Abstract

Human serum albumin (HSA) has three strong affinity sites for dicoumarol (K = 7.7 x 10⁵ M^-1) as well as an indeterminate number of much weaker sites. The binding of dicoumarol to HSA generates a large negative ellipticity band at 305 mµ in the normal circular dichroic spectrum of the protein. This extrinsic Cotton effect results from the perturbation of π → π^* transitions in the drug molecule by an asymmetrical center at or near the binding site for HSA. These results suggest that dicoumarol takes up a preferred orientation with respect to its HSA-binding site. The dicoumarol-HSA complex must also be rigid enough to preserve such a spatial relationship. In contrast, the binding of warfarin, acenocoumarin, ethyl biscoumacetate, and 4-hydroxycoumarin to HSA does not generate any extrinsic Cotton effects. The unique binding of dicoumarol to HSA would appear to involve the hydrophobic and electrostatic interactions of both coumarin rings.

The binding of either dicoumarol or warfarin to HSA quenches the native tryptophan fluorescence of the protein. The binding of warfarin to HSA is also accompanied by a 7-fold increase in the fluorescent yield of the drug and a shift of its fluorescence emission maximum from 400 mµ to 390 mµ. This suggests that warfarin is bound to a hydrophobic area of the HSA molecule.

Light absorbed by the HSA tryptophan groups and nonradiatively transferred to bound warfarin is re-emitted by the drug as fluorescence. This made it possible to estimate that the mean effective transfer distance between the single HSA tryptophan group and the bound warfarin molecule was 34.5 Å. Similar experiments gave a value of 23.7 Å for the mean effective transfer distance between dansylglycine and the same tryptophan group. While these distances can be considered only approximate, it is obvious that if the HSA molecule has a diameter of 56 Å (calculated assuming au anhydrous sphere of mol wt 69,000), dansylglycine and warfarin must be bound to different sites. This was confirmed by showing that warfarin does not displace dansylglycine from its binding site on HSA.