Assay method for urokinase activity by capillary-tube isotachophoresis using a synthetic substrate

Abstract

Urokinae activity was determined by capillary-tube isotachophoresis using N- a-acetyl- l-lysine methyl ester as a synthetic substrate. The resulting N- a-acetyl- l-lysine was separated and detected isotachophoretically using methanolic potassium hydroxide solution adjusted to pH 5.32 by adding a-ketoglutaric acid as a leading electrolyte and methanolic betaine hydrochloride solution as a terminating electrolyte. The enzymatic reaction was stopped by addition of tannic acid and the resulting supernatant solution was injected into an isotachophoretic analyser. Linearity was observed at urokinase activities in the range 1–30 I.U. The urokinase activities in commercial products determined by the method were in good agreement with those determined by Walton's modified plate method. The coefficient of variation of the method was less than 3.4%.