Quantitative activation and determination of inactive renin by high performance liquid chromatography

Assay of inactive renin in unfractionated mouse plasma is difficult and often impossible because of high concentrations of active renin and plasma protease inhibitors. Therefore, 0.025 to 0.05 ml of plasma or amniotic fluid from mice was separated by high performance liquid chromatography (HPLC) using a silica-based size exclusion column. The eluates were examined for enzymatically active renin before and after limited proteolysis with trypsin. Since inactive renin eluted as a single peak corresponding to a molecular weight of 38,000 daltons and the elution of active renin was markedly retarded, inactive and active renin were partially separated. compared with plasma, inactive renin in amniotic fluid eluted as a broader and sometimes diphasic peak, suggesting heterogenicity. The rapid and reliable separation by HPLC provided a more than 300-fold purification of inactive renin. Despite low concentrations of plasma protease inhibitors, a 1 000 000-fold molar excess of trypsin (1 mg/ml) was needed for optimal activation. The necessity for high trypsin concentrations for activation may partly be explained by enzyme kinetic considerations. By combining HPLC with trypsin activation, inactive renin was readily measured and found to be 9.4 (5.1-13.2) GU (Goldblatt units)/l in normal and 8.0 (5.1-12.2) GU/l in sialoadenectomized and nephrectomized mouse plasma, which is higher than previously determined in our laboratory. The concentration was 12.4 (8.8-16.1) GU/l in amniotic fluid. Thus, the concentration of inactive renin in plasma is almost as high as active renin in normal mice (17.6 GU/l) and is uninfluenced by the decrease of active renin to 0.3 GU/l after sialoadenectomy and nephrectomy.