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Purification of a benzo[a]pyrene binding protein by affinity chromatography and photoaffinity labeling

Academic Article
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Overview

authors

  • Collins, S.
  • Marletta, Michael

publication date

  • July 1986

journal

  • Biochemistry  Journal

abstract

  • Binding proteins for the polycyclic aromatic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) have been purified from C57B1/6J mouse liver. Following affinity chromatography on aminopyrene-Sepharose, a single polypeptide of 29,000 daltons was isolated. The photolabile compound 1-azidopyrene was developed as a photoaffinity labeling agent to identify the protein during its purification. 1-Azidopyrene was found to be a competitive inhibitor of [3H]B[a]P binding. Affinity labeling studies with [3H]-1-azidopyrene in unfractionated cytosol, and in purified preparations, yielded a single covalently labeled protein of 29,000 daltons. The formation of this labeled species was blocked by preincubation with excess unlabeled B[a]P. A native molecular weight of 30,000 was estimated by gel filtration chromatography of [3H]B[a]P- and [3H]-1-azidopyrene-labeled cytosol proteins. An equilibrium dissociation constant of 2.69 +/- 0.66 nM and a maximum number of binding sites of 2.07 +/- 0.10 nmol of [3H]B[a]P bound/mg of protein were estimated for the pure protein. Two-dimensional gel electrophoresis further resolved the purified 29,000-dalton protein into three major isoelectric variants, each of which was specifically labeled by [3H]-1-azidopyrene.

subject areas

  • Affinity Labels
  • Animals
  • Benzo(a)pyrene
  • Binding, Competitive
  • Carrier Proteins
  • Chromatography, Affinity
  • Cytosol
  • Electrophoresis, Polyacrylamide Gel
  • Kinetics
  • Liver
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Pyrenes
  • Tritium
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Identity

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi00363a022

PubMed ID

  • 3756142
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Additional Document Info

start page

  • 4322

end page

  • 4329

volume

  • 25

issue

  • 15

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