From the desk of Dr. Fred Drewe: Important Information Concerning Renin

Published: Apr 27th, 2006

Renin that is supplied by Lee Biosolutions, Inc is purified from human kidneys and is fully activated. Its molecular weight is 40 KDa but you can find on some SDS-PAGE electrophoresis systems two additional bands at 18 and 22 kDa. Both of the smaller peptides will react with antibodies against Renin and could be artifacts created in the electrophoresis system as well as a fully functional cleaved Renin protein. The protein concentration is determined by the Lowry method.

Renin activity is determined in the clinical chemistry laboratory by two common methods. The first method, which we do not use, is an immunoassay that measures the Renin directly by a mass method. The older and more common method is the PRA (Plasma Renin Activity) assay which measures the enzymatic activity of the Renin on its natural substrate, Angiotensinogen, and its conversion to Angiotensin I.

The substrate used is what is normally found in a plasma/serum sample that has been pHed to the range between 5.0 to 7.0, of which we use the pH of 6.0. The plasma derived reagent is treated with various protease inhibitors and other inhibitors to eliminate other proteolytic activity and also the conversion of Angiotensin I to Angiotensin II. The reaction is run at 37 C for 1 hour with different amounts of Renin added and then the Angiotensin I produced is measured by an EIA assay. The PRA activity unit can be expressed as nanograms of Angiotensin I produced per mL per hour. Or as we do, the PRA unit is defined as the amount of Renin that produces 0.2 milligrams of Angiotensin I per mL per hour. That is one PRA unit per mL will produce approximately 500,000 nanograms of Angiotensin I per hour at 37 C.

The other commonly used assay, particularly in research laboratories, is based on the cleavage of a synthetic peptide that results in a fluorescence signal. The chemical called Renin substrate 1, which is synthesized around the normal substrate, can be purchased from Molecular Probes, via Invitrogen, as Catalog #R-2931 or Sigma as R8276 or Bachem as M-2050. This reaction is run at a higher pH than the PRA assay at 8.0. A common formulation of the assay reagent is 50 mM Tris-HCl, pH 8.0 containing 100 mM sodium chloride with the substrate concentration being 2 micromolar. The assay is run at temperatures of 25, 30, and 37 C dependent on the fluorometer that is used. Our assay is run at 37 C. The sensitivity of the assay is dependent on the sensitivity of the fluorometer used and can as low as 30 ng per mL.

Since the PRA assay is derived from the amount of substrate contained in plasma/serum material as the reagent, its unit is somewhat variable from lab to lab. In general, Our estimation is that 1 PRA unit is equivalent to 1 unit in this assay, where one unit is defined as the amount of enzyme that will cleave one nanomole of Renin Substrate 1 per minute, at the same temperature.

Normally it is not recommended to freeze the Renin at all and definitely freeze/thaw cycles should be avoided.

Fred Drewe, PhD
January, 2005