Vg. Omelyanenko et al., ROLE OF ELECTROSTATIC INTERACTIONS IN THE BINDING OF FLUORESCEIN BY ANTIFLUORESCEIN ANTIBODY 4-4-20, Biochemistry, 32(39), 1993, pp. 10423-10429
Anti-fluorescein antibodies are excellent model systems for studying t
he biochemical basis of molecular recognition because a prodigious amo
unt of both physico-chemical and structural information is available f
or these antibodies. Furthermore, recombinant single-chain antibodies
have been produced for several anti-fluorescein antibodies, and site-s
pecific mutagenesis studies have defined the energetic contributions o
f a number of key active-site residues. In previous studies, we determ
ined the three-dimensional structure of an antigen-binding fragment of
a high-affinity anti-fluorescein antibody (4-4-20) in complex with fl
uorescein. These studies showed that fluorescein binds tightly in an a
romatic slot and participates in a network of electrostatic interactio
ns. 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 s
tudies 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 (DELTADELTAG-degrees is-approximately-
equal-to kcal mol-1). We attributed this decrease to the ionization of
Tyr-L32, which probably disrupts a hydrogen bond between tyrosine's h
ydroxyl group and fluorescein's phenylcarboxylate group. The fluoresce
nce life time 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. Titration experiments showed that the longer lifetime component was
not due to unbound fluorescein. This led to the hypothesis that at le
ast two conformers exist for the 4-4-20/fluorescein complex at pH 10.6
.