OXYLUCIFERIN FLUORESCENCE IS A MODEL OF NATIVE BIOLUMINESCENCE IN THE FIREFLY LUCIFERIN LUCIFERASE SYSTEM

The steady state and subnanosecond time-resolved fluorescence properties of oxyluciferin and its structural analogues luciferin (LH2), 6'-methoxyluciferin (MeOLH2) and 2-cyano-6-hydroxybenzothiazole (BT) were investigated in aqueous (pH 1-10) and ethanol solutions. The structures of the three fluorescence emitters were identified as follows: blue, enol-phenolate; yellow-green, phenol; red, phenolate of keto form. The dissociation rate of the excited luciferin 6'-hydroxyl group is shown to be lower than the rate of emission by the phenol form itself and, consequently, although ground and excited pK values differ considerably (8.5 and - 0.5 respectively), blue fluorescence is observed in non-polar and strong acidic aqueous solutions. Fluorescence decay curves for oxyluciferin and its analogues (blue and yellow-green emission maxima) were obtained in water (pH 7.8) and ethanol (96%).