The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography(SEC).It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L...The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography(SEC).It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride(GuHCl)or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase.Although the oxidized and reduced glutathione(GSSG and GSH)were employed in the oxidized mobile phase,the reduced-denatured insulin still could not be renatured.However,in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed,the reduced-denatured insulin can be refolded with SEC,and the aggregation of denatured insulin can be diminished by urea.In addition,the disulfide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase.The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely.The results were further tested with reversed-phase liquid chromatography(RPLC)and hydrophobic interaction chromatography(HIC).展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.29675017&39880003)the Foundation of the Young Teacher in the University of Ministry of Education,China(Grant No.DF00308)+1 种基金the Natural Science Foundation in Shaanxi Province(Grant No.2001H02)the Foundation of the Committee of Education in Shaanxi Province(Grant No.99JK101).
文摘The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography(SEC).It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride(GuHCl)or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase.Although the oxidized and reduced glutathione(GSSG and GSH)were employed in the oxidized mobile phase,the reduced-denatured insulin still could not be renatured.However,in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed,the reduced-denatured insulin can be refolded with SEC,and the aggregation of denatured insulin can be diminished by urea.In addition,the disulfide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase.The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely.The results were further tested with reversed-phase liquid chromatography(RPLC)and hydrophobic interaction chromatography(HIC).