Cytochrome c oxidase assay in minute amounts of human skeletal muscle using single wavelength spectrophotometers.
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 spectrophotometer (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 microg) 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).
Miró
O
O
Department of Internal Medicine, IDIBAPS, Hospital Clinic, University of Barcelona, Spain.
Cardellach
F
F
Barrientos
A
A
Casademont
J
J
Rötig
A
A
Rustin
P
P
D004579:Electron Transport / Q000502:physiology; D003576:Electron Transport Complex IV / Q000032:analysis*; D006801:Humans; D008931:Mitochondria, Muscle / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology*; D012120:Respiration Disorders / Q000175:diagnosis; D013053:Spectrophotometry / Q000295:instrumentation / Q000379:methods*
D003576:Electron Transport Complex IV