Immunological identification of two sulfhydryl-containing fragments of human plasma fibronectin

We report studies of the free sulfhydryl groups of human plasma fibronectin and the use of mouse monoclonal antibodies in determining the localization of free sulfhydryl groups in fibronectin fragments. Immunoaffinity chromatography and a competitive inhibition assay established that the antigenic determinant recognized by monoclonal antibody 2-8 is located in a 31-kilodalton early tryptic fragment of fibronectin, whereas the antigic determinant recognized by monoclonal antibody 180-8 is located in an 80-kilodalton late tryptic fragment. Gelatin-agarose affinity chromatography of early and late tryptic digests localized the 31- and 80-kilodalton fragments in the carboxyl-terminal one-half of the fibronectin subunit. Titration of fibronectin in 3 M guanidine or 6.3 M urea with 5,5'-dithiobis(2-nitrobenzoic acid) or 2,2'-dipyridyl disulfide indicated that there are 1.3 to 1.6 free sulfhydryl groups/200-kilodalton subunit. Titration of 31- and 80-kilodalton fragments suggested the presence of 0.7 and 1.0 sulfhydryl groups/fragment, respectively. The sulfhydryl groups were not accessible to titration in the absence of denaturant. Both the 31- and 80-kilodalton tryptic fragments bound to thiol-agarose under denaturing conditions. Trypsin digestion of iodo[2-³H] acetic-labeled fibronectin followed by immunoaffinity chromatography confirmed the presence of free sulfhydryl in the 80-kilodalton fragment. Trypsin digestion of S[³H]CM-fibronectin produced traces of radiolabeled 31-kilodalton fragment, a nonlabeled 23-kilodalton fragment recognized by monoclonal antibody 2-8, and radiolabeled trichloroacetic acid-soluble material of <10 kilodaltons. This finding suggests that alkylation of the free sulfhydryl changes the tryptic sensitivity of the 31-kilodalton region of fibronectin. Thus, the experiments define two sulfhydryl-containing regions of fibronectin.