HEPARIN-BINDING TO MONODISPERSE PLASMA FIBRONECTIN INDUCES AGGREGATION WITHOUT LARGE-SCALE CHANGES IN CONFORMATION IN SOLUTION

Plasma fibronectin was purified by gelatin affinity chromatography in the absence of urea and studied by photon correlation spectroscopy. Po lydispersity in the observed translational diffusion coefficient (D-20 ,D-w) was minimized by subsequent gel permeation fast protein liquid c hromatography (FPLC) on Superose 6, which separated fibronectin monome rs (D-20,D-w = 2.15 +/- 0.03 x 10(-7) cm(2) sec(-1); polydispersity 5. 2%) from aggregates. Addition of heparin to FPLC-purified fibronectin, at physiological pH, ionic strength and temperature, induced fibronec tin aggregation, as shown by an increase of up to 60% in the static li ght-scattering intensity. Additional changes induced by heparin were a n approximate 40% decrease in D-20,D-w and an increase in polydispersi ty to 33%. After removal of aggregates by FPLC, the translational diff usion coefficient for fibronectin monomers was unaffected by the prese nce of heparin, in conditions where fluorescence polarization with flu oresceinamine-labelled heparin showed that 80% of the available hepari n 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-pu rified fibronectin, without subsequent removal of aggregates. The resu lts demonstrate the importance of minimizing polydispersity in the bio physical analysis of fibronectin in solution. We conclude that heparin binding to monomeric fibronectin occurs without large-scale changes i n the conformation of the fibronectin molecule, although the possibili ty of more extended conformations in aggregated forms of fibronectin c annot be excluded.