Improved method for hydrolyzing proteins and peptides without inducing racemization and for determining their true D-amino acid content

A new method of hydrolyzing proteins and peptides without racemizing the amino acids has been developed. This method consists of performing a brief partial chemical hydrolysis for 15 min in 6 M HCl at 80-90°C, followed by an enzymatic hydrolysis with pronase for 12-16 h at 50°C, and finally an enzymatic hydrolysis with leucine aminopeptidase and peptidyl-D-amino acid hydrolase for 24 h. Using this new method the time required for complete hydrolysis of proteins is less than 3 days. The total hydrolysis averages 97-100%, and the amount of racemization of the amino acids is less than 0.002%. This method may then be used as a tool to easily determine the intrinsic D-amino acid content of peptides or proteins from animal or vegetable tissues.