Sy. Tetin et al., ELUCIDATION OF ANTI-SSDNA AUTOANTIBODY BV-04-01 BINDING INTERACTIONS WITH HOMOOLIGONUCLEOTIDES, Biochemistry, 32(35), 1993, pp. 9011-9017
Binding interactions of various synthetic oligohomonucleotides with an
ti-ssDNA autoantibody BV 04-01 (IgG2b, kappa) and the corresponding si
ngle-chain antibody (SCA) 04-01/212 were studied. Oligonucleotide bind
ing to IgG or SCA resulted in quenching of the protein's tryptophan fl
uorescence permitting direct assessment of ligand binding under equili
brium conditions. The effect of oligothymidylate length, (dT)n, on try
ptophan quenching was evaluated. The equilibrium dissociation constant
s (K(d)) for the binding of (dT)6 and (dT)8 were the same [(1.3 +/- 0.
02) x 10(-7) M], while decreasing the length of the oligothymidylate t
o (dT)3 increased the K(d) an order of magnitude. To assess base speci
ficity, the comparative binding of other hexahomonucleotides was exami
ned. Neither (dA)6 nor (dC)6 showed measurable binding, while the diss
ociation constant for (dG)6 was (7.1 +/- 0.3) x 10(-7) M. Fluorescence
lifetime quenching data correlated with the steady-state binding resu
lts and indicated that the quenching process contains both dynamic and
static components. The ability of BV 04-01 to bind (dT)6 and (dG)6 nu
cleotides was further supported by fluorescence anisotropy studies wit
h fluorescein-labeled hexadeoxynucleotides. Various levels of tryptoph
an fluorescence quenching upon titration with oligothymidylates of dif
ferent length, as well as the similar affinities for (dT)6 and (dG)6,
supported the concept that the groove-type binding pocket in BV 04-01
consists of binding subsites that cooperatively adapt for efficient bi
nding of oligonucleotides.