Purification of nucleotide-linked peptide.
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.
Pandey
V N
VN
Department of Biochemistry, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark 07203-2757.
Modak
M J
MJ
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification*; D002848:Chromatography, DEAE-Cellulose; D002373:Cyclic AMP Receptor Protein; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D010455:Peptides / Q000302:isolation & purification*; D010777:Photochemistry; D014357:Trypsin; D014466:Ultraviolet Rays
D000345:Affinity Labels; D000596:Amino Acids; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D010455:Peptides; D014357:Trypsin