Proteolytically derived fragments of human plasma fibronectin and their localization within the intact molecule.

Abstract

Plasma fibronectin, which is also known as cold-insoluble globulin, consists of two polypeptide chains of approximately 250,000 daltons joined by disulfide bonds located near one end of the molecule.Plasma fibronectin was isolated b y gelatin-Sepharose affinity chromatography and ion exchange chromatography on DEAE-cellulose.Purified plasma fibronectin was cleaved with human a-thrombin, and three major fragments were recovered: two large ones with molecular weights of approximately 230,000 and 235,000, and a small one with a molecular weight of 29,000.The fragments were separated on a gelatin-Sepharose affinity column.The smaller piece was not retained, while the larger fragments and the remaining undigested molecules were bound and could be eluted with 2 M guanidine HC1.NHz-terminal analysis was performed on intact plasma fibronectin and on the fragments using the dansyl (5-dimethylaminonaphthalene-1-sulfonyl) chloride technique.No NHz-terminal residue was detected in either the intact molecule or the small fragment, whereas analysis of the large fragments yielded dansylalanine.Treatment of the intact molecule and the small fragment with pyroglutamyl peptide hydrolase resulted in the appearance of NHz-terminal alanine.These results were confirmed by automated amino acid sequence analysis; furthermore, the intact molecule and the small fragment were shown to contain identical NHz-terminal sequences.The data indicate that the smaller proteolytically derived fragment is located at the NH2 terminus of plasma fibronectin, while the large fragments constitute the carboxyl portion of the molecule.Plasma fibronectin is a noncoagulable component of vertebrate plasma.Highly purified preparations of plasma fibronectin were first obtained from human plasma by Mosesson and Umfleet (1).This molecule was found to be a glycoprotein composed of two disulfide-linked polypeptide chains with molecular weights of approximately 220,000 (2).Results of proteolytic digestion of plasma fibronectin indicate that all of the interchain disulfide bonds are located close to one end of the molecule (3, 4).The NH, termini of this protein are *