Partial purification of dog angiotensinogen.

Abstract

Dog angiotensinogen was purified 450-fold from the plasma of nephrectomized dogs by a simple four-step procedure involving precipitation between 1.5 and 2.3 M ammonium sulfate, gel filtration on Sephadex G-150, ion-exchange chromatography on DE-52 cellulose, and affinity chromatography on Concanavalin A-Sepharose. The purity of the final preparation was over 50%. The preparation of dog angiotensinogen had an apparent molecular weight of 80,000 determined by gel filtration on Sephadex G-100. Kinetic studies indicated that the Km of the reaction of dog renin with partially purified dog angiotensinogen (1,840 pmol/ml) was similar to that for the reaction with angiotensinogen in diluted dog plasma (1,820 pmol/ml). Thus the purification procedures employed did not alter the affinity of dog renin for the Leu10-Leu11 bond of dog angiotensinogen. Because the concentration of angiotensinogen in dog plasma is about 700 pmol/ml, a first order reaction with respect to substrate is indicated in vivo.