ISSN:
1573-4943
Keywords:
Circular dichroism
;
FTIR
;
disulfide exchange
;
G-CSF
;
sulfhydryl titration
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract Oh-edaet al. have shown instability of granulocyte-colony stimulating factor (G-CSF) upon storage abovepH 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 lowpH where the cysteine is protonated, both proteins have much greater stability and that a Cys17 → Ser analog is extremely stable at neutralpH and 37°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 pK a 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 bypH, showed an identical course, indicating comparable conformational stability. Contribution of conformational changes to the observed instability at higherpH is unlikely, since little difference in fluorescence spectrum occurs betweenpH 6.0 and 8.0. Based on these observations, G-CSF, whether glycosylated or not, should not be stored above pH 7.0 in solution. On the other hand, G-CSF is extremely stable in acidic solution as expected from the proposed mechanism.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01025117
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