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 phos phorescence in the stopped-flow technique. Time-resolved Trp phosphore scence can now be detected following rapid mixing of protein solutions with a time resolution of IO ms and a sensitivity in terms of chromop hore 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 o f trace impurities along the flow lines or deformation of the macromol ecules by the shear stress of laminar flow, To illustrate the potentia l of Trp phosphorescence in the stopped-flow to detect the time evolut ion of protein conformation the interaction of urea and guanidine hydr ochloride (GdnHCl) with the native structure of horse liver alcohol de hydrogenase (LADH) has been re-examined under conditions of rapid dena turation. Remarkable differences in the action of the two denaturing a gents has been confirmed by the phosphorescence lifetime (tau p) of th e internal Trp residue (W314), Whereas in urea, up to 8 M, tau p is no t minimally perturbed, in GdnHCl it decreases sharply and progressivel y 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 becom e highly loose and flexible prior to the major unfolding transition. T herefore, denaturation of LADH in GdnHCl, as opposed to urea, proceeds from a partly unfolded intermediate conformation of the protein. Othe r characteristics of this intermediate state are a partial loss of ter tiary structure, as revealed by the circular dichroism of the aromatic s, and an almost complete inhibition of the catalytic activity. Contro l experiments with equimolar NaCl demonstrate that tau p, the tertiary structure and the catalytic activity are affected to a much smaller e xtent and that, therefore, salt effects do not account for the differe nce between urea and GdnHCl, Finally, measurements of the unfolding re action emphazise that the kinetics of LADH denaturation are heterogene ous with both denaturing agents. From the constancy of tau p during th e course of the reaction it is concluded that the multiphasic behavior is a manifestation of multiple unfolding pathways owing to a pluralit y of stable LADH conformations.