RENILLA-RENIFORMIS BIOLUMINESCENCE - LUCIFERASE-CATALYZED PRODUCTION OF NON-RADIATING EXCITED-STATES FROM LUCIFERIN ANALOGS AND ELUCIDATION OF THE EXCITED-STATE SPECIES INVOLVED IN ENERGY-TRANSFER TO RENILLA GREEN FLUORESCENT PROTEIN

A number of coelenterate-type luciferin analogues with structural changes in the p-hydroxyphenyl and p-hydroxybenzyl substituents have been synthesized. During chemiluminescence, each of the analogues produces a blue emission arising from the singlet excited state of the corresponding oxyluciferin monoanion. During bioluminescence two emissions are observed with coelenterate-type luciferin and some of its analogues. One of these arises from the amide monoanion (λmax⋍ 480 nm) and the other arises from the neutral species of oxyluciferin (λmax⋍ 395 nm). Certain analogues produce both emissions, while others produce only the near-ultraviolet emission. Structural changes in the p-hydroxyphenyl substituent result in complete or nearly complete elimination of emission from the monoanion, resulting in over a 100-fold reduction in bioluminescence quantum yield. Structural changes in the p-hydroxybenzyl substituent do not have a significant effect on the emission spectrum but decrease the luciferase turnover number approximately 25-fold. The large decrease in the bioluminescence quantum yield observed with some of the analogues can be overcome by addition of green fluorescent protein (GFP). GFP forms a rapid equilibrium complex with luciferase and is known to function in this system as an efficient energy-transfer acceptor [Ward, W. W., & Cormier, M. J. (1978) Photochem. Photobiol. 27, 389-396], Spectral analyses have shown that radiationless energy transfer occurs from the singlet excited state of the oxyluciferin monoanion and not from the neutral excited species. The energy-transfer data suggests that the luciferase-bound monoanion singlet excited state can be quenched by solvent and/or protein functional groups. Energy transfer to GFP can apparently favorably compete with this quenching process. Lifetime measurements have shown that the rate of energy transfer must be at least 3 x 108 s_1. © 1979, American Chemical Society. All rights reserved.