Stopped-flow studies on drug-protein binding

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Abstract
Summary We have studied the binding of warfarin to human serum albumin (HSA) with the stopped-flow method. At 37°C the rate constant for the velocity of dissociation of the stable warfarin-HSA complex is 10s−1 (t 50%=0.07 s). Concentration and temperature dependent association constants for warfarin binding to HSA have been measured (2.5·105 M−1 s−1 at 6°C, 9.8·105 M−1 s−1 at 22°C and 15.3·105 M−1 s−1 at 37°C). Our experimentally obtained relaxation constants are best explained by the existence of 5 equivalent low affinity binding sites for warfarin on the HSA molecule, each capable of conversion into a high affinity site. The measured energy of activation for this conversion is 57.5 kJ M−1.