Bovine serum albumin observed by infrared spectrometry. I. Methodology, structural investigation, and water uptake.

The results of preliminary infrared (IR) spectrometry experiments on bovine serum albumin (BSA) films are presented. An analysis of spectral variations due to raising the temperature and deuteration of N--H groups leads to the assignment of most IR bands of BSA. From this analysis we furthermore deduce that at 115 degrees C only hydrogen bonds established by N&bond;H groups on the still present H(2)O molecules, which are so strongly bound to the protein that they do not evaporate, are weakened, some of which are broken. These N--H...OH(2) groups represent some 5% of all N--H groups in the dried protein. Spectral changes due to hydration by water vapor are also analyzed and a precise method to measure the water-vapor pressure of the atmosphere surrounding the BSA film, or equivalently the relative humidity, is described. Various procedures to measure the number of H(2)O molecules embedded in BSA are then presented and evaluated. One of them is selected as the best one for proteins, because it matches previous measurements based on gravimetric methods. This procedure is subsequently used in a study that is devoted to the determination of the various hydrogen-bond configurations, or interaction configurations, which are adopted by H(2)O molecules during the various steps of hydration of BSA. This first analysis of hydration spectra allows the completion of the assignment of IR bands. The various spectral components of the amide I band, which are interchanged during the hydration process, cannot be assigned to various secondary structures, as is usually proposed. It suggests that this usual assignment should be used with care, especially by taking into account the state of hydration, when one wishes to obtain structural information from it.

Bovine serum albumin observed by infrared spectrometry. II. Hydration mechanisms and interaction configurations of embedded H(2)O 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 H(2)O molecule and consequently requires somewhat severe steric conditions. The second mechanism occurs at all hydration levels and, because it involves more H(2)O 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 H(2)O molecules and one carboxyl COO(-) group by eight H(2)O molecules. The third mechanism is simpler and consists of the introduction of H(2)O 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 H(2)O molecules makes this hydrogen-bond network well developed. This analysis also shows that 80 H(2)O 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 alpha helices in BSA.

FT infrared and Raman investigation of saccharide-phosphatidylcholine interactions using novel structure probes.

Conformational consequences of adduct formation between saccharides (trehalose, glucose, raffinose) and sorbitol with dipalmitoylphosphatidylcholine (DPPC) in multibilayers are revealed by relative intensity changes of the band components corresponding to the nu asN(CH3)3 and nu sC-N(CH3)3 stretching modes of the choline chain terminal and those of the nu C = O band. The conformational sensitivity of those modes was demonstrated previously (J. Grdadolnik et al., Chem. Phys. Lipids 65 (1993) 121) and used to demonstrate the effects of stepwise hydration of phosphatidylcholines. The latter are compared with the effects of saccharide binding and found to be qualitatively similar, but not identical. The same is true of the low frequency shifts of the nu asPO2- vibration: the shifts due to saccharide binding correspond to the binding of six to seven water molecules per phosphate which is about 20 cm-1 less than the shift caused by full hydration. A particularly interesting finding concerns the appearance of two bands in the nu asPO2- region of the DPPC-saccharide adducts. The relative intensities of the two bands (1243 and 1223 cm-1) change on additional hydration; it is the one at 1223 cm-1 that prevails at high hydration levels. Major changes in saccharide conformation are not detectable but minor differences between the DPPC bound and crystal spectra are observed.