A kinetic study of the mechanism of radiation induced agglomeration of ovalbumin in aqueous solution

The effect of concentration on the protein radiolytic damage resulting in a change in molecular mass was measured in the concentration range from 0.2 to 2 mmol x dm(-3) ovalbumin in phosphate buffered solutions saturated with N2O. The electrophoretic analysis of samples on discontinuous SDS-polyacrylamide gets in the presence or absence of 5% beta -mercaptoethanol showed an expected result, i.e. that the protein scission did not take place in the absence of oxygen. Only ovalbumin agglomerates, bonded by covalent bonds other than S-S bridges, were observed. The G-value for the formation of ovalbumin agglomerates increased linearly from 1.1 to 2.4 by increasing the ovalbumin concentration from 0.2 to 2 mmol x dm-3. The result is interpreted as to be owing to the competition between ovalbumin agglomeration and some intramolecular reactions which did not lead to the change in the molecular mass. It was also found that the G-value is independent of irradiation dose rate. The result was rationalized as a kinetic evidence that the agglomeration is not a cross-linking process, i.e. it does not occur via recombination of the protein radicals produced in the interaction of ovalbumin and (OH)-O-. radical. The result suggested that the agglomeration takes place via the process of grafting, i.e. it occurs in the reaction of ovalbumin radical and an intact ovalbumin molecule. The time-resolved light scattering experiments provided an additional proof, supporting the reaction scheme of radiation-induced protein agglomeration. The biological consequences of the proposed mechanism of protein agglomeration are also discussed. (C) 2001 Elsevier Science Ltd. All rights reserved