Ribonuclease inhibitor from pig brain: Purification, characterization, and direct spectrophotometric assay

The ribonuclease inhibitor from pig brain has been purified 1500-fold by a combination of ammonium sulfate fractionation, ion-exchange chromatography, hydroxylapatite chromatography, and gel filtration. The inhibitor has a M r 50,000. It is a noncompetitive inhibitor for pancreatic ribonuclease A with a K i of 1 n m, forming a 1:1 complex. Both ribonuclease A and B, but not ribonuclease U 1 and T 1, are inactivated by the inhibitor. The inhibition capacity was abolished by sulfhydryl reagents such as p-chloromercuribenzoate. Incubation of the enzyme-inhibitor complex with the sulfhydryl reagent caused dissociation into active ribonuclease and inactive inhibitor. Dithiothreitol was required during purification to retain the activity of the inhibitor.