(131e) Binding Site Analysis and Plasma Protein Inhibition Study for the Recombinant Protein Tp0483 and Human Plasma Fibronectin Using Surface Plasmon Resonance (SPR) | AIChE

(131e) Binding Site Analysis and Plasma Protein Inhibition Study for the Recombinant Protein Tp0483 and Human Plasma Fibronectin Using Surface Plasmon Resonance (SPR)


Dickerson, M. T. - Presenter, University of Kentucky
Abney, M. - Presenter, University of Kentucky
Osborne, K. - Presenter, University of Kentucky
Bachas, L. - Presenter, University of Kentucky
Anderson, K. W. - Presenter, University of Kentucky

When a foreign body enters the bloodstream it elicits a swift immune response predicated on the adsorption of plasma proteins followed by a cascade of events that results in thrombosis. Though it is a critical function of the body's immune system, when blood contacting medical devices are involved it can lead to many undesirable outcomes including heart attack or stroke. To resolve this issue new ways to enhance material hemocompatibility are necessary. One such technique is the use of antigenic disguise proteins found in various microorganisms. Antigenic disguise is a term sometimes used to describe the ability of a microorganism to circumvent the host immune system. The exact method varies widely in each organism. One example is Treponema pallidum, which is believed to bind host plasma fibronectin (FN) and use it to shield potential antigen sites on its surface from attack. This ability is thought to originate in a number of surface proteins, one of which is labeled Tp0483. This research focuses on the nature of the bond between a fragment of Tp0483 and FN and also on its potential application in hemocompatibility enhancement. Tp0483/FN interactions were observed on a COOH terminated self-assembled monolayer (SAM). COOH was chosen because it has been shown to best facilitate binding between the two proteins. First, Tp0483 was added to the COOH surface then the remaining exposed SAM was blocked against nonspecific binding using BSA. The FN binding site was explored by blocking the RGD peptide sequence with an antibody and then examining the effect on binding to Tp0483 using surface plasmon resonance (SPR). Results showed that when anti-RGD was added to FN at either a 20:1 or a 50:1 ratio of anti-RGD to FN, binding of FN to Tp0483 was decreased by greater than 81%. This strongly suggests that RGD is the main binding site for FN on Tp0483. Two likely binding sites on Tp0483 have been identified using software based on hydrophobicity and charge and peptide sequences corresponding to these postulated sites are currently being synthesized to validate their role in binding to FN. Finally, the effect of Tp0483 alone and Tp0483/FN on plasma protein binding was investigated. Fibrinogen (Fg), human serum albumin (HSA), and vitronectin (Vn) were added to a COOH surface containing Tp0483 or Tp0483/FN and the binding compared to an unmodified COOH surface. Inhibition was measured as a percentage based on binding to the unmodified COOH SAM. Significant inhibition of plasma protein binding was seen on both surfaces for all three proteins. Binding of Fg and HSA were both inhibited by approximately 60% on Tp0483 alone and by approximately 80% on a surface containing both Tp0483 and FN. Binding of Vn was inhibited by 32% on the surface with Tp0483 alone and 51% on the surface with both Tp0483 and FN. These results support the use of Tp0483/FN to improve hemocompatibility. The mechanisms for plasma protein inhibition and optimization of this inhibition are currently being investigated.