Journal
JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS
Volume 68, Issue 2, Pages 101-111Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbbm.2006.04.003
Keywords
mitochondria; malate dehydrogenase; phenazine methosulfate; lipoamide dehydrogenase; 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT); NADH
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A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 mu g protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application. (c) 2006 Elsevier B.V. All rights reserved.
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