Spectrometric determination of urokinase in urine after gel filtration, using the chromogenic substrate S-2444.

Abstract

Using the chromogenic substrate S-2444, the spectrometric determination of urokinase (EC 3.4.21.31) in urine subjected to gel filtration was evaluated. Pure urokinase solutions were used to standardize analytical conditions. A low molecular mass (relative molecular mass < 5000) heat resistant (60 min, 97 degrees C) activity could be removed from urine by gel filtration (Sephadex G 25 Medium). In the analysis of the high molecular mass fraction (relative molecular mass > 5000) of urine, amidolysis remained linear during a period of 3 hours. The relation between enzyme activity and substrate turnover was linear in the range from 0.05-12 U/l. The coefficients of variation for within-run precision ranged from 1.4-3.8%. The analytical recovery was 98-104%. Average urokinase excretion in morning urines (collection period 1-3 hours) of 10 healthy males and 10 healthy females was respectively 0.82 and 0.68 U/g creatinine.