A simple fluorescence labeling method for studies of protein oxidation, protein modification, and proteolysis

Andrew M. Pickering, Kelvin J.A. Davies

Research output: Contribution to journalShort survey

10 Scopus citations

Abstract

Proteins are sensitive to oxidation, and oxidized proteins are excellent substrates for degradation by proteolytic enzymes such as the proteasome and the mitochondrial Lon protease. Protein labeling is required for studies of protein turnover. Unfortunately, most labeling techniques involve 3H or 14C methylation, which is expensive, exposes researchers to radioactivity, generates large amounts of radioactive waste, and allows only single-point assays because samples require acid precipitation. Alternative labeling methods have largely proven unsuitable, either because the probe itself is modified by the oxidant(s) being studied or because the alternative labeling techniques are too complex or too costly for routine use. What is needed is a simple, quick, and cheap labeling technique that uses a non-radioactive marker, binds strongly to proteins, is resistant to oxidative modification, and emits a strong signal. We have devised a new reductive method for labeling free carboxyl groups of proteins with the small fluorophore 7-amino-4-methycoumarin (AMC). When bound to target proteins, AMC fluoresces very weakly but when AMC is released by proteinases, proteases, or peptidases, it fluoresces strongly. Thus, without acid precipitation, the proteolysis of any target protein can be studied continuously, in multiwell plates. In direct comparisons, 3H-labeled proteins and AMC-labeled proteins exhibited essentially identical degradation patterns during incubation with trypsin, cell extracts, and purified proteasome. AMC-labeled proteins are well suited to studying increased proteolytic susceptibility after protein modification, because the AMC-protein bond is resistant to oxidizing agents such as hydrogen peroxide and peroxynitrite and is stable over time and to extremes of pH, temperature (even boiling), freeze-thaw, mercaptoethanol, and methanol.

Original languageEnglish (US)
Pages (from-to)239-246
Number of pages8
JournalFree Radical Biology and Medicine
Volume52
Issue number2
DOIs
StatePublished - Jan 15 2012

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Keywords

  • Fluorescent label
  • Free radicals
  • Non-radioactive label
  • Oxidative stress
  • Protein degradation
  • Protein labeling
  • Protein modification
  • Protein oxidation
  • Protein turnover
  • Proteolysis
  • Ubiquitin-proteasome system

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

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