Fluorescence quenching studies of bovine growth hormone in several conformational states.
The use of steady-state fluorescence quenching methods is reported as a probe of the accessibility of the single fluorescent tryptophan residue of bovine growth hormone (bGH, bovine somatotropin, bSt) in four solution-state conformations. Different bGH conformations were prepared by using previous knowledge of the multi-state nature of the equilibrium unfolding pathway for bGH: alterations in denaturant and protein concentration yielded different bGH conformations (native, monomeric intermediate, associated intermediate and unfolded). Because the intramolecular fluorescence quenching which occurs in the native state is reduced when the protein unfolds to any of the other conformations, steady-state fluorescence intensity measurements can be used to monitor bGH unfolding as well as the formation of the associated intermediate. These steady-state intensity changes have been confirmed with fluorescence lifetime measurements for the different conformational states of bGH. Fluorescence quenching results were obtained using the quenchers iodide (ionic), acrylamide (polar) and trichloroethanol (non-polar). Analysis of the results for native-state bGH reveals that the tryptophan environment is slightly non-polar (in agreement with the emission maximum of 335 nm) and the tryptophan is more exposed to acrylamide than most native-state tryptophan residues which have been studied. The tryptophan is most accessible to all quenchers in the unfolded state, because no steric restrictions inhibit quencher interaction with the tryptophan residue. The iodide quenching results indicate that the associated intermediate tryptophan is not accessible to iodide, probably due to negative charges inhibiting iodide penetration. The associated intermediate tryptophan is less accessible to all three quenchers than the monomeric intermediate tryptophan, due to tight packing of molecules in the associated intermediate state.
Havel
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Control Research and Development, Upjohn Company, Kalamazoo, MI 49001.
Kauffman
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Elzinga
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PA
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000465:Algorithms; D000818:Animals; D002417:Cattle; D013006:Growth Hormone; D007454:Iodides / Q000494:pharmacology; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000178:Acrylamides; D007454:Iodides; D020106:Acrylamide; D013006:Growth Hormone