Protein gamma-radiolysis in frozen solutions is a macromolecular surface phenomenon: fragmentation of lysozyme, citrate synthase and alpha-lactalbumin in native or denatured states

Abstract

Purpose : To test whether radiolysis-induced fragmentation in frozen aqueous protein solution is dependent on solvent access to the surface of the protein or to the molecular mass of the polypeptide chain. Materials and methods : 60 Co gamma-irradiation of three proteins at -78°C: lysozyme, citrate synthase and alpha-lactalbumin in their native state, with or without bound substrate, or denatured (random coil in urea/acid-denatured state). Results : By SDS-polyacrylamide gel electrophoresis/analysis of the protein-fragmentation process, it was found that for a given protein D 37 values (dose to decrease the measured amount of protein, with an unaltered polypeptidic chain, to 37% of the initial amount) varied according to the state of the protein. D 37 for denatured proteins was always much smaller than for native states, indicating a greater susceptibility to fragmentation. In urea, contrary to the native state, no well-defined fragments were observed. Radiolysis decay constants (K = 1/D 37) increased with solvent-accessible surface area of these proteins estimated from their radii of gyration in the various states. This is shown also in previous data on native or SDS-denatured proteins. Denatured proteins which have a large surface area exposed to the solvent compared with native ones are more fragmented at equal doses. Conclusions : It is concluded that D 37 is directly related to the exposed surface area and not to the molecular mass of the polypeptide chain.