STATE OF HEMOGLOBIN AFTER ITS FREEZING-THAWING WITH GLYCEROL

Abstract

We have studied the influence of glycerol upon HbA. The cryoprotector effect on conformation of hemoglobin A was investigated using methods of solvent-perturbation differential spectrophotometry and analysis of the IDAS (first derivatives of absorption spectra). Linear nature of the E/E dependence (the ratio of the difference between absorption of hemoglobin in glycerol and absorption of hemoglobin in saline to absorption of hemoglobin in saline) on glycerol concentration for hemoglobin A solutions with glycerol corresponds to literature data and means that if glycerol concentration is raised up to 40%, no changes of protein conformation are recorded by the method of solvent-perturbation differential spectrophotometry. An analysis of the IDAS of HbA in saline (control solution) and HbA solutions with glycerol was carried out. After freeze-thawing of HbA solutions with glycerol, no changes in the conformational state of proteins were recorded. The dependences of E/E on concentration of glycerol for HbA solutions with glycerol after freeze-thawing did not change. An analysis of the IDAS of hemoglobin A solutions with glycerol before and after freezing-thawing, was carried out. There is a permanence of negative maxima of the IDAS of hemoglobin A solutions with glycerol after freeze-thawing from corresponding samples till freeze-thawing. Intensities of negative maxima within 284-286 and 292 nm on the IDAS of hemoglobin A solutions with glycerol after freeze-thawing slighly differ from corresponding samples before freeze-thawing. The results obtained may be related to a change in the solvate shell of biopolymers. Providing hydroxyl groups, glycerol molecules are capable to occupy part of solvate shell of HbA with corresponding change in structure and energy parameters of the macromolecule, since the structure of the macromolecule in solution is known to be supported by a solvent in the immediate environment of the macromolecule. Stabilization occurs due to hydrogen bonds between nonelectrolytes and biopolymers, with nonelectrolytes acting as water substitutes, although influence effects of solvent not necessarily constitute a monotone function of the solvent composition.