COMPLETE PURIFICATION OF DOG RENAL RENIN

Canine renal renin was purified to homogeneity by using an 8-step procedure. Proteolytic enzyme inhibitors in all buffers utilized during the procedure improved the stability of renin. Separation of renin from other proteolytic enzymes by carboxymethylcellulose and pepstatin affinity chromatography was essential for optimal yield and stability of the product. A 600,000-fold purification was obtained with a 16% overall recovery. The specific activity of the pure enzyme was 4200 Goldblatt units/mg of protein. The criteria for homogeneity were the following: a symmetrical peak of renin activity eluted from the final gel filtration chromatography with uniform activity across the peak; a single stained band of protein by polyacrylamide disc gel electrophoresis, pH 8.9 and 7.8, assay of the slices from the pH 7.8 gel revealed that renin activity eluted as a symmetrical peak and corresponded to the stained band; sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis yielded a single protein band; isoelectric focusing yielded a single band. The molecular size of purified renin was 36,000 daltons by NaDodSO4 gel electrophoresis and 42,000 daltons by gel filtration chromatography; the isoelectric point was pH 5.7. The amino acid composition was similar to that reported for hog renal renin and mouse submaxillary gland renin. Pure canine renin was stable at 4 or -20.degree. C for 8 wk. The availability of purified renin permits specific antibody production for application in immunoassay and physiologic studies.