ERYTHRINA PROTEASE INHIBITOR - KINETICS OF INTERACTION WITH TISSUE-PLASMINOGEN ACTIVATOR

Erythrina caffra seeds contain a Kunitz-type inhibitor (ETI) of trypsi n and tissue plasminogen activator, which forms sodium dodecyl sulphat e labile complexes with trypsin and tissue plasminogen activator (t-PA ). Using kinetic experimental approaches designed to study enzyme inhi bitors defined as 'moderately tight-binding',1 we have obtained result s from which we have calculated a k(on) rate of 1.59 x 10(4) M-1 s-1 a nd a k(off) rate of 6.24 x 10(-4) s-1 for the t-PA-ETI interaction. Th e ratio of k(off)/k(on) values gave a K(i) of 4.06 x 10(-8) M. From st eady state kinetics a value of 2.88 X 10(-8) M was obtained. The K(i) of the reaction of two-chain t-PA with ETI did not differ markedly fro m that of the one-chain enzyme. Fibrinogen, poly-L-lysine or heparin d id, not affect the reaction significantly. The kinetic parameters we h ave defined and the reversibility of the ETI-t-PA interaction account for the efficacy of the inhibitor as a solid phase affinity reagent fo r the purification of t-PA.