Comparison of two methods for assaying complex I activity in mitochondria isolated from rat liver, brain and heart.
OBJECTIVE
To establish a more sensitive, reliable, and convenient assay for complex I activity.


METHODS
Two of the most widely used methods - the conventional NADH method and a newly developed 2, 6-dichlorophenolindophenol (DCIP)-coupled method - were compared and optimized.


RESULTS
The DCIP method gave a higher enzyme sensitivity and comparable rotenone sensitivity in heart mitochondria while the NADH method gave higher rotenone sensitivity with a relatively lower enzyme activity in the brain and liver mitochondria. Addition of bovine serum albumin (BSA) to the reaction mixture greatly improved the accuracy of the NADH assay. The reaction conditions were optimized for use with a microplate reader that requires only small amounts of mitochondria or tissue.


CONCLUSIONS
Considering the important contribution of the non-specific rotenone-insensitive activity in the complex I assay, it is suggested that the NADH method with BSA addition should be adopted for assaying complex I activity in the brain or liver samples, while the DCIP method is the better choice for heart samples.
Long
Jiangang
J
Institute of Mitochondrial Biology and Medicine, Department of Biological Science and Engineering, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, Xi'an Jiaotong University School of Life Science and Technology, China.
Ma
Jiangang
J
Luo
Cheng
C
Mo
Xiaoyan
X
Sun
Lei
L
Zang
Weijin
W
Liu
Jiankang
J
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003470:Culture Media; D042967:Electron Transport Complex I / Q000378:metabolism*; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine; D013010:Sonication
D003470:Culture Media; D012710:Serum Albumin, Bovine; D042967:Electron Transport Complex I