Purification and characterization of prorenin from porcine kidney

Prorenin was purified extensively from porcine kidney by a procedure involving extraction at pH 6.5 in the presence of protease inhibitors, ammonium sulfate fractionation, affinity chromatography on pepstatin-aminohexyl-Sepharose, acetone fractionation, chromatography on DEAE-cellulose, gel filtration on Ultrogel AcA 44, chromatography on concanavalin A-Sepharose, and chromatography on CM-Sepharose. The resulting preparation was essentially homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis. The molecular weight of the preparation was estimated to be 39,000 by SDS-gel electrophoresis and 44,000 by gel filtration on Ultrogel AcA 44, and the isoelectric point was at pH 5.1. The purified prorenin was stable for 60 min at 37 degrees C between pH 3.0 and 9.0 and on storage for 4 weeks at--80 degrees C. The prorenin did not show any renin activity which appeared after trypsinization. The molecular weight of trypsin-activated prorenin was estimated to be 40,000 by gel filtration on Ultrogel AcA 44. The specific renin activity was 2.06 mg angiotensin I/mg of prorenin per h, and the optimal pH for the renin activity was in the pH region from 6.0 to 6.5 with hog and rat plasma angiotensinogens as the substrates. Two distinct isoelectric points were identified at pH 4.9 and pH 5.0 with the trypsin-activated prorenin.