Regular fragmentation of hydrogen peroxide-treated fibronectin

• Published in: The Journal of biological chemistry Vol. 264; no. 8; pp. 4471 - 4475
• Main Author: Vartio, T
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15.03.1989; American Society for Biochemistry and Molecular Biology
• Subjects: Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Deferoxamine - pharmacology; Electrophoresis, Polyacrylamide Gel; Fibronectins - metabolism; Free Radicals; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen Peroxide - pharmacology; Hydroxides - metabolism; Hydroxyl Radical; Immunoblotting; Macromolecular Substances; man; Miscellaneous; Molecular Weight; Pentetic Acid - pharmacology; Peptide Fragments - metabolism; Polystyrenes - pharmacology; Polyvinyls - pharmacology; Proteins; Thiourea - pharmacology; Degradation; Human; Cell culture; Peroxides Hydrogen; Immunoblotting assay; Cleavage; Respiratory burst; Fibroblast; Fibronectin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)83767-X

In the presence of low concentrations (< 0.5 mM) of hydrogen peroxide Mr 350,000 and 170,000 fragments were generated from plasma and fibroblast medium fibronectins (Fns). No other major fragments were detected when H2O2 concentration was raised or the incubation time prolonged. A 200–300-fold concentration of H2O2 was needed for a complete degradation of the protein. The degradation was inhibited or completely prevented by deferoxamine, diethylene-triaminepentaacetic acid, and thiourea or by Chelex-pretreatment of the Fn solution suggesting a Fenton-type reaction to produce ·OH radicals from H2O2. In immunoblotting the Mr 170,000 fragment reacted with monoclonal antibodies against the NH2 terminus and mid-molecule but not with those against the cell-binding site and the COOH terminus of Fn. Reduction of the Mr 350,000 fragment produced α- and β-monomers of Fn as well as Mr 95,000 and 85,000 fragments which reacted with monoclonal antibodies against the cell-binding site and the COOH terminus of Fn. These results suggest that the Mr 170,000 fragment is derived from the NH2-terminal part of both subunits of Fn. The rest of the subunits, the Mr 95,000 (from α-chain) and Mr 85,000 (from β-chain), thus remain disulfide-bonded to an intact Fn subunit to form the nonreduced Mr 350,000 polypeptide. The results show that oxygen radical action may generate defined and reproducible fragments from Fn. The high susceptibility of Fn to the radical induced degradation makes it plausible to occur also in vivo.