Determination of Plasminogen by Means of a Chroniogenic Peptide Substrate

Plasmin splits the chromogenic substrate B2-Phe-Val-Arg-pNA (S-2160, Bofors) at a relatively high rate. Standard plasmin in glycerol obtained from Nat. Inst, for Biol. Stand, and Contr., London, was tested in a system with Tris buffer of varying pH and ionic strength. The pH optimum for the reaction was found to be 7.4. Variations in ionic strength between 0.05–0.1 had insignificant influence but at higher ionic strength there was a slight inhibition. A linear relationship was found between plasmin and AOD/min. At optimum pH and a final substrate concentration of 0.2 mM 0.1 CTA unit corresponds to approximately 0.10 nkat. Purified plasminogen (AB Kabi, Stockholm, Sweden) in the concentrations 0.02–0.2 CU/ml was activated optimally with streptokinase (Kabikinase® ) in the concentrations 500–2000 IU/ml. Higher concentration gave inhibition. The activity of streptokinase activated plasminogen increased with a decreasing ionic strength. A linear relationship was found between streptokinase activated plasminogen and AOD/min. Approximately 3,000 Plong/units per ml of urokinase was needed to obtain the same activation as with optimal streptokinase concentration. The method has been used for determination of plasminogen in plasma. With final dilution of plasma in the range 1/20–1/200 activated by streptokinase (2000 IU/ml) in a system of pH 8.2, I = 0.05, a linear relationship was found between plasma dilution and AOD/min. The reproducibility in a series of tests is good (variation coefficient < 3%) and with insignificant interference by inhibitors. The determinations were easily carried out in a simple spectrometer (405 nm) and in an automatic reaction rate analyzer (LKB 8600, 410 nm).