Anti-fluorescein antibodies are excellent model systems for studying the biochemical basis of molecular recognition because a prodigious amount of both physico-chemical and structural information is available for these antibodies. Furthermore, recombinant single-chain antibodies have been produced for several anti-fluorescein antibodies, and site-specific mutagenesis studies have defined the energetic contributions of a number of key active-site residues. In previous studies, we determined the three-dimensional structure of an antigen-binding fragment of a high-affinity anti-fluorescein antibody (4-4-20) in complex with fluorescein. These studies showed that fluorescein binds tightly in an aromatic slot and participates in a network of electrostatic interactions. In this report, we examine the role of electrostatic interactions in the 4-4-20 antigen-combining site by observing the effects of pH on the fluorescence of fluorescein and antigen-binding affinity. These studies showed that the salt link between fluorescein and Arg-L34 in 4-4-20 probably accounts for about -1.5 kcal/mol-1 of the observed free energy of interaction. Furthermore, at pH 10 and higher, the affinity decreases by more than 100-fold (delta delta G degrees approximately equal to 3 kcal mol-1). We attributed this decrease to the ionization of Tyr-L32, which probably disrupts a hydrogen bond between tyrosine's hydroxyl group and fluorescein's phenylcarboxylate group. The fluorescence lifetime of the 4-4-20/fluorescein complex was determined at both pH 8 and pH 10.6. Only one lifetime component (0.38 ns) was observed at pH 8, while two components (0.3 and 3.4 ns) were observed at pH 10.6.(ABSTRACT TRUNCATED AT 250 WORDS)