A new variant of human growth hormone was recently found [Pavlu, B. & Gellerfors, P. (1993) Bioseparation 3, 257-2651. We report here the identification and the structural determination of this variant. The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography. The rhGH hydrophobic variant (rhGH-HV) was isolated and subjected to trypsin digestion and RP-HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH. This tryptic peptide constitutes the C-terminus (aa 179-191) of hGH and contains one of the two disulfide bridges in hGH, viz.Amino acid sequences and composition analyses of the tryptic peptide from rhGH-HV (T&+ 19) and the corresponding tryptic peptide from rhGH (TI, + 19) were identical. Electrospray mass spectrometry (ES/MS) of TY8+ 19 isolated from rhGH-HV revealed a monoisotopic mass increase of 32.7, as compared to T18+19 from rhGH. A synthetic T18+19 peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragments in ES/MS compared to Tr8+19 isolated from rhGH-HV, i.e. m/z 617.7 and 682.9. These fragments are formed through a unique cleavage in the trisulfide (Cysl82-SSS-Cys189) bridge not found in the corresponding T18+ 19 disulfide peptide. Furthermore, the synthetic TY8+19 co-eluted in RP-HPLC with TI8 + 19 isolated from rhGH-HV. Two-dimensional NMR spectroscopy of the synthetic and T78+1g peptides were performed. Using these data all protons were assigned. The major chemical shift changes (A6>0.05 ppm) observed were for the P-protons of Cys182 and Cys189 in T;8+1g as compared to T18+19. CD spectroscopy data were also in agreement with the above results. Based on these physico-chemical data, rhGH-HV has been structurally defined as a trisulfide variant of rhGH.The receptor binding properties of rhGH-HV was studied by a biosensor device, BIAcoreTM. The binding capacity of rhGH-HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1 : 1.6 and 1 : 1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties. 0 Munksgaard 1996.Abbreviations: GHD, growth hormone deficiency; hGH, human growth hormone; rhGH, recombinant human growth hormone; rhGH-HV, recombinant human growth hormone hydrophobic variant; rhGHBP, the recombinant extracellular domain of the hGH receptor otherwise called the rhGH binding protein; RAMFc, immunosorbent-purified rabbit anti-mouse IgGFc; TIs+ tryptic peptide T,,,,, from rhGH having no peptide bond between Arg183 and Ser184: T;,,,,, tryptic peptide from rhGH-HV having no peptide bond between Argl83 and Ser184; hGH[ 179-1911, C-terminal tridecapeptide from rhGH; hGH-HV[ 179-1911, C-terminal tridecapeptide from rhGH-HV; RP-HPLC, reversed-phase high performance liquid chromatography; HIC, hydrophobic interaction chromatography; EDTA, ethylenediaminetetraaceti...

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