Purification of nucleotide-linked peptide

J Chromatogr. 1988 Jul 1;444:133-9. doi: 10.1016/s0021-9673(01)94016-6.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Affinity Labels
  • Amino Acids / analysis
  • Carrier Proteins / isolation & purification*
  • Chromatography, DEAE-Cellulose
  • Cyclic AMP Receptor Protein*
  • Escherichia coli / metabolism
  • Hydrolysis
  • Peptides / isolation & purification*
  • Photochemistry
  • Trypsin
  • Ultraviolet Rays

Substances

  • Affinity Labels
  • Amino Acids
  • Carrier Proteins
  • Cyclic AMP Receptor Protein
  • Peptides
  • Trypsin