Conformational Changes in Human Serum Albumin as Revealed by Hydrogen-Deuterium Exchange Studies

The H-D exchange in aqueous solutions of defatted human serum albumin (HSA) has been investigated by the infrared spectroscopic technique. Data on the exchange rates are presented as plots of the fraction of unexchanged peptide hydrogen atoms (X) at a given time (t) versus log(k0t), where k0 is the pH dependent exchange rate constant of solvent exposed peptide groups. When no changes in the stability of a protein takes place as a function of pH, this presentation of the exchange, measured at a series of values of pH, gives a complete of the protein. If it is assumed that the motility of the protein is so high that the exchange mechanism is of the so-called EX2 type, the relaxation spectrum may be taken as an illustration of the distribution of changes in free energy, ΔG, of the transitions of the protein, exposing the peptide groups to the solvent. In the case of HSA at 25° the plot of X versus log(k0t) is a discontinuous function of pH, illustrating the changes in the stability of the conformation of the protein which are provoked by changes in pH. Only a fragment of the relaxation spectrum can be obtained at any given value of pH, but from the relative displacement of the fragments, estimates can be made of changes in the above mentioned values of ΔG. The pH of maximum stability of defatted HSA at 25° is found at pH ∼ 7. At this pH less than 20% of the peptide groups are protected from solvent exposure by conformations stabilized by values of ΔG larger than 8 kcal mole-1; for half of the peptide groups ΔG is smaller than 5 kcal mole-1. Experiments made at pH 6.2 and at different temperatures indicate that the temperature of maximum stability of the protein at this pH is below 5°. A decrease in the exchange rates in the presence of palmitate is observed at pH 7.6.