THE USE OF NI-NITRILOTRIACETIC ACID AGAROSE FOR ESTIMATION OF AFFINITIES OF HEXAHISTIDINE-TAGGED FAB TO SINGLE-STRANDED-DNA

The complex formed between P-32-labeled (dT)(15) and a hexahistidine ( 6-His)-tagged anti-single-stranded DNA (ssDNA) Fab, DNA-1, was trapped by addition of nickel-chelating nitrilotriacetic acid (Ni-NTA) agaros e that led to efficient separation of bound ligand from free. High sta bility of the immobilized complex (half-life of 4 h) and low nonspecif ic binding of [P-32](dT)(15) allowed for a rapid estimation of the dis sociation constant (K-d) and was found to be similar to 130 nM. Oligon ucleotide bound DNA-1 preimmobilized on Ni-NTA agarose with the same K -d as the Fab/(dT)(15) complex formed in solution, indicating that the interaction of the 6-His tag with the resin did not interfere with bi nding, Addition of unlabeled (dT)(15) led to a fast exchange with boun d [P-32](dT)(15). Mutant versions of DNA-1 were also examined and resu lts obtained were in agreement with data from equilibrium gel filtrati on and fluorescence titration [A. A. Komissarov, M. J, Calcutt, M. T. Marchbank, E.N, Peletskaya, and S.L. Deutscher (1996) J. Biol. Chem. 2 71, 12241-12246], These results demonstrate that the Ni-NTA assay is a n efficient and accurate method to examine B-His-tagged protein-nuclei c acid complexes. Furthermore, a competition modification of this assa y may be used for detection of anti-ssDNA antibodies in serum. (C) 199 8 Academic Press.