Conformational changes of recombinant human granulocyte-colony stimulating factor induced by pH and guanidine hydrochloride.
Fluorescence and circular dichroism were used to follow the pH-dependent conformational changes of granulocyte colony stimulating factor (G-CSF). Tryptophan fluorescence of the spectra monitored at 344 nm, or after deconvolution of the emission spectra, at 345 nm, showed a decrease in intensity on going from pH 7 to 4, with a midtransition pH of 5.8. On the other hand, tyrosine fluorescence measured either by the ratio of intensity at 308 nm to that at 344 nm, or by the fluorescence intensity at 303 nm after deconvolution of the spectra, increased in intensity as the pH was changed from 6 to 2.5, with a midtransition pH of 4.5. Near UV circular dichroic spectra also showed changes between pH 7.5 and 4.5, which correlated with the transition monitored by the tryptophan fluorescence. The guanidine hydrochloride-induced conformational changes of G-CSF at five pH values from 2.5 to 7.5 were also studied. Circular dichroic and fluorescence spectra revealed minor conformational changes by the addition of 1 or 2 M guanidine HCl at all pH values examined, while the major conformational transition occurred between 2 and 4 M guanidine hydrochloride. The secondary structure of the protein was most stable between pH 3.3 and 4.5. The guanidine HCl-induced denaturation of G-CSF involved more than a two-state transition, with detectable intermediate(s) present, and the structure of the intermediate(s) appeared to depend on the pH used. These results are consistent with the pH dependence of the structure described above, and demonstrate the complex conformational properties of G-CSF.
Narhi
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Amgen Inc., Thousand Oaks, California 91320.
Kenney
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Arakawa
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D002942:Circular Dichroism; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D006146:Guanidines; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D019791:Guanidine