Cytochrome c oxidase assay in minute amounts of human skeletal muscle using single wavelength spectrophotometers

Òscar Miró, Francesc Cardellach, Antoni Barrientos, Jordi Casademont, Agnés Rötig, Pierre Rustin

Research output: Contribution to journalArticle

64 Scopus citations

Abstract

Determination of cytochrome c oxidase (COX; EC 1.9.3.1) activity in human mitochondria presents several technical difficulties which result in a large intra- and interlaboratory variability, especially when a single wavelength spectrophotometer (SWS) is used, as is generally done in most laboratories in the context of screening procedures for the detection of respiratory chain deficiencies. We studied the experimental conditions of COX assay in human skeletal muscle mitochondria using a SWS in order to define the optimal conditions for the assay and compared these results with those obtained using a double wavelength spectraphotometer (DWS). We demonstrate that a low intra-individual variability of COX assay can be obtained with SWS by: (1) using manual stirrers to avoid the formation of bubbles in the mixture; (2) preincubating mitochondria and laurylmaltoside before the addition of cytochrome c, which prevents light scattering secondary to mitochondrial swelling; and (3) using low amounts (1-2 μg) of mitochondrial protein to extend and linearize the reaction rate. Under these experimental conditions, the concordance between SWS and DWS was very good (R = 0.975).

Original languageEnglish (US)
Pages (from-to)107-111
Number of pages5
JournalJournal of Neuroscience Methods
Volume80
Issue number1
DOIs
StatePublished - Mar 13 1998

    Fingerprint

Keywords

  • Cytochrome c oxidase
  • Electron transport chain
  • Mitochondria
  • Skeletal muscle
  • Spectrophotometry

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this