AI Article Synopsis
- This study investigated thiol-disulfide exchange in recombinant human growth hormone (hGH) and model peptides to understand how higher-order structures impact this reaction.
- Different free thiol peptides were used at pH 7.0 and 37°C to examine disulfide exchange, revealing that both cyclic and linear peptides lost native disulfides faster than hGH itself.
- Glutathione showed the highest reactivity in forming mixed disulfides, while longer reactions allowed for the regeneration of native disulfides, particularly influenced by the presence of certain amino acids like Arg next to Cys.
Article Abstract
Purpose: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854756 | PMC |
| http://dx.doi.org/10.1007/s11095-016-1879-3 | DOI Listing |
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