Combination of bovine carbonic anhydrase with a fluorescent sulfonamide.

Bovine erythrocyte carbonic anhydrase forms a highly fluorescent complex with 5-dimethylaminonapbthalene-lsulfonamide (DNSA). The binding, studied either by enhancement of ligand fluorescence or by the quenching of protein ultraviolet fluorescence, shows that only 1 mole of DNSA is bound per mole of protein; the dissociation constant at pH 7.4 is 2.5 X lo-’ M. The fluorescence of free DNSA in water has peak emission at 580 rnp and a quantum yield of only 0.055, but bound DNSA has an emission maximum at 468 rnp and a yield of 0.84. Arguments are presented to explain the large emission blue shift on the basis that the binding site is extremely hydrophobic and that the -S02NH2 group of the ligand loses a proton upon binding to the enzyme. The binding appears specifically to involve the sulfonamide site known to exist in carbonic anhydrase; several other “fluorescent probe” compounds showed no evidence of binding to the enzyme. Calculation of the energy transfer efficiency gave the surprising result that 85% of the photons absorbed by the 7 tryptophan residues are transferred to the single bound DNSA molecule. The transfer efficiency is much higher than hitherto observed for a protein having only one 5dimethylaminonaphthalene-I-sulfonyl group. Although the diameter of the protein is roughly 51 A, the bound DNSA group is probably within the critical transfer distance R. (= 21.3 A) of all the tryptophans. The effective average distance between DNSA and tryptophan was found to be 16 A. The fluorescence properties of the complex were quite different from those of a conjugate prepared by reaction of 5-dimethylaminonaphthalene-1-sulfonyl chloride with carbonic anhydrase. Various considerations lead to the conclusion that the sulfonamide-binding site and the tryptophan residues are in the interior of the protein. The tryptophan fluorescence of the protein was 73% quenched by the binding of 1 DNSA molecule. Although large, this degree of quenching was less than the over-all efficiency of energy transfer of photons absorbed by the protein. This result indicates that the fluorescence effi-