The preparation of rabbit renin-substrate for the assay of minute amounts of renin.
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ABSTRACT:
SUBMITTER: Cook WF
PROVIDER: S-EPMC1207055 | biostudies-other | 1965 Aug
REPOSITORIES: biostudies-other
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Project description:1. Rabbit plasma enzymes that degrade angiotensin I are inhibited completely by the combination of 2,3-dimercaptopropan-1-ol (10mm), EDTA (10mm) and chlorhexidine gluconate (0.005%, w/v). These compounds do not modify the reaction of renin with renin substrate and are termed the selective inhibitors. 2. The renin substrate concentration of plasma can be measured as angiotensin I content by incubating plasma plus the selective inhibitors with renin for a time sufficient to allow complete utilization of renin substrate. 3. This reaction obeys first-order kinetics to substrate concentrations of at least 1000ng. of angiotensin I content/ml. In general, the renin substrate concentrations of normal rabbit plasmas are less than 1000ng. of angiotensin I content/ml. Thus the time required for the complete release of angiotensin I from normal plasma is inversely related to renin activity and is independent of renin substrate concentration. 4. A method for the assay of renin substrate, taking these reaction kinetics into account, is presented.
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Project description:1. EDTA (10mm), 2,3-dimercaptopropan-1-ol (10mm) and chlorhexidine gluconate (0.005%, w/v) cause complete inactivation of plasma enzymes that degrade angiotensin I, but have no effect on the reaction of renin with its substrate. The reagents were termed the selective inhibitors. 2. Thus it is possible to measure renin in plasma by its ability to catalyse the release of angiotensin I. 3. Sterile plasma, treated with the selective inhibitors, is incubated with renin substrate (500-1000ng. of angiotensin content/ml.) at pH6 at 42 degrees for 6hr. 4. Under these conditions the reaction obeys first-order kinetics. Renin activity is calculated in terms of the percentage release of the angiotensin content/hr. 5. As described, the assay is sufficiently sensitive to measure renin in the plasma of all normal rabbits. By extending the length of the incubation, much lower activities can be measured.
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