2ND DERIVATIVE SPECTROSCOPY OF ENOLASE AT HIGH HYDROSTATIC-PRESSURE -AN APPROACH TO THE STUDY OF MACROMOLECULAR INTERACTIONS

Second derivative spectroscopy in the ultraviolet region of proteins h as been used to study the polarity of the regions surrounding tyrosine residues. We show here that it can also be a tool to study the degree to which proteins associate and that it can be effectively combined w ith hydrostatic pressure in order to evaluate equilibrium dissociation constants and reaction volumes. Hydrostatic pressure causes yeast eno lase to dissociate. Clear changes in the second derivative spectra of enolase were observed as pressure was increased. At enolase concentrat ions of about 20 mu M, the midpoint of the transition is about 1800 ba r. All aspects of the transition are reversible up to 2700 bar. It is likely that the transition observed is the result of enolase dimers di ssociating into monomers. The second derivative spectra indicate that one or more tyrosine residues is in an unusually polar environment in the dimer, an environment that is less polar in the monomer. Three tyr osines (6, 11, 130) are near the dimer interface. Tyrosines 6 and 11 a re pointing into the water-filled crevice between the subunits and are close to several immobilized waters. All three are close to a network of intersubunit salt bridges,sand hydrogen bonds. We believe that the average tyrosine polarity in the dimer reflects the exposure of these tyrosines to immobilized water and the fixed dipole of the salt bridg e. The water in the crevice between the subunits should be more mobile in the monomer; the salt bridge does not exist in the monomer. In con trast to the behavior of native enolase under pressure, the same prote in in guanidine hydrochloride shows no obvious changes with pressure. Similarly, the small protein hen egg-white lysozyme shows no change in the second derivative as a function of pressure.