Association of Near-Infrared Dyes with Bovine Serum Albumin

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Abstract
The binding of five near-infrared (near-IR) dyes to bovine serum albumin (BSA) has been evaluated by absorption, fluorescence, and circular dichroism. These dyes have a common squarilic acid polymethine backbone but differ in the substituents on two sites, R1 and R2, symmetrically located on the chromophore. The magnitude of the association constants for the dye–albumin complexes depended on the nature of the side chains and ranged from 34,000 to 1 × 107 M−1. Listed by the kind of side chain and in increasing binding order, the association constants are: [R1 = R2 = butyl phthalimide] < [R1 = R2 = cetyl] < [R1 = R2 = ethyl] < [R1 = butyl phthalimide, R2 = butyl sulfonate] < [R1 = R2 = butyl sulfonate]. The chromophore seems to interact mainly with a hydrophobic cavity on BSA. However, the association constants increase substantially when the side chain or chains are butyl sulfonate. This observation suggests that near the hydrophobic region there is at least one positively charged site with which the butyl sulfonate chain or chains interact. The molar ratio of the dye–albumin complex determined was 1:1. The optical constants of the dye–albumin complexes cannot be determined directly; therefore, a method is described for determining these complexes' optical parameters and thus their association constants.