Evaluation of protein cross-linking and biodegradability by determination of tryptophan released by pronase using reversed-phase HPLC with photodiode array detection

A bioanalytical method for evaluating the extent of protein cross-linking was developed. The method is based on reversed-phase HPLC determination of tryptophan released from the cross-linked proteins by Pronase, a nonspecific proteolytic enzyme. The amounts of tryptophan released from un-cross-linked bovine serum albumin (BSA), a nanopeptide, and Trp-Phe in control experiments were consistent with the tryptophan content of the protein and peptides. However, the amount of tryptophan released decreased significantly when BSA was cross-linked extensively by heating at 120 o C in the presence of glucose