Exposure to solvent of tyrosyl and tryptophanyl residues of bovine antithrombin in the absence and presence of high-affinity and low-affinity heparin.

The exposure to solvent of the aromatic amino acid residues of free bovine antithrombin was probed by three different methods, which gave comparable results. Solvent perturbation suggested that about 35% of the tyrosyl residues and about 40% of the tryptophanyl residues of the protein are exposed to solvent. Fluorescence quenching experiments indicated that 60% of the tryptophanyl fluorescence of antithrombin arose from exposed residues. These values are similar to those previously reported for the human protein. In spectrophotometric titrations, two out of a total of ten tyrosyl residues titrated with a normal pKa and therefore, presumably are accessible to solvent. The binding of low-affinity of high-affinity heparin to antithrombin had neglibible effects on the solvent perturbation spectra and on the spectrophotometric titration curves. Iodide quenching experiments indicated an altered quenching pattern of the fluorescence of the protein in the presence of either form of heparin, but this effect could not be interpreted as a change of the exposure of trytophanyl residues. The binding of heparin to antithrombin therefore apparently leads to, at most, minimal changes of the exposure of the aromatic amino acids of the protein to the surrounding solvent.