Effect of various environments on the intrinsic fluorescence polarization spectra of horse liver alcohol dehydrogenase

The intrinsic fluorescence polarization spectra of horse liver alcohol dehydrogenase in neutral buffer, various molarities of guanidine and urea, low pH, and neutral salt are presented. Correlation of polarization changes at two exciting wave-lengths with enzymatic activity changes is shown for horse liver alcohol dehydrogenase. Correlations with sedimentation coefficient changes and intrinsic viscosity changes were observed in some cases. Lowering the pH to 2.5 caused an approximately 30–35% lowering of the polarization at all wavelengths > 255 nm. It is suggested that this type of polarization change reflects subunit dissociation without unfolding. The effect of the more drastic solvents is to “flatten out” the spectrum by a rise in polarization at wavelengths below 250 nm, and a fall in polarization at wavelengths above 250 nm, the percentage change varying across the spectrum. This type of polarization change must reflect unfolding, with rupture of H-bonds, and changes in hydrophobic interactions. Restoration of the initial (native) polarization was observed in dilution experiments from guanidine and urea solutions of the enzyme. The fluorescence quantum yield per mole of tryptophan was determined for horse liver alcohol dehydrogenase and found to be 18%, a value higher than that for free tryptophan. A spectrophotofluorometer suitable for measuring polarization spectra is described.