Two assay procedures have been developed for measuring the ratio of the two molecular weight forms of urokinase commonly found in either urine or the fluid of cultured human kidney cells. The first method is a mini gel filtration technique which rapidly separates the high molecular weight (HMW) and low molecular weight (LMW) urokinases. This procedure can be used for urokinase samples of any purity. The biological active fractions are assayed by the fibrin plate technique. These biological activities are converted to relative molar amounts by the use of the molar activity constant for each molecular weight form. The second procedure is based on the differing responses of the two molecular species in the fibrin plate and the Ploug clot lysis assay systems. The comparison of assay responses of an unknown with a predetermined curve obtained from known mixtures of HMW and LMW urokinases results in a measure of the relative amounts of the two molecular forms. This system is not applicable to samples of low purity.The two systems gave comparable results. For example, the WHO First International Reference Preparation (66/46) of Urokinase is 31% HMW urokinase by the differential method and 32% HMW urokinase by the gel filtration technique. Both methods require small amounts of sample (400 IU or less) and should replace the use of sodium dodecylsulfate polyacrylamide gel electrophoresis for urokinase molecular species determinations.
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