × 100) was lower than 0.015% for aspartic acid and lower than 0.01% for other amino acids. d-Amino acids released from peptides or proteins were estimated with enzymatic methods based on the use of octopus d-aspartate oxidase or hog kidney d-amino acid oxidase; with these enzymes, 0.05 nmol of a d-amino acid was determined in the presence of up to 20 μmol of a mixture of l-amino acids (ratio % d/d + l= 0.00025). The method allows the determination of d-amino acids either in tissues in which they are present in high concentrations (as human cataract lenses, tooth enamel, etc.) or in those with low enantiomer content (as brain, erythrocytes, etc.). Using the method described, we hydrolyzed several synthetic peptides consisting of d- and l-amino acids and determined the amount of d-amino acids. In addition, we totally hydrolyzed all the nuclear proteins of human cataractous lenses. The amount of d-aspartic acid was 0.026 μmol/mg in lenses of woman aged between 71 and 76 years and 0.0256 μmol/mg in lenses of men aged between 55 and 72 years. The d-aspartic acid measured corresponds to about 12% with respect to total aspartic acid.