The rapid reaction of human serum albumin with trinitrobenzenesulfonate (I) and the location of the reactive site were investigated to characterize the chemical modification of albumin by I. The modification proceeds through trinitrophenylation of a lysine residue of albumin and monoaddition of the byproduct, sulfite ion, to the trinitrophenylalbumin, as reported previously. The individual kinetic parameters for both reactions were determined at various pH values and 25 degrees. The epsilon-amino group of the lysine residue which has a pKa value of approximately 8.9 was the reactive group involved in the trinitrophenylation. The dissociation constant of the sulfite monoadduct was about 10-fold smaller than that of the monoadduct of the model compound trinitrophenyl alpha-acetyllysine. The modification of albumin by I reduced the fluorescence intensity of the tryptophan-214 residue in the albumin amino acid sequence. Acetylation of the lysine-199 residue with aspirin and 5-nitroaspirin decreased the trinitrophenylation rate of albumin with I. These results on the fluorescence spectroscopy and the effect of the acetylation suggest that the reactive group for I is the lysine-199 residue located near the tryptophan-214 residue.