Circular dichroism spectroscopy of heparin-antithrombin interactions

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Abstract
Circular dichroism spectroscopy was used to examine the interaction of [human and bovine] antithrombin with heparin-derived oligosaccharides and mucopolysaccharides of various sizes. The various complexes exhibited 2 major types of chiral absorption spectra. The 1st of these patterns is seen when octasaccharide, decasaccharide, dodecasaccharide or tetradecasaccharide fragments bind to the protease inhibitor. The circular dichroism spectra of these complexes, when compared to the spectrum of free antithrombin, show several distinguishing characteristics. There is a marked general increase in positive chiral absorption that is maximal at 296 and 288 nm and 290 and 282.5 nm. These observations indicate perturbation of buried and exposed tryptophan residues. A significant augmentation in circular dichroism that peaks at 269.5 and 263 nm is noted. These findings are probably due to the summed positive and negative contributions arising from tryptophan residue(s), disulfide bridge(s) and phenylalanine residue(s). Given that these heparin fragments are able to accelerate factor Xa-antithrombin interactions but not thrombin-antithrombin interactions, these spectral transitions must be associated with either the binding of a critical domain of the oligosaccharides to the protease inhibitor or the activation of the protease inhibitor with respect to factor Xa neutralization. The 2nd of these patterns is apparent when octadecasaccharide, low MW heparin (6500), and high MW heparin (22,000) interact with antithrombin. The circular dichroism spectra of these complexes, compared to the spectrum of free protease inhibitor, are similar to the 1st pattern except for changes within the 292- to 282-nm and 275- to 255-nm regions. The subtraction of the 1st pattern from the 2nd pattern reveals a shallow negative band between 300 and 275 nm with potential negative minima at 290 and 283 nm and a deep negative band between 275 and 255 nm with possible negative minima at 268 and 262 nm. This chiral absorption profile is most likely to arise from conformational changes of a disulfide bridge(s). This circular dichroism difference curve might be explained on the basis of transitions originating from a tryptophan residue(s). Given this method for generating the data, these spectral alterations must be associated with the binding of a 2nd critical domain of the mucopolysaccharide to antithrombin that is required for rapid complex formation with thrombin or the activation of the protease inhibitor with respect to the neutralization of the latter enzyme.