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

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