The Spectrophotometric Determination of Human Serum Carboxypolypeptidase with Angiotensin Converting Enzyme-Like Activity

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Abstract
A colorimetric method was developed for the direct chemical assay of human carboxypeptidase A [EC 3.4.12.2] with angiotensin converting enzyme-like activity in serum or plasma, using the substrate analogue glycyl-L-histidylglycine and the angiotensin converting enzyme substrate angiotensin I (A-I). This method was based on the spectrophotometric determination of histidylglycine and histidyl-leucine, products of the hydrolysis of glycyl-L-histidylglycine and A-I respectively. o-Phthalaldehyde reacted with the imidazole moiety of N-terminal histidyl peptides to produce a yellow chromophore. Inhibitors were tested for their effects on carboxypolypeptidase activity. The hydrolysis of Gly-His-Gly and A-I was inhibited by histidyl-leucine and angiotensin II, both products of the hydrolysis of A-I. Bothrops jararaca venom extract EDTA, p-chloromercuribenzoate, 8-hydroxyquinoline and 2,3-dimercaptopropanol, previously reported as converting enzyme inhibitors, also inhibited carboxypolypeptidase activity. Angiotensin converting enzyme activity in the serum of 66 adults ranged from 10-37 nmol of glycyl-L-histidylglycine hydrolysed in 10 min by 10 .mu.l of serum at 37.degree. C and pH 7.25.

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