Growth hormone variants

GH can occur in a variety of forms, and there has been much debate as to whether these forms vary significantly in their biological actions [1,95,123,124], 

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G. Teshima and E. Canovadavis, Separation of oxidized human growth hormone variants by reversed-phase HPLC-effect of mobile phase pH and organic modifier, J. Chromatogr., 625 201 (1992). 

Kohler, M., Thomas, A., Puschel,K.,effl/. (2009) Identification of human pituitary growth hormone variants by mass spectrometry. Journal of Proteome Research, 8,1071-1076. 

Fig. 1. Primary stmcmre of the human growth hormone family of proteins. GH-N = pituitary GH GH-V = placental GH variant ...

Lastly, it has been shown that in rare cases trisulfide bridges (Cys-SSS-Cys) can be found as hydrophobic variants of recombinant proteins. The experimental mass of such a trisulfide-bridged peptide would be increased by 32 amu. Andersson et al.125 have described this phenomenon for recombinant human growth hormone during expression in E. coli. However, the receptor binding properties were not affected by this trisulfide modification. 

Andersson C., Edlund P.O., Gellerfors P, Hansson Y., Holmberg E., Hult C., Johansson S., Kordel J., Lundin R., Mendel-Hartvig I., Noren B., Wehler T., Wid-malm G., and Ohman J. (1996), Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli, Int. J. Peptide Protein Res. 47(4), 311-321. 

Abdel-Meguid, S. S., Shieh, H.-S., Smith, W. W., Dayringer, H. E., Violand, B. N. and Bentle, L. A. (1987). Three-dimensional structure of a genetically engineered variant of porcine growth hormone. Proc. Natl. Acad. Sci. USA, 84, 6434-6437. 

Proteolytic secretion variants of recombinant human growth hormone were isolated by anion-exchange HPLC but compared analytically to the reference standard using reversed-phase (C4) HPLC with an ammonium bicarbonate/acetonitrile mobile phase rather than the more commonly used TFA/acetonitrile mobile phase [297]. 

Fig. 7. Diagram showing the pathways by which a precursor of the mRNA for human GH (the product of a single gene) could give rise to mRNAs coding for either the 20K or the 22K variants of the molecule. A, B and C are the three introns. II, III, IV are three exons (coding region) the position of exon I is not shown. The shaded region represents the nucleotide sequence that codes for residues 32 to 46 in the 22K form of growth hormone, but which is included as part of intron B in processing to the mRNA for the 20K form.

The idea of using CIEF to separate bound from free antibody was realized for the first time by Shimura et al. [25], These authors quantified human growth hormone (hGH) using tetramethylrodamine/iodoacetamide-labeled anti-hGH Fab fragment. Excess fluorescendy labeled Fab was added to solutions with variable amounts of hGH and, upon incubation, Fab -Ag complex was separated from the excess Fab by CIEF. LIF detection was used, which provided a very low detection limit of 5 x 10 12 M of methionyl hGH. In this report different forms of Ag were detected simultaneously in one run, since complexes of labeled Fab with non-, mono-, and di-deaminated variants of met-hGH exhibited different isoelectric points and hence could be resolved by CIEF. 

Pal, G., Kossiakoff, A. A., and Sidhu, S. S. (2003). The functional binding epitope of a high affinity variant of human growth hormone mapped by shotgun alaninescanning mutagenesis Insights into the mechanisms responsible for improved affinity./ Mol. Biol. 332(1), 195-204. 

Schiffer, C., Ultsch, M., Walsh, S., Somers, W., De Vos, A. M., and Kossiakoff, A. (2002). Structme of a phage display-derived variant of human growth hormone complexed to two copies of the extracellular domain of its receptor Evidence for strong structural coupling between receptor binding sites./. Mol. Biol. 316(2), 277-289. 

As an illustration of HIC technique, the recombinant human growth hormone (hGH) and methionyl hGH (met-hGH) were well-separated by the HIC technique [14]. The optimized conditions were found to be IM ammonium phosphate dibasic, pH 8.0/propanol (99.5 0.5) and 0.1 M sodium phosphate dibasic, pH 8.0/propanol (97.5 2.5) for mobile phase A and B, respectively, with a descending gradient from 100% A to 100% B in 30 minutes at a column (TSK-phenyl 5PW, 75 x 7.5 mm) temperature of 30°C. Note that the addition of a small amount of propanol as organic modifiers significantly decreases elution time while maintaining resolution and efficiency. This HIC method allowed separation of several hGH variants from the main hGH peak while retaining their native structures. 

The classical parameters of >90% talc adsorption and TCA precipitation, with 3% agreement between the talc and TCA values, and <25% binding to resin are appropriate for polypeptide hormones with a certain chemical composition, including most anterior pituitary hormones. We have found, so far, two exceptions to the above parameters the S variant form of human growth hormone (hGH-S) and human FSH (all preparations). These are acidic proteins and show a decreased adsorption to talc and an increased binding to resin. 

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