Nanosecond Time-resolved Proton Transfer Studies with Dehydroluciferin and Its Complex with Luciferase

Fluorescence lifetime measurements have been used to obtain nanosecond time-resolved emission spectra of dehydroluciferin in various solvents and when bound to luciferase. The blue fluorescence caused by the phenol decreases with decay time relative to the green emission caused by the phenolate. The time course of excited state ionization may thus be measured directly. The rate of proton transfer is very fast in aqueous solution but slower in 80% ethanol. Addition of imidazole increases the rate of proton transfer. Dehydroluciferin when bound to luciferase shows a slow rate of proton transfer, suggesting that the binding site is hydrophobic.