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: 60Co gamma-irradiation of three proteins at -78 degrees 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 D37 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. D37 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/D37) 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 D37 is directly related to the exposed surface area and not to the molecular mass of the polypeptide chain.
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: 60Co gamma-irradiation of three proteins at -78 degrees 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 D37 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. D37 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/D37) 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 D37 is directly related to the exposed surface area and not to the molecular mass of the polypeptide chain.
Authors: Matthew Warkentin; Jesse B Hopkins; Jonah B Haber; Gregor Blaha; Robert E Thorne Journal: Acta Crystallogr D Biol Crystallogr Date: 2014-10-23