Cysteine 17 of recombinant human granulocyte-colony stimulating factor is partially solvent-exposed.
Oh-eda et al. have shown instability of granulocyte-colony stimulating factor (G-CSF) upon storage above pH 7.0 [J. Biol. Chem. (1990) 265, 11,432-11,435]. To clarify the mechanism of this instability, the accessibility of a free cysteinyl residue at position 17 for disulfide exchange reaction was examined using a sulfhydryl reagent. The results show that the cysteine is partially solvent-exposed in both glycosylated and nonglycosylated forms, suggesting that the exposure of the cysteine plays a critical role in the instability of the protein. This is supported by the facts that at low pH where the cysteine is protonated, both proteins have much greater stability and that a Cys17-->Ser analog is extremely stable at neutral pH and 37 degrees C. It was observed that the rate of sulfhydryl titration is slower for the glycosylated form than for the nonglycosylated form, suggesting that the cysteine residue is less solvent-exposed for the former protein or that the pKa is somewhat more basic. In either case, the carbohydrate appears to affect the reactivity of the sulfhydryl group through steric hindrance or alteration in local conformation. Both the glycosylated and nonglycosylated proteins showed essentially identical conformation as determined by circular dichroism, fluorescence, and infrared spectroscopy. Unfolding of these two proteins, induced either by guanidine hydrochloride or by pH, showed an identical course, indicating comparable conformational stability. Contribution of conformational changes to the observed instability at higher pH is unlikely, since little difference in fluorescence spectrum occurs between pH 6.0 and 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)
Arakawa
T
T
Amgen, Inc., Amgen Center, Thosand Oaks, California 91320.
Prestrelski
S J
SJ
Narhi
L O
LO
Boone
T C
TC
Kenney
W C
WC
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003545:Cysteine; D004220:Disulfides; D004926:Escherichia coli; D006031:Glycosylation; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012694:Serine; D012997:Solvents; D013055:Spectrophotometry, Infrared; D014162:Transfection
D004220:Disulfides; D006146:Guanidines; D011994:Recombinant Proteins; D012997:Solvents; D016179:Granulocyte Colony-Stimulating Factor; D012694:Serine; D019791:Guanidine; D003545:Cysteine