NOVEL SERINE-PROTEASE INHIBITOR FROM THE AUSTRALIAN BROWN SNAKE, PSEUDONAJA-TEXTILIS TEXTILIS - INHIBITION-KINETICS

A 7 kDa protein component, isolated from the venom of the Australian b rown snake, Pseudonaja textilis textilis, was found to be an inhibitor of the serine protease plasmin. Its mode of action, inhibitory effici ency and specificity were determined, and compared with those displaye d by aprotinin (a Kunitz inhibitor), Using aprotinin as a model small protein protease inhibitor, and with plasmin as the enzyme, the observ ed slow-onset inhibition was consistent with the two-stage reversible mechanism, E+I reversible arrow EI reversible arrow EI'. Formation of the initial complex (EI) was fast, but binding was relatively loose wi th an initial K-i=3.78 nM, while transition from EI to EI', was slow a nd binding was tight, with a final K-i'=53.2 pM. With snake inhibitor, and in contrast to the above standard mechanism, the observed inhibit ion was consistent with a competitive, single-stage reversible mechani sm, prior to cleavage of the inhibitor to an inactive product. Plasmin and trypsin bound the snake inhibitor via this mechanism, with K-i va lues of 0.15 mu M and 0.30 mu M, respectively. Snake protein concentra tions up to 1.0 mu M failed to inhibit a number of serine proteases, i ncluding recombinant two-chain tissue plasminogen activator, high mole cular weight urokinase (55 kDa), alpha thrombin, elastase and a chymot rypsin. Results demonstrate that the small protein protease inhibitor from the Australian brown snake does not act via the standard slow tig ht-binding mechanism common to other small protein serine protease inh ibitors.