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Complex I and ATP synthase enzyme activity measurements

AE A. C. Van Erp
RR R. A. Rebolledo
DH D. Hoeksma
NJ N. R. Jespersen
PO P. J. Ottens
RN R. Nørregaard
MP M. Pedersen
CL C. Laustsen
JB J. G. M. Burgerhof
JW J. C. Wolters
JC J. Ciapaite
HB H. E. Bøtker
HL H. G. D. Leuvenink
BJ B. Jespersen
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Mitochondria were isolated as previously described62, then diluted in PBS, lysed by sonication, and centrifuged at 600 g for 10 min at 4 °C. Protein concentration was determined in the supernatant using a BCA protein assay kit (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA).

Activity of complex I was monitored spectrophotometrically at 600 nm and 37 °C, as previously described68. Rotenone-sensitive complex I activity was calculated using the molar extinction coefficient of DCIP, equal to 21000 M−1cm−1 and expressed as nmol/min/mg protein68. The activity of ATP synthase was measured spectrophotometrically at 340 nm and 37 °C, as previously described64. Oligomycin-sensitive ATP synthase activity was calculated using the molar extinction coefficient of NADH, equal to 6220 M−1cm−1 and expressed as nmol/min/mg mitochondrial protein69.

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