Title: An Infrared Spectroscopy Technique for Measuring Relative
Changes in Conformation of Proteins Adsorbed to Biomaterials Surfaces

 

Presenter:  Jack R. Smith

NIST Polymers Division (854)

Materials Science and Engineering Laboratory
100 Bureau Drive

Room A121, Bldg. 224

MailStop 5843

+1.301.975.6903 (phone)
+1.301.975.4977 (fax)

email:  jrsmith@nist.gov

Presenter is not a Sigma Xi member

Mentors:  Marcus T. Cicerone (Polymers Division), Curtis Meuse (Biotechnology Division)

Category: Biotechnology

The interaction of cells with biomedical implant materials is mediated by adsorbed proteins. Therefore, engineering cellular response to biomaterials via manipulation of surface chemistry, roughness, or patterning requires understanding how protein adsorption changes with surface properties. An infrared (IR) spectroscopy assay to measure the relative conformational changes of surface-adsorbed proteins has been developed and applied to synthetic, polymeric biomaterials. The extent of protein

denaturation upon adsorption has been quantified via hydrogen-deuterium exchange (HDX) by obtaining the equilibrium fractionation number (f) as the ratio of the integrated areas of the amide II (N-H in plane bend) and amide I (amide C=O stretch) bands in the IR spectrum. This number can be related directly to the free energy of unfolding of the protein, and is inversely related to its structural stability. In the experiment, the polymer

is spin-coated onto an Au surface and then exposed to 50 % deuterated (i.e., 50 - 50 % D2O-H2O), protein-containing 0.1 M NaPO4 buffer solution. Subsequently, the buffer is removed and an IR beam is reflected from the polymer/protein-coated Au substrate. The experiment is performed in reflection in order to maximize the signal from the monolayer of adsorbed protein (via reflection-absorbtion from protein layer) and to provide an experimental configuration that is amenable to exploring a wide range of proteins, polymers and surface structures. Preliminary results have been obtained for three proteins [Bovine Serum Albumin (BSA), Porcine Trypsin (PT) and Human Fibronectin (HFn)] interacting with two different surfaces, Au control and a tyrosine-derived polycarbonate[poly(DTO carbonate)]. In the case of BSA, adsorbtion to the surfaces of Au and poly(DTO carbonate) was found to cause a decrease in f (from the solution value) by 52 % and 71 %, respectively. This indicates substantial denaturation of the protein upon adsorption to either surface. The equilibrium fractionation number for PT, on the other hand, was found to increase by 23 % upon adsorption to Au but to decrease by 23 % upon adsorption to poly(DTO carbonate). This indicates a decrease in the structural stability of trypsin upon adsorption to the hydrophobic polymer, but possibly not upon adsorption to relatively hydrophilic Au. In the case of HFn, f was found to increase by 74 % and to decrease by 55 % upon adsorption to Au and poly(DTO carbonate), respectively. Again, this indicates a decrease in structural stability of the protein upon adsorption to the hydrophobic polymer, but possibly not upon adsorption to Au. The effect of surface roughness on conformation will also be explored. It seems plausible that these kinds of large, qualitative structural changes in proteins upon adsorption to surfaces influences cellular response to them. This will be discussed in some detail.