Stopped-flow studies on drug-protein binding

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Abstract
Binding curves obtained by the stopped-flow method for the association of warfarin and human serum albumin (HSA) at pH 6.0 and 9.0 have been analysed with digital- and analog computers. The first association product (WHSA') at pH 6–9 does not contribute to the observed fluorescence enhancement during warfarin-HSA complex formation. A similar consecutive relaxation process leads to a more stable warfarin-HSA complex, with HSA in the neutral (N)-form (pH 6) and base (B)-form (pH 9). This rearrangement can be measured by stopped-flow (k2=31 s−1 at pH 6; k′2=63 s−1 at pH 9). At pH 6 a further concentration dependent relaxation process has been observed indicating that the complex of warfarin with the N-form of HSA gets partially converted into its B-form with a half-time for this N→B transition in the range of 0.2–0.4 s. A drug such as warfarin can act as effector molecule for conformational changes of the HSA tertiary and quaternary structure during the formation of a high affinity complex.