Determination of active human α-, β- and γ-thrombin and human Glu1-, Lys77, Val442- and Val561-plasmin concentration in the presence of cognate serine proteinase

A spectrophotometric method for the determination of active human α-, β- and γ-thrombin concentration, in the presence of human Glu¹, Lys⁷⁷-, Val⁴⁴²-, and Val⁵⁶¹-plasmin, as well as of active human Glu¹-, Lys⁷⁷-, Val⁴⁴²- and Val⁵⁶¹-plasmin concentration, in the presence of human α-, β- and γ-thrombin, is reported. This assay method has been developed from the quantitative analysis of kinetics for the hydrolysis of the N-α-carbobenzoxy-L-lysine p-nitrophenyl ester catalyzed by human α-, β- and γ-thrombin, in the presence of human Glu¹-, Lys⁷⁷-, Val⁴⁴²- and Val⁵⁶¹-plasmin selectively inhibited by the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor), and by human Glu¹-, Lys⁷⁷-, Val⁴⁴²- and Val⁵⁶¹-plasmin, in the presence of human α-, β- and γ-thrombin selectively inhibited by hirudin, the thrombin-specific inhibitor from leech Hirudo medicinalis. The active enzyme concentration has been estimated, under conditions where [S](o) >> K(m), from the dependence of the initial rate for substrate hydrolysis (i.e., V) on the enzyme concentration (i.e., [E](o)) taking into account the proportionality constant k(cat) (i.e., V = k(cat) · [E](o)). The minimum active human α-, β- and γ-thrombin and human Glu¹-, Lys⁷⁷- Val⁴⁴²- and Val⁵⁶¹-plasmin concentration which may be evaluated from the analysis of the experimental data corresponds to 2.5 x 10^-3 μM.