Effect of sulfhydryl reagents on the enzymatic activity of human renin: implications for renin assay.

Sulfhydryl (SH) reagents are sometimes used in renin assays, yet their effects on the enzymatic activity of human renin are not clearly understood. We have employed radioimmunoassay of angiotensin I (AI) to assess the effects of dithiothreitol (DTT) and of dimerecaptopropanol (DMP) on the formation of AI at pH 5.5 and at pH 7.4. When ethylenediamine tetra-acetate, phenylmethylsulfonyl fluoride, and 8-hydroxyquinoline were used as angiotensinase inhibitors, both DTT and DMP decreased the rate of AI formation in each of two human plasma pools at both pH values. In contrast, in a system of semi-purified human kidney renin and hog renin substrate, DTT enhanced the formation of AI, increasing Vmax without changing Km at both pH values. The reaction of human kidney renin with synthetic tetradecapeptide renin substrate was stimulated by DTT at pH 7.4 but inhibited by DTT at pH 5.5. In all three systems, SH reagents altered the ratio of reaction velocity at pH 5.5 to reaction velocity at pH 7.4. We conclude that SH reagents affect the assay of human renin in complex ways which depend upon the pH of incubation and upon the subcomplex ways which depend upon the pH in incubation and upon the substrate utilized. Although the mechanism of these effects is not known, such effects probably contribute to the lack of agreement among many of the procedures for renin assay.