AVS 47th International Symposium
    Biomaterial Interfaces Wednesday Sessions
       Session BI-WeP

Paper BI-WeP8
Use of Bacterial Adhesion Related and Collagen Related Peptides to Bind and Orient Fibronectin on Surfaces

Wednesday, October 4, 2000, 11:00 am, Room Exhibit Hall C & D

Session: Poster Session
Presenter: U. Klueh, University of Connecticut, Schools of Medicine and Dentistry
Authors: U. Klueh, University of Connecticut, Schools of Medicine and Dentistry
D.L. Kreutzer, University of Connecticut, Schools of Medicine and Dentistry
J.D. Bryers, University of Connecticut, Schools of Medicine and Dentistry
Correspondent: Click to Email
Although small molecular weight proteins and peptides have been bound and oriented on surfaces, little is known about orienting large molecular weight proteins, (e.g. FN) on surfaces. Recently two classes of peptides have been shown to bind to FN in vitro, i.e. Collagen Related Peptides (CRP) and Bacterial Adhesion Related Peptides (BARP). We hypothesized that if these peptides could be used to not only bind, but also orient FN on surfaces. We further hypothesized that antibodies to specific regions of the FN molecule can be used to demonstrate the orientation of the peptide bound FN. CRP and BARP peptides were synthesized commercially and immobilized on polystyrene surfaces. FN was nonspecifically bound to polystyrene by physisorbtion. FN binding to the immobilized peptides was quantitated using monomeric/functional @super125@I-FN and polyclonal antibodies to FN. Orientation of the bound FN was demonstrated using antibodies specific to the amino (anti-N) and carboxyl (anti-C) termini of FN. Polystyrene immobilized CRP and BARP bound 125 ng/cm@super2@ and 94 ng/cm@super2@ of FN. Little FN bound to control (non-peptide containing) surfaces 5 ng/cm@super2@. FN bound to CRP and BAR peptide bound anti-FN and anti-C antibodies but did not bind significant levels of anti-N antibodies, compared to randomly bound FN (i.e. physisorbed FN). Additionally, we demonstrated that although CRP did inhibit FN binding to immobilized collagen, BRAP did not. Finally we demonstrated that the uses of monomeric/functional FN was critical in establishing FN monolayers on CRP or BARP coated surfaces. Our results not only demonstrate the ability of CRP and BAR peptide to specifically bind and orient fibronectin in monolayers, but also underscore the usefulness of specific polyclonal and monoclonal antibodies to characterize the binding and orientation of FN on these surfaces.