Bovine serum albumin observed by infrared spectrometry. II. Hydration mechanisms and interaction configurations of embedded H2O molecules

The hydration mechanism of bovine serum albumin (BSA) is studied, and we analyze (de)hydration spectra displayed previously. We first determine the three elementary (de)hydration spectra on which all these (de)hydration spectra can be decomposed. They correspond to three different hydration mechanisms for the protein, which we define after a quantitative analysis performed in a second step. The first mechanism, which involves ionization of carboxylic COOH groups, occurs at low hydration levels and rapidly reaches a plateau when the hygroscopy is increased. It is a mechanism that involves a single H2O molecule and consequently requires somewhat severe steric conditions. The second mechanism occurs at all hydration levels and, because it involves more H2O molecules, requires less severe steric conditions. It consists of the simultaneous hydration of one amide NH group and one carbonyl-amide CO group by four H2O molecules and one carboxyl COO− group by eight H2O molecules. The third mechanism is simpler and consists of the introduction of H2O molecules into the hydrogen-bond network of the hydrated protein. It becomes important at a high hydration level, when the presence of an appreciable number of H2O molecules makes this hydrogen-bond network well developed. This analysis also shows that 80 H2O molecules remain embedded in one dried protein made of 604 peptide units. They are held by hydrogen bonds established by NH groups and at the same time they establish two hydrogen bonds on two carbonyl-amide CO groups. The proportion of free NH groups can be determined together with that of carbonyl-amide CO groups accepting no hydrogen bonds and that of carbonyl-amide CO groups accepting two hydrogen bonds. The proportion of NH groups establishing one hydrogen bond directly on a carbonyl-amide CO group is 65%, which is the proportion of peptide units found in α helices in BSA. © 2000 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 54–67, 2001