Quantitative evaluation of the contribution of ionic interactions to the formation of the thrombin-hirudin complex

Abstract

The effect of ionic strength on the kinetics of inhibition of human alpha-thrombin has been examined by using genetically engineered forms of hirudin that differed only in the number of negatively charged residues in the carboxyl-terminal region of the molecule. Analysis of the data obtained allowed the binding energy for the thrombin-hirudin complex to be divided into contributions from ionic and nonionic interactions. The contribution of nonionic interactions to the binding energy was the same for each of the forms whereas the ionic contribution varied with the charge of the molecule. Each of the negatively charged residues made an approximately equal contribution of -4kJ mol-1 to the binding energy. For native hirudin, ionic interactions accounted for 32% of the binding energy at an ionic strength of zero.

Changes in interactions in complexes of hirudin derivatives and human α‐thrombin due to different crystal forms

“…A number of mutants have been made in the C-terminal tail of hirudin to investigate its interactions with thrombin (Braun et al, 1988;Stone et al, 1989;Betz et al, 1991a,b;Yue et al, 1992). These are summarized in Table 2.…”

Electrostatic interactions in the association of proteins: An analysis of the thrombin–hirudin complex

“…The binding energy (AGb) for rhir has previously been determined at different ionic strengths and estimates for AG;' were obtained by fitting these data to a modified Debye-Huckel equation (Stone et al, 1989 Stone et al (1989), and experimental estimates of the contribution of electrostatic interactions to binding energy (AGf (exp)) were calculated from data given in the same paper. Estimates of the electrostatic term of binding energy calculated by the MTK and FD methods are represented by AG;' (MTK) and AGf (FD), respectively.…”

“…The C-terminal region of recombinant hirudin (rhir) between residues 55 and 65 contains five negatively charged residues, and results from studies using site-directed mutagenesis indicate that each of these negatively charged residues (Asp 55: Glu 57: Glu 58: Glu 61', and Glu 62') contributes to the stabilization of the complex (Braun et al, 1988a;Betz et al, 1991). In addition, the effect of ionic strength on the interaction between thrombin and hirudin suggests that electrostatic interactions with the C-terminal region of hirudin are important for the rate of complex formation and the stability of the complex (Stone et al, 1989). However, although protein engineering studies indicate that each of the negatively charged residues in the C-terminal region of hirudin makes about the same contribution to binding energy (Braun et al, 1988a;Stone et al, 1989;Betz et al, 1991), 727…”

“…In addition, the effect of ionic strength on the interaction between thrombin and hirudin suggests that electrostatic interactions with the C-terminal region of hirudin are important for the rate of complex formation and the stability of the complex (Stone et al, 1989). However, although protein engineering studies indicate that each of the negatively charged residues in the C-terminal region of hirudin makes about the same contribution to binding energy (Braun et al, 1988a;Stone et al, 1989;Betz et al, 1991), 727 …”

Electrostatic Interactions in the Binding Pathway of a Transient Protein Complex Studied by NMR and Isothermal Titration Calorimetry

“…In typical stopped-flow binding measurements, the protein and ligand are rapidly mixed, and the extent of the reaction is monitored as a function of time using techniques including spectroscopic absorbance (21), fluorescence (10,26,27), enzymatic activity (1,28,29), and surface plasmon resonance (30,31). These techniques are limited by the requirement that the lifetimes of the free and bound states must be severalfold longer than the time required to completely mix the protein and ligand solutions; otherwise, the reaction proceeds nearly to completion before the start of the measurement period.…”