CYTOCHROME-C-OXIDASE ASSAY IN MINUTE AMOUNTS OF HUMAN SKELETAL-MUSCLEUSING SINGLE-WAVELENGTH SPECTROPHOTOMETERS

Determination of cytochrome c oxidase (COX; EC 1.9.3.1) activity in hu man mitochondria presents several technical difficulties which result in a large intra-and interlaboratory variability, especially when a si ngle wavelength spectrophotometer (SWS) is used, as is generally done in most laboratories in the context of screening procedures for the de tection 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 compa red these results with those obtained using a double wavelength spectr ophotometer (DWS). We demonstrate that a low intra-individual variabil ity of COX assay can be obtained with SWS by: (1) using manual stirrer s 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. a nd (3) using low amounts (1-2 mu g) 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). (C) 19 98 Elsevier Science B.V. All rights reserved.