Peptide Esters and Nitroanilides as Substrates for the Assay of Human Urinary Kallikrein

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Abstract
Ac-Phe-ArgOMe is hydrolyzed much faster than are Bz-ArgOEt, Z-ArgOMe or Ac-Gly-ArgOMe by the kallikrein from human urine. The synthesis of Ac-Phe-ArgOEt is described. Hydrolysis of this substrate can be conveniently monitored by a coupled spectrophotometric procedure. Increase in absorbance [A] was linear with time and proportional to the amount of kallikrein up to a .DELTA.A366 of a least 0.22/10 min. This assay for human urinary kallikrien was 46-fold more sensitive than that based on Bz-ArgOEt and 38-fold more sensitive than that with D-Val-Leu-Arg-p-nitroanilide. A number of Arg p-nitroanilides are hydrolyzed by this enzyme at still lower rates. The assay of human urinary kallikrein with D-Val-Leu-ArgOEt was about a factor of 2 less sensitive than the assay with Ac-Phe-ArgOET. This also held for Z-TyrONp, which displayed rapid spontaneous hydrolysis. The rate of the enzymic reaction with Z-TyrONp dropped off rapidly.