Differential behavior of the two free sulfhydryl groups of human plasma fibronectin: Effects of environmental factors

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Abstract
We report here a novel approach to label specifically one of the two cryptic, free sulfhydryl groups per subunit of human plasma fibronectin with either an 15N, 2H‐maleimide spin label or a coumarinylphenyl maleimide fluorescent label. This permits the use of electron spin resonance (ESR) or fluorescence techniques to study molecular dynamics of fibronectin with the label attached to a single site per chain on the protein molecule. The method is based on our observation that upon adsorption of fibronectin to a gelatin‐coated surface, the SH1 site, located between the DNA‐binding and the cell‐binding domains, is partially exposed, while the SH2 site, located within the carboxyl‐terminal fibrin‐binding domain, remains buried and unreactive. The procedures for the preparation of the selectively labeled fibronectins are described in detail. The physicochemical properties of these single‐site labeled fibronectins, particularly as affected by high salt, heparin, surface binding, and temperature, were characterized by ESR spin‐label and steady‐state fluorescence techniques. The steady‐state fluorescence measurement indicates that both local environments of SH1 and SH2 sites are relatively hydrophobic, and that the SH2 site is more hydrophobic than the SH1 site. The ESR results show that heparin or high salt induces an increase in the domainal flexibility in both SH1 and SH2 regions, perhaps through the disruption of domain–domain interactions in the fibronectin molecule, and that the former is more effective than the latter in producing such an effect. The observed heparin effect is reversible by addition of calcium ions in the SH2 regions but not in the SH1 regions. In addition, at temperatures above 44°C, both type III homologous regions containing the free sulfhydryl groups are shown to undergo denaturation and aggregation processes. The data presented here suggest that the newly developed method for differential labeling of the free sulfhydryl groups in fibronectin should be useful for mapping the spatial arrangement of structural domains in the protein molecule using spin‐label–spin‐probe and fluorescence energy transfer techniques.