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Thiol-Disulfide Exchange in Human Growth Hormone

Pharmaceutical Research volume 33, pages 1370–1382 (2016)Cite this 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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Scheme 1
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Abbreviations

ACN:

Acetonitrile

Cys:

Cysteine

DTT:

Dithiotreitol

FA:

Formic acid

GSH:

Glutathione (reduced form)

hGH:

Human growth hormone

LC/MS:

Liquid chromatography / mass spectrometry

MWCO:

Molecular weight cut-off

PB:

Phosphate buffer

qTOF:

Quadrupole time-of-flight

TIC:

Total ion chromatogram

XIC:

Extracted ion chromatogram

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors gratefully acknowledge financial support from NIH R01 GM085293. We also thank Dr. Jennifer Laurence (University of Kansas) for providing us with plasmid coding for hGH.

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Authors and Affiliations

  1. Department of Industrial and Physical Pharmacy, Purdue University, 575 Stadium Mall Drive, Room 124D, West Lafayette, Indiana, 47901-2091, USA

    Saradha Chandrasekhar, Balakrishnan S. Moorthy, Ruichao Xie & Elizabeth M. Topp

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  1. Saradha Chandrasekhar
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  2. Balakrishnan S. Moorthy
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  3. Ruichao Xie
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  4. Elizabeth M. Topp
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Corresponding author

Correspondence to Elizabeth M. Topp.

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Chandrasekhar, S., Moorthy, B.S., Xie, R. et al. Thiol-Disulfide Exchange in Human Growth Hormone. Pharm Res 33, 1370–1382 (2016). https://doi.org/10.1007/s11095-016-1879-3

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  • DOI: https://doi.org/10.1007/s11095-016-1879-3

KEY WORDS

  • human growth hormone
  • kinetics
  • peptide
  • protein
  • thiol-disulfide exchange