FT-IR spectroscopy of interacting biomolecules: Antithrombin/high-affinity heparin.

Abstract

The thromboresistance of some heparin-coated surfaces may be mediated by a heterogeneous catalytic mechanism similar to that by which high-affinity heparin (HAH) catalyzes the inhibition of coagulation cascade proteases in homogeneous solution. If so, then the physiological serine protease inhibitor, antithrombin III (AT), must bind to the immobilized heparin molecules at specific catalytically active high-affinity sites. Indirect spectroscopic and kinetic evidence of such binding was sought in this study. A multifaceted approach--involving short path length Fourier transform infrared (FT-IR) absorbance, FT-IR attenuated total reflection (FT-IR/ATR), fluorescence enhancement, and protease activity assays--was employed. Quantitative analysis of multicomponent FT-IR spectra was facilitated by: (a) designing and applying a long-pass spectral filter to reduce broad-b and noise from the spectral data; (b) eliminating Fresnel interference artifacts from absorbance difference spectra; and (c) applying a new quantitative analysis algorithm--designed for multicomponent systems having r and om error in all path length, concentration, and absorbance measurements--to the AT/HAH system. A number of test systems were analyzed to demonstrate the efficacy of each of these analytical tools. The first FT-IR indication of hydrogen bonding between HAH and AT in aqueous solution was obtained here by correlating fluorescence enhancement data with FT-IR absorbance difference spectra. FT-IR/ATR spectroscopy of systems having HAH ionically bound to polyvinyl chloride impregnated with tridodecylmethyl ammonium chloride (PVC/TDMAC) yielded information about the stability, total concentration, and AT-binding activity of the immobilized HAH. These results, in conjunction with measurements of apparent AT activity in the presence of PVC/TDMAC/HAH, indicate that this surface catalyzes the inhibition of thrombin (the final protease of the intrinsic coagulation cascade) by AT. Spectral perturbations associated with the binding of AT to PVC/TDMAC/HAH suggest that AT is oriented on the surface and that its secondary structure is altered by this binding.