Chromogenic substrate recovery from enzyme assay mixts. - by denaturing enzyme and extracting with solvents

摘要

Recovery of unreacted chromogenic substrate (I) from an aq. reaction mixt. used for quantitative enzyme determn., where the mixt. contains (I), reaction prods. of (I) and a proteolytic enzyme, is effected by (a) inhibiting the reaction between the enzyme and (I), (b) separating the resulting ppted. enzyme, (c) extracting the aq. reaction mixt. first with diisopropyl ether (DIPE) and then with ethyl acetate (EtOAc), and (d) recovering (I) from the EtOAc extract. The process may be applied to p-nitroaniline-peptide conjugates of the type used for determn. of thrombin or trypsin. Since a large excess of such conjugates is generally used, their recovery provides considerable cost savings.