Isolation of the in vivo emitter in bacterial bioluminescence

Abstract

A blue fluorescence protein was isolated and purified from extracts of the luminous bacterium Photobacterium phosphoreum. It is a single polypeptide of MW 22,000 with absorption maxima at 274 and 418 nm. It is efficiently fluorescent (.vphi.F [fluorescence quantum yield] 0.45), with a fully corrected spectral maximum (476 nm) and distribution identical to the in vivo bioluminescence from this same type of bacterium. At low concentration this fluorescence shifts towards the red and becomes identical to the in vitro bioluminescence emission. This spectral shift apparently results from a change in the protein pulled by dissociation of the chromophore (Kd .simeq. 10-7 M). If the blue fluorescence protein is included in the in vitro bioluminescence reaction with reduced FMN, oxygen, aldehyde and luciferase (P. phosphoreum), the bioluminescence spectrum is shifted towards the blue from its maximum at 490 nm to one at 476 nm, where it is again identical in all respects to the in vivo bioluminescence spectrum. This is accompanied by an increase in the initial light intensity by an order of magnitude at saturating levels of blue fluorescence protein, and the specific light yield of the luciferase is increased 4-fold. The blue fluorescence protein probably acts as a sensitizer of the bacterial bioluminescence reaction.