PH-DEPENDENCE OF THE INTERACTION OF HIRUDIN WITH THROMBIN

The kinetics of the inhibition of human alpha-thrombin by recombinant hirudin have been studied over the pH range from 6 to 10. The association rate constant for hirudin did not vary significantly over this pH range. The dissociation constant of hirudin depended on the ionization state of groups with pK(a) values of about 7.1, 8.4, and 9.2. Optimal binding of hirudin to thrombin occurred when the groups with pK(a) values of 8.4 and 9.0 were protonated and the other group with a pK(a) of 7.1 was deprotonated. The pH kinetics of genetically engineered forms of hirudin were examined in an attempt to assign these pK(a) values to particular groups. By using this approach, it was possible to show that protonation of His51 and ionization of acidic residues in the C-terminal region of hirudin were not responsible for the observed pK(a) values. In contrast, the pK(a) value of 8.4 was not observed when a form of hirudin with an acetylated alpha-amino group was examined, and, thus, this pK(a) value was assigned to the alpha-amino group of hirudin. The requirement for this group to be protonated for optimal binding to thrombin is discussed in terms of the crystal structure of the thrombin-hirudin complex. Examination of this structure allowed the other pK(a)values of 7.1 and 9.2 to be tentatively attributed to His57 and the alpha-amino group of Ile16 of thrombin.