Irradiation of Proteins in the Solid State: I. Aggregation and Disorganization of Secondary Structure in Bovine Serum Albumin

Abstract

Solid bovine serum albumin was irradiated in the absence of oxygen with 2-Mev electrons. Changes in solubility, average molecular weight from light-scattering, sedimentation, and accessibility of disulfide groups were measured. On irradiation the protein becomes insoluble in water but remains soluble in salt solutions. There is a threshold in the relation between water solubility and dose. With high doses the protein becomes insoluble in salt solutions but still dissolves in 4 [image] guanidine hydrochloride. The loss of water solubility is associated with an increase in the average molecular weight as determined by light-scattering. The aggregates are not dissociated by 4 [image] guanidine hydrochloride or by the breaking of disulfide bonds. Changes occur in the sedimentation behavior at doses where the average molecular weight is not altered, and the amount of material changed follows an all-exponential (one-hit) relationship with dose. In the native protein all 17 disulfide groups per molecule are hidden and can neither be oxidized nor reduced. On irradiation 50% are revealed in the fraction which has become insoluble in water. In the fraction which retains water solubility but which has an altered sedimentation behavior, 25% of the disulfide bonds are reactive. Quantitative considerations indicate 3 stages of radiation damage. One event (primary ionization) changes the shape as shown by revelation of 25% of the disulfide bonds and by the ultracentrifuge. The solubility and molecular weight are not affected. Two events per molecule (i.e., threshold relationship with dose) change solubility and give small aggregates. Further irradiation causes extensive aggregation by intermolecular hydrogen bonds.