GSH/GSSG对新型降糖药淀粉酶抑肽折叠过程的影响

Effects of GSH/GSSG on the Folding Process of New Antidiabetic Tendamistat

目的观察还原型谷胱甘肽(GSH)与氧化型谷胱甘肽(GSSG)对治疗2型糖尿病药物淀粉酶抑肽(Tendamistat)小分子蛋白质氨基酸链折叠过程的影响。方法设计四组Tendamistat的复性折叠实验,按照不同浓度及比例加入GSH与GSSG,进行Tendamistat的再氧化复性折叠反应。然后采用非变性聚丙烯酰胺凝胶电泳的方法,分离在复性折叠过程中出现的并且用碘乙酰胺捕捉的折叠中间体及复性产物,显示其在不同实验条件下的变化,观察GSH与GSSG的浓度配比变化对Tendamistat再氧化复性折叠的影响。结果当GSH与GSSG的实验浓度分别为1mmol·L^(-1)和0.1mmol·L^(-1)时,出现规范型折叠中间体和复性产物;保持GSSG的浓度0.1mmol·L^(-1)不变,GSH的浓度在0.1mmol·L^(-1)到10mmol·L^(-1)之间变化,可促进折叠,但若其浓度继续增加,则折叠中间体消失,折叠过程被抑制中断;当GSH的浓度为0.1mmol·L^(-1)不变时,逐渐增加GSSG的浓度,会导致出现不规范及错误折叠中间体;根据以上三组实验结果选定的1mmol·L^(-1)GSH和0.1mmol·L^(-1)GSSG作为复性反应条件,可使变性还原Tendamistat在2h内再氧化重折叠复性。结论变性还原Tendamistat的复性重折叠涉及到其4个半胱氨酸残基经再氧化形成两个二硫键的过程,GSH/GSSG氧化还原电对不可缺少,其正确复性折叠及天然二硫键的重新连接,则取决于GSH/GSSG的浓度及其比例关系,GSH与GSSG对Tendamistat复性折叠的最佳浓度为1mmol·L^(-1)和0.1mmol·L^(-1),比例为10:1。

Objective To investigate the effects of reduced glutathione（GSH） and oxidized glutathione （GSSG） on the amino acid chain folding process of small molecule protein in the drug Tendamistat which was used to treat type 2 diabetes.Methods The four Tendamistat experiments were designed in the renaturation folding tests,and then different concentrations and proportions of GSH and GSSG were added to cause Tendamistat reacting in the reoxidation and renaturation folding.By using the method of native - denaturing polyacrylamide gel electrophoresis,folding intermediates and the renatured products were separated which were caught by iodoacetamide in renaturation folding process to display their changes under different experimental conditions,and to observe the effects of different concentrations of GSH and GSSG ratio changes on the Tendamistat reoxidation and renaturation folding.Results When GSH and GSSG were tested with the concentrations of 1mmol·L-1 and 0.1mmol·L-1,in the electrophoresis diagram standard type folding intermediates and renaturation products appeared,which showed they promoted renaturation folding respectively.When the concentration of GSSG was 0.1mmol·L-1,GSH concentration varied from 0.1mmol·L-1 to 10mmol·L-1, which facilitated folding.However,if concentrations continued to increase,the folding intermediates disappeared and the folding process was suppressed and interrupted.When the concentration of GSH was 0.1mmol·L-1,with the gradual increase in the concentration of GSSG,this produced unstandardized and misfolded intermediates.According to the test results in the above three groups,for the selected 1mmol·L-1 GSH and 0.1mmol·L-1 GSSG as the refolding reaction conditions,the denatured and reduced Tendamistat in 2 hours could cause reoxidation and refolding.Conclusion The denatured and reduced Tendamistat renaturation folding involves its 4 cysteine residues by reoxidation to form disulfide.GSH/GSSG redox electronic pairs are indispensable.The correct refolding and natural disulfide bond reconnections depend on the concentrations of GSH / GSSG and their proportional relationships.The study shows that the optimal concentrations of Tendamistat refolding caused by GSH and GSSG are 1mmol·L-1 and 0.1 mmol·L-1 respectively,and their ratio is 10:1.