Stopped flow fluorescence energy transfer measurement of the rate constants describing the reversible formation and the irreversible rearrangement of the elastase-alpha1-proteinase inhibitor complex
Research article published in The Journal of biological chemistry (1998)
Abstract
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-alpha1-protease inhibitor. A moderately tight initial Michaelis-type complex EI1 (Ki = 0.38-0.52 microM) forms and dissociates rapidly (k1 = 1.5 x 10(6) M-1 s-1, k-1 = 0.58 s-1). EI1 then slowly converts into EI2 (k2 = 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). EI2 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 time-dependent change in fluorescence resonance energy transfer between fluorescein-elastase and rhodamine-eglin 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.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
Research article relevant to leech therapy and its derived compounds.
Por qué esto importa para la hirudoterapia
El resumen utilizó transferencia de energía de resonancia de fluorescencia para observar directamente el mecanismo de inhibición secuencial de dos pasos de la elastasa por el inhibidor de alfa1-proteinasa, midiendo la formación rápida de un complejo inicial tipo Michaelis (Ki = 0,38–0,52 µM) seguido de una reorganización más lenta hacia una especie inhibidora estable. El estudio también examinó la interacción de la elastasa con eglina c, descrita en el resumen como un 'inhibidor canónico', que formó un complejo inicial sin experimentar ninguna transformación detectable por fluorescencia. El resumen no aporta información sobre sanguijuelas, hirudoterapia ni el origen biológico de la eglina c. No existe un enlace defendible con las sanguijuelas a partir de este resumen.
Citación
Stopped flow fluorescence energy transfer measurement of the rate constants describing the reversible formation and the irreversible rearrangement of the elastase-alpha1-proteinase inhibitor complex
Mellet P · The Journal of biological chemistry, 1998
Contexto clínico relacionado
Explore cómo esta investigación se conecta con la práctica clínica
Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: 18 de junio de 2026