Raman spectroscopic studies of a dimeric form of recombinant bovine growth hormone.
Lyophilized dimeric recombinant bovine growth hormone (r-bGH) produced through incubation of r-bGH at 37 degrees C and 96% relative humidity for 8 days was examined by Raman spectroscopy. The secondary structure of the dimeric material is comparable to that of nonincubated r-bGH, due to the high similarity of the amide I, III, and V vibrational envelopes of the two samples. The dimeric material exhibits disulfide stretching that is indicative of the presence of only one disulfide bond (Cys53-Cys164). No sulfhydryl S-H stretching vibrations are observed, suggesting that cysteines from the cleaved disulfide bridge (Cys181-Cys189) are bound to nonsulfur atoms. Either high humidity (96%) or mild heat (37 degrees C) alone will cleave only one disulfide bond, but the final products are different. Incubation at ambient temperature and high humidity leads to a significant secondary structural change, while mild heat at very low humidity does not alter r-bGH secondary structure. Spectral data for incubations solely in mild heat are consistent with r-bGH structures that have lost the small loop (Cys181-Cys189) disulfide bridge, while incubations under only high humidity conditions are compatible with what would be expected if the large loop (Cys53-Cys164) cystine link was broken. Mild heat and high humidity are both present in dimer formation, yet only the small loop bridge is severed. The data suggest that heat may be the primary factor in determining which cystine link is broken. More severe heating (75 degrees C) cleaves both cystines and alters both secondary and tertiary structure.
Thamann
T J
TJ
Research and Development, Pharmacia and Upjohn, Inc., Kalamazoo, Michigan, 49001, USA.
D000818:Animals; D002417:Cattle; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D013006:Growth Hormone / Q000737:chemistry*; D017433:Protein Structure, Secondary; D013059:Spectrum Analysis, Raman
D004220:Disulfides; D013006:Growth Hormone; C108583:sometribove