Hydrolysis of haemoglobin surveyed by infrared spectroscopy: I. solvent effect on the secondary structure of haemoglobin

The hydrolysis of bovine haemoglobin in an acetic acid/sodium acetate buffer enables to produce peptides of major importance in biomedical research. The global objective is to survey this reaction by infrared spectroscopy. This article concerns the first step: the evaluation by spectroscopy of the effect on protein secondary structure of the addition of ethanol in the buffer.Conformational changes are related to solvent-protein interactions as the protein folding is driven by the entropy of removing hydrophobic groups from contact with the solvent. Therefore, the stability of haemoglobin in an ethanol-water mixture results in a competition between the water structure, which is strengthened by the presence of the alcohol, and the solubility of hydrophobic residues. Previous non infrared experiments, based on mass spectrometry for example, have been reported for the investigation of the denaturation of haemoglobin by an organic solvent.The use of vibrational spectroscopy for protein secondary structure determination has proved its efficiency. We focus here on the study of the denaturing of haemoglobin in a water medium by addition of ethanol, As our investigations deal with very low concentrated haemoglobin, we apply a technique that uses films dried from dilute solution. Although it is not generally accepted that the protein conformation is retained when the solvent evaporates off, we validate this method comparing some results to previous infrared study made at upper concentrations with liquid sampling. We observe that Fourier Transform Infrared (FTIR) Spectroscopy, combined with few mathematical treatments, permits to estimate that haemoglobin remains in a native form unless a concentration of more or less 20% of ethanol is reached. For greater values modifications are perceptible on the infrared spectra. (C) 1999 Elsevier Science B.V, All rights reserved.