Affinity Chromatography on Immobilized Fibrin Monomer, IV. Two Fibrin-Binding Peptides of a Chymotryptic Digest of Human Plasma Fibronectin

Abstract

If a chymotryptic digest of fibronectin was passed over fibrin-Sepharose at 4.degree. C, 2 fragments of MW 30,000 and MW 60,000 were retained. The 2 peptides could be separated on gelatin-Sepharose which left the smaller one unaffected but adsorbed the larger one. The peptide of MW 30,000 was rich in cystine and showed an isoelectric point (pI) of 8.2. The same pI and a similar amino-acid composition were found for an earlier described N-terminal fragment of the same size which was available by plasminolysis of a cathepsin D-derived gelatin-binding fragment of MW 70,000. The fibrin-binding fragment of MW 30,000 was correlated to the N-terminal domain of the 2 nearly identical fibronectin subunits. The fibrin- and gelatin-binding fragment of MW 60,000, if cleaved by cathepsin D, yielded a gelatin-binding fragment of MW 40,000 and a fragment of MW 18,000 nearly devoid of cyst(e)ine. The former showed an amino-acid composition related to that of a gelatin-binding fragment of the same size representing the 2nd plasminolysis fragment from the above mentioned N-terminal MW 70,000 peptide. The 2 gelatin-binding fragments separated by electrofocusing into 4 bands in the region of pI 4.8-5.2. Consequently, the gelatin-binding region of the MW 60,000 fragment was correlated to a position adjacent to the N-terminal domain. The basic fragment of MW 18,000 was considered from the specificity of cathepsin D to represent an extension of the gelatin-binding region on its C-terminal side. This was corroborated by an unsuccessful attempt to cleave the MW 60,000 fragment with plasmin, being specific for a site at the N-terminus of the gelatin-binding domain. The affinity for fibrin was lost after cathepsin D cleavage of the MW 60,000 fragment. There was a 2nd, so far unrecognized fibrin affinity site extending from the C-terminal part of the gelatin-binding domain into the subsequent peptide region. Fragments from the C-terminal half of the fibronectin subunits had considerably less affinity to fibrin-Sepharose than the 2 peptides mentioned of MW 30,000 and MW 60,000.