A novel serine protease inhibitor from the Australian brown snake, Pseudonaja textilis textilis: Inhibition kinetics

Abstract

A 7kDa protein component, isolated from the venom of the Australian brown snake, Pseudonaja textilis textilis, was found to be an inhibitor of the serine protease plasmin. Its mode of action, inhibitory efficiency and specificity were determined, and compared with those displayed by aprotinin (a Kunitz inhibitor). Using aprotinin as a model small protein protease inhibitor, and with plasmin as the enzyme, the observed slow-onset inhibition was consistent with the two-stage reversible mechanism, E+IEIEI′. Formation of the initial complex (EI) was fast, but binding was relatively loose with an initial K=3.78 nM, while transition from EI to EI′, was slow, and binding was tight, with a final K′=53.2 pM. With snake inhibitor, and in contrast to the above standard mechanism, the observed inhibition was consistent with a competitive, single-stage reversible mechanism, prior to cleavage of the inhibitor to an inactive product. Plasmin and trypsin bound the snake inhibitor via this mechanism, with K values of 0.15 μM and 0.30 μM, respectively. Snake protein concentrations up to 1.0 μM failed to inhibit a number of serine proteases, including recombinant two-chain tissue plasminogen activator, high molecular weight urokinase (55 kDa), α thrombin, elastase and α chymotrypsin. Results demonstrate that the small protein protease inhibitor from the Australian brown snake does not act via the standard slow, tight-binding mechanism common to other small protein serine protease inhibitors.