A Simple Radioassay for Human Urinary Kallikrein
We have developed a sensitive, highly selective assay for human urinary kallikrein (HUK) that uses Pro-Phe-Arg-[3H]benzylamide as substrate. The substrate was prepared from Pro-Phe-Arg-3-iodo-benzylamide by dehalogenation in 3H2 gas. HUK is measured by its ability to release [3H]benzylamine. The pH optimum is 9.5. Urokinase, plasmin and thrombin do not interfere. The assay can measure as little as 5 ng of HUK in a 15 min incubation at 37° C. Typically, we use 50 μl of dialyzed urine for HUK assays. Reactions are terminated by adding 0.1 M NaOH, and reaction product is separated from substrate by partitioning with an equal volume of toluene. A sample of the toluene phase is submitted for liquid scintillation counting. As judged by separations obtained on molecular sieve chromatography (Sephacryl), only one urinary enzyme possesses the ability to hydrolyze our substrate. The enzyme MW 45,000, is inhibited by Trasylol but not by soya bean trypsin inhibitor (SBTI). It is reactive with and is inhibited by antibodies prepared against pure HUK.
KeywordsAngiotensin Converting Enzyme Plasma Kallikrein Urinary Enzyme Urinary Kallikrein Toluene Phase
Unable to display preview. Download preview PDF.
- Amundsen, E., L. Svendsen, A.M. Vennerød and K. Laake, Determination of plasma kallikrein with a new chromogenic tripeptide derivative, 1976, in: Chemistry and Biology of the Kallikrein-Kinin System in Health and Disease, eds. J.J. Pisano and K.F. Austen (U.S. Government Printing Office, Washington, D.C. ) p. 215.Google Scholar
- Beaven, V.H., J.V. Pierce and J.J. Pisano, 1971, A sensitive isotopic procedure for the assay of esterase activity: Measurement of human urinary kallikrein, Clin. Chico. Acta, 32, 67.Google Scholar
- Claeson, G., L. Aurell, P. Friberger, S. Gustaysson and G. Karlsson, Designing of peptide substrates. Different approaches exemplified by new chromogenic substrates for kallikreins and urokinase, Haemostasis, 7, 62.Google Scholar
- Ryan, J.W., A. Chung, L.C. Martin and U.. Ryan, 1978, New substrates for the radioassay of angiotensin converting enzyme of endothelial cells in culture, Tissue & Cell, 10, 555.Google Scholar
- Ryan, J.W., N.B. Oza, L.C. Martin and G.A. Pena (this volume), Components of the kallikrein-kinin system in urine.Google Scholar