Acrylamide-quenching of Rhizomucor miehei lipase

Abstract

Steady-state and time-resolved fluorescence-quenching measurements have been performed to study multitryptophan lipase from filamentous fungus Rhizomucor miehei. Using the steady-state acrylamide fluorescence quenching data and the fluorescence-quenching-resolved-spectra (FQRS) method, the total emission spectrum of native (“closed-lid”) lipase has been decomposed into two distinct spectral components accessible to acrylamide. According to FQRS analysis, more quenchable component has a maximum of fluorescence emission at about 352 nm whereas less quenchable component emits at about 332 nm. The redder component participates in about 60–64% of the total lipase fluorescence and may be characterized by the dynamic and static quenching constants equal to K 1 = 3.75 M −1 and V 1 = 1.12 M −1, respectively. The bluer component is quenchable via dynamic mechanism with K 2 = 1.97 M −1. Significant difference in the values of acrylamide bimolecular rate quenching constants estimated for redder and bluer component (i.e., k q = 1.2 × 10 9 M −1 s −1 vs. k q = 4.3 × 10 8 M −1 s −1, respectively), suggests that tryptophan residues in fungal lipase are not uniformly exposed to the solvent.