No abstract

Between pH 6 and 9 in the kinetics of the binding of warfarin to human serum albumin a two-step mechanism operates: a diffusion-controlled step, followed by a much slower step during which the stable warfarin-albumin complex is formed. The association rate constant for the formation of the wadarin-albumin complex depends on the transition between neutral and basic forms of the albumin.A pH-dependent conformational change in bovine and human serum albumin around physiological pH is often mentioned in the literature (see Wilting et al. [1,2] for references). There is evidence for the existence of two conformational states, the N form occurring mainly below neutral pH and the B form at higher pH. The conformational change between these two states is therefore called the neutral-to-base or N-B transition. In this paper we describe the effect that this N-B transition has on the kinetics of the binding of warfarin (W) to human serum albumin (P).In a recent paper [3] we reported on the kinetics of the binding of warfarin to human serum albumin at pH 8.7, where the albumin is in the B conformation. We produced evidence that the kinetics of the binding proceed in two steps, i.e., a diffusion-controlied step, followed by the considerably slower step, during which the stable warfarin-albumin complex (WP) is formed. This binding process can be summarized in the following reaction scheme for the reaction between warfarin and albumin; for preparation and pre-treatment of this human serum albumin, see Refs. 3 and 8; the stopped-flow experiments were carried out as described previously [3]; ratio warfarin to albumin (r)=0.01; 25°C; pH 6.2 (O), 7.5 (Q) and 8.7 (D), in phosphate or borate buffer, l=0.1; drawn curves are calculated by assuming a two-step reaction model as described previously [3]: intercept on the ordinate axis corresponds to k_2; plateau level for [P]> 1.5.10 -5 M corresponds to k 2 qk_E; K l can be estimated from the point of intersection of the two straight lines, which can be drawn through the experimental points [3]; the binding constant (Keq) can be calculated by using Keq = K 1 • k 2/k_ 2, as described previously [3].