Purification of nucleotide-linked peptide.


Affinity labeling of nucleotide-binding enzymes/proteins with ...
Affinity labeling of nucleotide-binding enzymes/proteins with 32P-labeled nucleotides is a powerful technique to identify nucleotide-binding proteins as well as to radiolabel the specific binding site. We have used this approach for labeling a nucleotide-binding domain in DNA polymerase and have isolated peptides bearing the linked nucleotides. The method used for separating tryptic peptides on hydrophobic matrices with an acetonitrile gradient in 0.1% trifluoroacetic acid as eluent results in loss of radioactivity, presumably through dissociation of the cross-linked nucleotide. This can be averted by the use of a non-acidic medium in the peptide purification protocol. We have devised a relatively simple procedure to concentrate the nucleotide-linked peptides by chromatography on DEAE-Sephadex A25. Most neutral and basic peptides as well as free nucleotides are removed by eluting the DEAE-Sephadex column with 0.2 M ammonium bicarbonate. The nucleotide-linked peptide is then eluted with 0.6 M ammonium bicarbonate. Radioactivity in the collected fractions is conveniently determined by scintillation counting. Labeled peptide in the 0.6 M ammonium bicarbonate eluate can be purified on a C4 reversed-phase column with an acetonitrile gradient in phosphate buffer (pH 6.8). By this procedure, 32P-labeled nucleotide linked with protein/peptide can be quantitatively purified with minimum loss.




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