Time-resolved protein phosphorescence in the stopped-flow: Denaturation of horse liver alcohol dehydrogenase by urea and guanidine hydrochloride

This study reports the implementation of room temperature protein phosphorescence in the stopped-flow technique. Time-resolved Trp phosphorescence can now be detected following rapid mixing of protein solutions with a time resolution of IO ms and a sensitivity in terms of chromophore concentration down to 0.1 mu M. Calibration tests with monomeric and multimeric proteins proved that in all cases the delayed emission is not affected by artefacts that could arise from either enrichment of trace impurities along the flow lines or deformation of the macromolecules by the shear stress of laminar flow, To illustrate the potential of Trp phosphorescence in the stopped-flow to detect the time evolution of protein conformation the interaction of urea and guanidine hydrochloride (GdnHCl) with the native structure of horse liver alcohol dehydrogenase (LADH) has been re-examined under conditions of rapid denaturation. Remarkable differences in the action of the two denaturing agents has been confirmed by the phosphorescence lifetime (tau p) of the internal Trp residue (W314), Whereas in urea, up to 8 M, tau p is not minimally perturbed, in GdnHCl it decreases sharply and progressively from 800 ms down to 23 ms in 6 M solutions. Such reduction of tau p implies that in the region of W314 the polypeptide structure has become highly loose and flexible prior to the major unfolding transition. Therefore, denaturation of LADH in GdnHCl, as opposed to urea, proceeds from a partly unfolded intermediate conformation of the protein. Other characteristics of this intermediate state are a partial loss of tertiary structure, as revealed by the circular dichroism of the aromatics, and an almost complete inhibition of the catalytic activity. Control experiments with equimolar NaCl demonstrate that tau p, the tertiary structure and the catalytic activity are affected to a much smaller extent and that, therefore, salt effects do not account for the difference between urea and GdnHCl, Finally, measurements of the unfolding reaction emphazise that the kinetics of LADH denaturation are heterogeneous with both denaturing agents. From the constancy of tau p during the course of the reaction it is concluded that the multiphasic behavior is a manifestation of multiple unfolding pathways owing to a plurality of stable LADH conformations.