2 - Direct Demonstration of Association of the Blood-Brain Barrier Proteins ZO-1 and Occludin Using Surface Plasmon Resonance Spectroscopy; Effect of SIN-1

Abstract

This chapter explores the surface plasmon resonance (SPR) spectroscopy approach for investigating the interactions of tight junction proteins directly and continuously for pharmacological studies. The application of SPR is a simple and direct method to determinate which tight junctions (TJ) proteins really associate and which of their domains are involved. Compared to coprecipitation studies and chromatographic binding procedures, the advantage of SPR is that the concentration dependence, association, and dissociation can be easily analyzed. Also, the immobilized occludin can be regenerated for different binding measurements over consecutive weeks. This shows that a complete series of binding experiments can be performed with the same chip under identical conditions. In addition to yielding qualitative comparisons, it allows the calculation of reaction kinetics and binding constants. These advantages make the usage of biomolecular interaction analysis to study the molecular composition of tight junctions a very effective approach. The chapter also argues that the interaction between the guanylate kinase-like (GuK) domain of zona occludens protein 1 (ZO-1) and occludin is strongly influenced by 3-morpholinosydnonimine (SIN-1).