Serpins are thought to inhibit proteinases by first forming a Michaelis-type complex that later converts into a stable inhibitory species. However, there is only circumstantial evidence for such a two-step reaction pathway. Here we directly observe the sequential appearance of two complexes by measuring the time-dependent change in fluorescence resonance energy transfer between fluorescein-elastase and rhodamine-␣ 1protease inhibitor. A moderately tight initial Michaelistype complex EI 1 (K i

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... ‬ 0.38 -0.52 M) forms and dissociates rapidly (k 1 ‫؍‬ 1.5 ؋ 10 6 M ؊1 s ؊1 , k ؊1 ‫؍‬ 0.58 s ؊1 ). EI 1 then slowly converts into EI 2 (k 2 ‫؍‬ 0.13 s ؊1 ), the fluorescence intensity of which is stable for at least 50 s. The two species differ by their donor-acceptor energy transfer efficiency (0.41 and 0.26, respectively). EI 2 might be the final product of the elastase ؉ inhibitor association because its transfer efficiency is the same as that of a complex incubated for 30 min. The timedependent change in fluorescence resonance energy transfer between fluorescein-elastase and rhodamineeglin c, a canonical inhibitor, again allows the fast formation of a complex to be observed. However, this complex does not undergo any fluorescently detectable transformation.