[Ultraviolet difference spectroscopy study of alpha- and beta gamma-thrombin binding to heparin]

The interaction of alpha- and beta gamma-thrombin with heparin was studied by ultraviolet difference spectroscopy within the wavelength range of 230-300 nm. The absorption difference spectrum of the thrombin-heparin complex was negative and had two maxima at 255 nm (5300 M-1 cm-1) and 282 nm (4700 M-1 cm-1) for alpha-thrombin and at 240 nm (4900 M-1 cm-1) and 282 nm (4100 M-1 cm-1) for beta gamma-thrombin. It is assumed that the conformational changes induced by heparin in the enzyme molecule involve the transfer of some tryptophan and tyrosine residues from the interior of the protein to the surface. The absorption changes during alpha-thrombin--heparin interaction at physiological ionic strength suggest binding of some alpha-thrombin molecules to a heparin molecule at the ligand-enzyme molar ratio lower than 1. Under the same conditions beta gamma-thrombin forms an equimolar complex with heparin with the dissociation constant equal to 7,0.10(-9) M. The ionic strength increase up to 0,217 M NaCl results in some disturbances in beta gamma-thrombin-heparin interaction and prevents the binding of additional alpha-thrombin molecules to an equimolar complex of alpha-thrombin with heparin. Therefore the kinetics of the two enzyme forms interaction with heparin are similar, the alpha-thrombin affinity for heparin being a little higher. The data obtained suggest that alpha-thrombin binding to heparin is essential for biological inactivation of thrombin.