Three spectrophotometric assays for the measurement of the five respiratory chain complexes in minuscule biological samples

Abstract

Background: The measurement of the activities of the five complexes comprising the respiratory chain has proven to be a major challenge when a limiting amount of biological material is available. Here we report a set of three convenient assays that allows this measurement under such circumstances.

Methods: One assay relies on the sequential addition of reagents to measure first complex IV activity, followed by complex II+III, and then glycerol-3-phosphate dehydrogenase+complex III activities and finally isolated complex III activity. A second assay measures the activity of complex II followed by glycerol-3-phosphate dehydrogenase and isocitrate dehydrogenase. A third assay measures rotenone-sensitive complex I activity and subsequently oligomycin-sensitive complex V activity.

Results: These assays have been successfully used on extracts of small numbers of human cells displaying various defects in the respiratory chain, and on frozen tissue homogenates of retina and very early mouse embryos.

Conclusions: The strength of this set of assays lies both in its rapid and simple execution and its capacity for immediate detection of partial defects, because each activity can be compared with one or two other activities measured in the same sample.