Disulfide isoform intermediates in the reoxidation of recombinant human basic fibroblast growth factor.
The reoxidation of human recombinant basic fibroblast growth factor was investigated following treatment of the protein with a mixture of reduced and oxidized glutathione, both in the absence and in the presence of protein disulfide isomerase. The oxidative process took place throughout the formation of two transient intermediates and yielded a stable bFGF derivative, GS2-bFGF. All of these components were separated by HPLC and accurately characterized at the molecular level by advanced mass spectrometric procedures. When the reoxidation was carried out in the presence of PDI, a 4-fold increase in the reaction rate was estimated. A mixed disulfide with a single glutathione molecule was shown to occur in the two transient intermediates, each of which has different cysteine residues involved in the linkage. The final product GS2-bFGF was structurally different from other bFGF derivatives previously described [Thompson, S. A. (1992) J. Biol. Chem. 267, 2269-2273; Caccia et al. (1992) Eur. J. Biochem. 204, 649-655]. The four cysteine residues are all involved in disulfide bridges; Cys 34 and Cys 78 are linked to exogenous glutathione, whereas Cys 91 and Cys 101 form an intramolecular S-S bridge.
Servizio di Spettrometria di Massa del CNR, Napoli, Italy.
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Ruoppolo
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Nitti
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Valsasina
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Malorni
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Marino
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Pucci
D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007535:Isomerases / Q000494:pharmacology; D013058:Mass Spectrometry; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment
D004220:Disulfides; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol