Protein Hydration Changes in the Formation of the Nicotinamide Adenine Dinucleotide Complexes of Glyceraldehyde 3-Phosphate Dehydrogenase of Yeast

The preferential hydrations of the glyceraldehyde 3-phosphate dehydrogenase of yeast and its (NAD)4 complex have been calculated from buoyant densities measured by an isopycnic sedimentation method in a concentration series of potassium phosphate solutions and by sedimentation equilibrium in cesium chloride gradients. In potassium phosphate solution at the isopycnic concentration and pH 7.4 and 25°, the preferential hydration of the apoenzyme is 0.480 g of water per g of protein. There is a 15.7% decrease in preferential hydration when the NAD complex is formed. This corresponds to a 6.3% decrease in volume of the solvated protein and is in accord with a reported 7% volume contraction measured by x-ray small angle scattering. The buoyant density increase associated with NAD complex formation is attributed to the elimination of water from cavities in the contracting protein structure that exclude the salt component of the solvent. Essentially the same hydration change occurs at pH 8.5, where the protein binds NAD with a high degree of positive cooperativity, and at pH 7.4, where the binding appears to occur at noninteracting sites. Chloride binding to the apo- and holoenzymes has been estimated from the pH shift that occurs when cesium chloride at its isopycnic concentration is added to a solution of the isoionic protein. The buoyant density increase associated with NAD complex formation does not involve an increased binding of salt, the heavy component of the solvent.