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-polyacry lamide gets in the presence or absence of 5% beta -mercaptoethanol showed a n expected result, i.e. that the protein scission did not take place in the absence of oxygen. Only ovalbumin agglomerates, bonded by covalent bonds o ther than S-S bridges, were observed. The G-value for the formation of oval bumin agglomerates increased linearly from 1.1 to 2.4 by increasing the ova lbumin concentration from 0.2 to 2 mmol x dm-3. The result is interpreted a s to be owing to the competition between ovalbumin agglomeration and some i ntramolecular reactions which did not lead to the change in the molecular m ass. It was also found that the G-value is independent of irradiation dose rate. The result was rationalized as a kinetic evidence that the agglomerat ion is not a cross-linking process, i.e. it does not occur via recombinatio n of the protein radicals produced in the interaction of ovalbumin and (OH) -O-. radical. The result suggested that the agglomeration takes place via t he process of grafting, i.e. it occurs in the reaction of ovalbumin radical and an intact ovalbumin molecule. The time-resolved light scattering exper iments provided an additional proof, supporting the reaction scheme of radi ation-induced protein agglomeration. The biological consequences of the pro posed mechanism of protein agglomeration are also discussed. (C) 2001 Elsev ier Science Ltd. All rights reserved.