BINDING OF 2-ACETYLPYRIDINE-5-[(2-CHLOROANILINO)THIOCARBONYL] THIOCARBONOHYDRAZONE (BW348U87) TO HUMAN SERUM-ALBUMIN

Dissociation constants and rate constants for the binding of 2-acetylpyridine-5-[(2-chloroanilino)thiocarbonyl]thiocarbonohydrazone (1, BW348U87), an agent that enhances the antiherpetic efficacy of acyclovir, to human serum albumin have been determined. I quenched the fluorescence of human serum albumin, whereas the absorbance of I al 370 nm increased upon binding to the protein. The absorbance change was attributed to preferential binding of anionic I (pK(a) 7.0). Titration data indicated multiple binding sites for I. The dissociation constant of the high-affinity site was 0.11 muM. The time course for binding of I to 100 nM human serum albumin was biphasic. The early and late phases were described by first-order rate constants that had maximal values of 100 and 11 sec-1, respectively. The rate constant for the dissociation of I from human serum albumin was estimated to be 6 sec-1. Dodecyl sulfate and octanoate displaced I from human serum albumin.I with rate constants of 4.5 and 7.3 sec-1, respectively. Since the fluorescence emission spectrum of human serum albumin and the absorption spectrum of I overlapped significantly (the spectral overlap integral, J, was 2.6 x 10(-14) M-1 cm3), the possibility of Forster dipole-dipole energy transfer was considered. However, a significant fraction of the fluorescence quenching by I resulted from a conformational change in the protein upon binding of I and was not the result of dipole-dipole energy transfer. Nonetheless, the distance between one of the binding regions for I on human serum albumin and a tryptophan residue in the protein was estimated to be 31 angstrom by this method. The high affinity of I for albumin could be related to its low hematological toxicity.