Inter-sulfhydryl Distances in Plasma Fibronectin Determined by Fluorescence Energy Transfer: Effect of Environmental Factors

Human plasma fibronectin, a dimeric glycoprotein, contains two cryptic free sulfhydryl groups per chain. Recent observations revealed that upon binding to a gelatin-coated surface the SH1 site, located between the DNA-binding and cell-binding domains, is partially exposed, while the SH2 site, situated within the carboxyl-terminal fibrin-binding domain, remains buried. Utilizing this newly discovered property of plasma fibronectin, we have developed a procedure to introduce maleimide derivatives of fluorescent probes such as N-(1-pyrenyl)maleimide, 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin, or fluorescein 5-maleimide selectively into either the SH1 or SH2 site of the fibronectin molecule and have measured the inter-sulfhydryl distances in fibronectin by fluorescence energy transfer methods. The results show that the distance between the SH1 site of one subunit and the SH1 site of the other subunit is between 35 and 44 A, indicating the close proximity of the two subunits near the SH1-containing regions. On the other hand, the distance between the SH2 site of one subunit and the SH2 site of the other subunit is found to be greater than 95 A, suggesting that the two SH2-containing regions are well separated. Additionally, the distance between the SH1 and SH2 sites within each subunit is estimated to be 42-53 A, assuming no intersubunit energy transfer between the probes. Heparin or high salt, which drastically affects the hydrodynamic properties of fibronectin, had virtually no effect on the distance between the SH1-SH1 or the SH1-SH2 pair.(ABSTRACT TRUNCATED AT 250 WORDS)