Acrylamide-quenching of Rhizomucor miehei lipase

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 K1 = 3.75 M−1 and V1 = 1.12 M−1, respectively. The bluer component is quenchable via dynamic mechanism with K2 = 1.97 M−1. Significant difference in the values of acrylamide bimolecular rate quenching constants estimated for redder and bluer component (i.e., kq = 1.2 × 109 M−1 s−1 vs. kq = 4.3 × 108 M−1 s−1, respectively), suggests that tryptophan residues in fungal lipase are not uniformly exposed to the solvent.