Extraction, purification, and acetylation of human renin and the production of antirenin to human renin

Abstract

Bioassay procedures employed for the quantitative determination of human renin; antirenin to human renin; angiotensin; and angiotensinase are described. All of these assays are based on the determination of the increase of the direct, mean systemic blood pressure of trained, unanesthetized dogs after intravenous injection of the test substances. Four preparations of commercially available angiotensin II (Hypertensin Ciba) were assayed and found to contain 2000–2200 dog units of angiotensin per milligram. The extraction and purification of renin were carried out on batches up to 35 kg of human kidneys. This resulted in an over-all yield of 60% and a 600-fold purification of the renin, including the complete removal of angiotensinase. The immunization of rats, rabbits, and a dog with human renin resulted in the production of antirenin to human renin with maximum titers of 7, 12, and 11 units per milliliter of serum, respectively. The acetylation of human renin induced a decrease of its enzymic (pressor) activity and an alteration of its antigenic structure, but not an impairment of its antigenicity. As a result, the immunization of a dog with acetylated human renin produced antirenin of a high titer, with cross-reactivity toward both untreated and acetylated human renin. In its chemical and immunological characteristics, human renin differs from that of other species: (a) Human renin was not acetylated under conditions found to be effective for renin of five other species; it required different conditions, such as a higher temperature and a more alkaline reaction. (b) Antirenin which had been produced with untreated human renin, or with acetylated human renin, failed to react with the renin of hog, dog, rabbit, beef, sheep, and rat. The acetylation of the renin preparation obtained after Step 8 of the new isolation procedure lowered the pressor activity to 36% and the amount of amino groups to approximately 24% of the original value.