The chromogenic peptide substrate H-D-Val-L-Leu-L-Lys-pNA (S-2251) has been chosen from a series of potent plasmin substrates. It is a highly specific and sensitive substrate towards plasmin (Km 2.5·10-4 mol/1, V 0.5·10-6 mol/min CTA-U) and its solubility in tris buffer (1·10-2 mol/1)is sufficient to obtain substrate saturation. Tris buffer pH 7.4 and I 0.15 serves as an optimal medium for the determination of plasmin. In this buffer, antiplasmins in plasma have been determined by using 0.05 CU of plasmin and 20 ul of plasma. At least two inhibitors have been found. One fast (<15 sec. incubation time) and the other(s)slow (5 min. incubation time). The substrate has also been used for plasminogen determinations after activation of 10 ul of plasma with UK (500 Ploug-U) or an excess (40 times) of SK. It was found that neither plasminogen free plasma (100% antiplasmin activity) nor an excess (>1000 times) of SBTI inhibited the substrate activity of the SK activated plasminogen. Optimal conditions for the SK-activated plasminogen was tris buffer pH 7.4, I 0.05. Km = 2.4.10-4 mol/1 for both purified plasminogen and plasma and V = 1.10-4 mol/min.CU or 3.10-6 mol/min-ml plasma.
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Friberger et al. (1977) studied this question.