Thiol-Disulfide Exchange in Human Growth Hormone.
Thiol-Disulfide Exchange in Human Growth Hormone.
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DOI：
10.1007/s11095-016-1879-3
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发表时间：
2016-06
影响因子：
3.7
通讯作者：
Topp EM
中科院分区：
文献类型：
--
作者：
Chandrasekhar S;Moorthy BS;Xie R;Topp EM
在重组人生长激素(HGH)及其衍生的模型胰蛋白酶多肽中监测了硫醇-二硫键交换，以考察高阶结构对反应的影响。在hGH中，不同长度和不同氨基酸序列的游离硫醇多肽在pH 7.0和37°C的条件下引发反应。用胰酶消化蛋白质样品，用LC/MS分析天然二硫键、杂乱二硫键和游离硫醇，并将天然二硫键和二硫键交换的损失与来自hGH的模型肽进行比较。在反应的前60min内，由hGH二硫键C端衍生的环状(cT20-T21)和线状多肽(T20-T21pep)中天然二硫键的损失大于hGH本身C端二硫键的损失。在被测试的硫醇中，谷胱甘肽(GSH)活性最强，在完整的hGH和模型多肽中形成混合二硫的比例最高。在较长的反应时间(>；240min)下，hGH和cT20-T21中的天然二硫键都得到了再生。半胱氨酸和与半胱氨酸相邻的含有Arg残基的二或三肽的再生速度最快，这表明它们在复性中可能是有用的。HGH和相关模型肽中的硫醇-二硫键交换反应受更高级结构、硫醇反应物的大小和硫醇反应物中与半胱氨酸相邻的Arg残基的影响。CD和HDX-MS测定表明，hGH中二硫键的还原不影响高阶结构。
Thiol-disulfide exchange was monitored in recombinant human growth hormone (hGH) and in model tryptic peptides derived from hGH to investigate the effects of higher-order structure on the reaction. Different free thiol-containing peptides, varying in length and amino acid sequence, were used to initiate the reaction at pH 7.0 and 37 °C in hGH. Protein samples were digested with trypsin and analyzed for native disulfides, scrambled disulfides and free thiols using LC/MS. The loss of native disulfide and disulfide exchange was compared with model peptides derived from hGH. Loss of native disulfide in cyclic (cT20-T21) and linear peptides (T20-T21pep) derived from the C-terminal hGH disulfide during the first 60 min of reaction was greater than loss of the C-terminal disulfide in hGH itself. Of the thiols tested, glutathione (GSH) was the most reactive, forming the highest percentage of mixed disulfides in intact hGH and in the model peptides. At longer reaction times (>240 min), native disulfides in both hGH and cT20-T21 were regenerated. The fastest rates of regeneration were observed for Cys and the di- or tripeptide containing an Arg residue adjacent to Cys, suggesting that they may be useful in refolding. Thiol-disulfide exchange reactions in hGH and related model peptides were influenced by higher order structure, by the size of the thiol reactant and by an Arg residue adjacent to Cys in the thiol reactant. Reduction of disulfide bonds in hGH did not affect higher order structure as measured by CD and HDX-MS.