Unfolding and breakdown of insulin in the presence of endogenous thiols.
Native insulin denatures and unfolds in the presence of thiol catalyst via disulfide scrambling (isomerization). It undergoes two transient non-native conformational isomers, followed by an irreversible breakdown of the protein to form oxidized A- and B-chain. Denaturation and breakdown of native insulin may occur under physiological conditions. At 37 degrees C, pH 7.4, and in the presence of cysteine (0.2 mM), native insulin decomposes with a pseudo first order kinetic of 0.075 h(-1). At 50 degrees C, the rate increases by 5-fold. GdnCl and urea induced denaturation of insulin follows the same mechanism. These results demonstrate that stability and unfolding pathway of insulin in the presence of endogenous thiol differ fundamentally from its reversible denaturation observed in the absence of thiol, in which native disulfide bonds of insulin were kept intact during the process of denaturation.
Center for Protein Chemistry, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas, Houston, TX 77030, USA.
Chuantao
C
Jiang
Jui-Yoa
JY
Chang
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008968:Molecular Conformation; D010179:Pancreas / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000494:pharmacology*; D013696:Temperature; D013997:Time Factors
D004220:Disulfides; D007328:Insulin; D013438:Sulfhydryl Compounds; D019791:Guanidine; D003545:Cysteine