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1 Center For Biophysical Sciences/Engineering, University of Alabama School of Medicine, Birmingham, Alabama 35294-4400, USA
2 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA
3 Department of Biology, Palo Alto College, San Antonio, Texas 78224, USA
4 Department of Biology, The University of Texas at San Antonio, San Antonio, Texas 78249-0609, USA
(RECEIVED August 10, 2004; FINAL REVISION December 1, 2004; ACCEPTED December 3, 2004)
Although a 22-kDa human growth hormone (hGH) is the predicted protein product of the hGH-N gene, a pleiotropic collection of uncharacterized molecular weight and charge isoforms is also produced. Using chromatography and preparative SDS-PAGE under reducing conditions we isolated an unusually stable mercaptoethanol-resistant (MER) 45-kDa hGH. A 5-h incubation at 100°C in the presence of 2-mercapto-ethanol was required to convert approximately 90% of MER-45-kDa hGH into a 22-kDa hGH. Other reductants were not as effective in splitting MER-45-kDa hGH. After fracturing MER-45-kDa hGH, the 22-kDa hGH fragments would spontaneously reassociate if the reductant was removed; however, alkylation of cysteine residues prevented their reassociation. Identical amino acid sequences for the first six N-terminal residues were obtained for MER-45-kDa hGH and its 22-kDa hGH cleavage product. Structural identity of MER-45-kDa hGH and 22-kDa hGH was demonstrated by MALDI-TOF mass spectrometry of tryptic digests. MER-45-kDa hGH did not break up upon incubation with EDTA and EGTA. The significance of this work to our understanding of the structure of hGH isoforms is that it demonstrates that MER-45-kDa hGH is not a single chain polypeptide but is instead a homodimer of 22-kDa hGH monomers. The MER-45-kDa hGH dimer is held together by interchain disulfide bonds and not by divalent metal cation bridges. Additionally, MER-45-kDa hGH’s interchain disulfide links are exceptionally resistant to reducing agents and thus confer extreme stability to the homodimer.
Keywords: human growth hormone; isoform; dimer; mass spectrometry; preparative electrophoresis; purification; protein structure
Abbreviations: SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis • MER-45-kDa hGH, mercaptoethanol-resistant 45-kDa human growth hormone • TEMED, N, N, N', N'-tetramethylethylenediamine • EPPS, N-[2-hydroxyethyl]piperazine-N'-[3-propanesulfonic acid] • CAPS, 3-[cyclohexylamino]-1-propanesulfonic acid • EGTA, ethylene glycolbis(
-aminoethylether)-N, N, N', N'-tetraacetic acid • EDTA, ethylenediaminetetraacetic acid • DTT, dithiothreitol • TCEP-HCl, tris(2-carboxyethyl)phosphine hydrochloride • hGH, human growth hormone • MALDI-TOF/MS, matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041048805.
Reprint requests to: Luis S. Haro, Department of Biology, The University of Texas at San Antonio, 6900 N. Loop 1604 W., San Antonio, TX 78249-0609, USA; e-mail: luis.haro{at}utsa.edu; fax: (210) 458-5658.
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