A sensitive radiometric assay for enkephalin convertase and other carboxypeptidase B-like enzymes

A sensitive radiometric assay for carboxypeptidase B-like enzymes has been developed using enkephalin convertase, an enkephalin synthesizing carboxypeptidase. The assay is based on the differential solubility of 3H-labeled substrate and product in chloroform. The substrates 3H-benzoyl-Phe-Ala-Arg or 3H-benzoyl-Phe-Leu-Arg are poorly soluble in chloroform due to the charged arginine. The products of carboxypeptidase B-like activity on these substrates, 3H-benzoyl-Phe-Ala or 3H-benzoyl Phe-Leu partition quantitatively into chloroform, allowing rapid separation of product from substrate. This assay is approximately 100 times more sensitive than a similar fluorometric assay utilizing dansyl-Phe-Ala-Arg as a substrate.