A stopped-flow kinetic study on the binding of phenylbutazone to human serum albumin using absorption and circular dichroism techniques.

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The mechanism of the binding of phenylbutazone(4-butyl-1,2-diphenyl-3,5-pyrazolidinedione) to human serum albumin has been investigated by kinetic experiments using both optical absorption and circular dichroism (CD) stopped-flow methods. Three distinct processes were found to exist by the absorption technique; one of these was within the dead time of the apparatus and other two slow processes were at around 10<SUP>−2</SUP> and 1 s, of which the relaxation times were determined. On the other hand, only two processes, which correspond in time range to the first and third processes seen by optical absorption, were found using CD technique. By examining the concentration dependence of the reciprocal relaxation time of each process and by taking account of the binding isotherm, a binding mechanism has been proposed. According to the elucidated reaction scheme, successive binding of two molecules of phenylbutazone is followed by a conformational change which destabilizes one of the bound species. The detailed nature is discussed in the light of the circular dichroism change of each process.
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