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Kinins—II pp 115-125 | Cite as

A Simple Radioassay for Human Urinary Kallikrein

  • Alfred Chung
  • James W. Ryan
  • Guillermo Pena
  • Narendra B. Oza
Part of the Advances in Experimental Medicine and Biology book series (AEMB)

Abstract

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.

Keywords

Angiotensin Converting Enzyme Plasma Kallikrein Urinary Enzyme Urinary Kallikrein Toluene Phase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

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Copyright information

© Springer Science+Business Media New York 1979

Authors and Affiliations

  • Alfred Chung
    • 1
  • James W. Ryan
    • 1
  • Guillermo Pena
    • 1
  • Narendra B. Oza
    • 1
  1. 1.Department of MedicineUniversity of Miami School of MedicineMiamiUSA

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