An acetyltransferase assay for CREB-binding protein based on reverse phase-ultra-fast liquid chromatography of fluorescent histone H3 peptides

Anal Biochem. 2015 Oct 1:486:35-7. doi: 10.1016/j.ab.2015.06.024. Epub 2015 Jun 20.

Abstract

CREB-binding protein (CBP) is a lysine acetyltransferase that regulates transcription by acetylating histone and non-histone substrates. Defects in CBP activity are associated with hematologic malignancies, neurodisorders, and congenital malformations. Sensitive and quantitative enzymatic assays are essential to better characterize the pathophysiological features of CBP. We describe a sensitive nonradioactive method to measure purified and immunopurified cellular CBP enzymatic activity through rapid reverse phase-ultra-fast liquid chromatography (RP-UFLC) analysis of fluorescent histone H3 peptide substrates. The applicability and biological relevance of the assay are supported by kinetic, inhibition, and immunoprecipitation studies. More broadly, this approach could be easily adapted to assay other lysine acetyltransferases or methyltransferases.

Keywords: CREB-binding protein; Fluorescent peptide; HPLC; Histone acetyltransferase; Histone methyltransferases; SETD2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • CREB-Binding Protein / metabolism*
  • Chromatography, High Pressure Liquid
  • Chromatography, Reverse-Phase
  • Enzyme Assays / methods*
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism*
  • Histones / chemistry*
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism*

Substances

  • Fluorescent Dyes
  • Histones
  • Peptide Fragments
  • CREB-Binding Protein