Paper BI-TuP16
Correlation of Changes in Protein Bioactivity Post-Adsorption to Adsorbed Orientation and Conformation

Tuesday, October 16, 2007, 6:00 pm, Room 4C

It has long been known that proteins rapidly and irreversibly adsorb onto biomaterial surfaces upon contact with bodily fluids. The structure and bioactivity of the adsorbed protein layer are recognized to be critical factors that influence subsequent cellular responses; however, the molecular mechanisms involved are mostly unknown. The bioactivity of an adsorbed protein could be inhibited due to adsorption-induced conformational changes, orientation effects causing the active site to be sterically blocked, or a combination of both. We have developed experimental methods to measure the bioactivity of an adsorbed protein layer and probe protein orientation and/or adsorption-induced conformational changes. Alkanethiol self-assembled monolayers (SAMs), with different surface chemistries, were used as model surfaces and standard spectrophotometric bioactivity assays were used to measure the percent of protein bioactivity retained post-adsorption. The secondary structure of the adsorbed protein layers was determined using circular dichroism (CD) and compared to the native structure of the proteins. Solvent accessible tryptophan residues were successfully modified using 2-hydroxy-5-nitrobenzyl bromide (Koshland’s Reagent) and quantified via spectrophotoscopy. The specific locations of the modified residues are being determined by mass spectrometry to further assess the adsorbed orientation and tertiary structure of the proteins for correlation with changes in their bioactivity.