Effects of pressure upon the fluorescence of the riboflavin binding protein and its flavin mononucleotide complex.

The effect of pressure in the range of 10(-3)-10 kbars upon the ultraviolet fluorescence of the riboflavin binding protein and the fluorescences of its complex with flavin mononucleotide has been studied. The fluorescence spectrum of the isolated protein showed a reversible red shift of 12nm (1000 cm-1) at high pressure, indicating the reversible exposure of the tryptophan to solvent. From the pressure dependence of the visible fluorescence of the protein-flavin complex in the region of 1-4 kbars the volume change in dissociation of the protein-ligand complex was estimated to be +3.3ml/mol. A very sharp increase in fluorescence-up to 30-fold of the low-pressure value-takes place in the region 5-8 kbars. This increase is due to release of the flavin from the complex and is assigned to pressure denaturation of the protein. The midpoint, rho 1/2, of this transition was found at 6.5 kbars and the change in volume, delta, in the reaction (native-to-denatured) was calculated to be -74ml/mol. Addition of up to 30% methanol results in a progressive decrease both in delta and rho 1/2, in agreement with the concept that hydrophobic bonding stabilizes the native structure.