title: Heparin binding to monodisperse plasma fibronectin induces aggregation without large-scale changes in conformation in solution.
abstract: Plasma fibronectin was purified by gelatin affinity chromatography in the absence of urea and studied by photon correlation spectroscopy. Polydispersity in the observed translational diffusion coefficient (D20,w) was minimized by subsequent gel permeation fast protein liquid chromatography (FPLC) on Superose 6, which separated fibronectin monomers (D20,w = 2.15 +/- 0.03 x 10(-7) cm2 sec-1; polydispersity 5.2%) from aggregates. Addition of heparin to FPLC-purified fibronectin, at physiological pH, ionic strength and temperature, induced fibronectin aggregation, as shown by an increase of up to 60% in the static light-scattering intensity. Additional changes induced by heparin were an approximate 40% decrease in D20,w and an increase in polydispersity to 33%. After removal of aggregates by FPLC, the translational diffusion coefficient for fibronectin monomers was unaffected by the presence of heparin, in conditions where fluorescence polarization with fluoresceinamine-labelled heparin showed that 80% of the available heparin binding sites on fibronectin were occupied. Small differences in the circular dichroism spectrum of gelatin affinity-purified fibronectin were observed before and after removal of aggregates by gel permeation FPLC, and similar changes were seen when heparin was added to FLPC-purified fibronectin, without subsequent removal of aggregates. The results demonstrate the importance of minimizing polydispersity in the biophysical analysis of fibronectin in solution. We conclude that heparin binding to monomeric fibronectin occurs without large-scale changes in the conformation of the fibronectin molecule, although the possibility of more extended conformations in aggregated forms of fibronectin cannot be excluded.
authors.affiliation: Department of Biochemistry, University of Edinburgh, UK.
authors.forename: L
authors.initials: L
authors.lastname: Vuillard
authors.forename: D J
authors.initials: DJ
authors.lastname: Hulmes
authors.forename: I F
authors.initials: IF
authors.lastname: Purdom
authors.forename: A
authors.initials: A
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005353:Fibronectins / Q000097:blood* / Q000737:chemistry; D005454:Fluorescence Polarization; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008027:Light; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D012542:Scattering, Radiation; D012996:Solutions
chemical_list: D005353:Fibronectins; D012996:Solutions; D006493:Heparin