The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding.Six major folding intermediates,named P1A,P2B,P3A,P4B,P5B,;P6B,were captured...The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding.Six major folding intermediates,named P1A,P2B,P3A,P4B,P5B,;P6B,were captured during the folding process.The refolding experiments indicate that all of these intermediates are on-pathway.Based on these intermediates;the formation of hypothetic transients,we propose a two-stage folding pathway of insulin.(1)At the early stage of the folding process,the reduced A chain;B chain individually formed the intermediates:two A chain intermediates(P1A;P3A),;four B chain intermediates(P2B,P4B,P5B,;P6B).(2)In the subsequent folding process,transient I was formed from P3A through thiol/disulfide exchange reaction;then,transients II;III,each containing two native disulfides,were formed through the recognition;interaction of transient I with P4B or P6B;the thiol group’s oxidation reaction mainly using GSSG as oxidative reagent;finally,transients II;III,through thiol/mixture disulfide exchange reaction,formed the third native disulfide of insulin to complete the folding.展开更多
基金Supported by the Chinese Academy of Sciences(Grant KJ951-B1-606)
文摘The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding.Six major folding intermediates,named P1A,P2B,P3A,P4B,P5B,;P6B,were captured during the folding process.The refolding experiments indicate that all of these intermediates are on-pathway.Based on these intermediates;the formation of hypothetic transients,we propose a two-stage folding pathway of insulin.(1)At the early stage of the folding process,the reduced A chain;B chain individually formed the intermediates:two A chain intermediates(P1A;P3A),;four B chain intermediates(P2B,P4B,P5B,;P6B).(2)In the subsequent folding process,transient I was formed from P3A through thiol/disulfide exchange reaction;then,transients II;III,each containing two native disulfides,were formed through the recognition;interaction of transient I with P4B or P6B;the thiol group’s oxidation reaction mainly using GSSG as oxidative reagent;finally,transients II;III,through thiol/mixture disulfide exchange reaction,formed the third native disulfide of insulin to complete the folding.
