title: Aggregates formed during storage of beta-galactosidase in solution and in the freeze-dried state.
abstract: Aggregates formed during storage of freeze-dried beta-galactosidase were compared with those formed in solutions. Freeze-dried beta-galactosidase aggregated during storage in the presence of moisture, producing a protein precipitate which was soluble in guanidine hydrochloride solution but not in buffer solution. The protein precipitate dissolved in guanidine solution exhibited a large molecular size by high-performance size exclusion chromatography and converted to proteins of original size in the presence of dithiothreitol. It is suggested that the aggregation involves chemical interaction via covalent disulfide bonding. In contrast, beta-galactosidase in aqueous solution aggregated without formation of protein precipitates. Soluble aggregates were converted to proteins of original size in guanidine solution without dithiothreitol, suggesting noncovalent bonding. The difference in aggregation behavior may be ascribed to the difference in the water:protein ratio. We propose that inactivation of beta-galactosidase is due to formation of thermally denatured (unfolded) protein, which aggregates dependent on the water:protein ratio, either via noncovalent interactions at a high water:protein ratio in solution or via covalent interaction at a low water:protein ratio in the freeze-dried state.
authors.affiliation: National Institute of Hygienic Sciences, Tokyo, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Yoshioka
authors.forename: Y
authors.initials: Y
authors.lastname: Aso
authors.forename: K
authors.initials: K
authors.lastname: Izutsu
authors.forename: T
authors.initials: T
authors.lastname: Terao
mesh_terms: D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D004356:Drug Storage; D005612:Freeze Drying; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D001616:beta-Galactosidase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002021:Buffers; D012996:Solutions; D001616:beta-Galactosidase