Complex I Enzymatic Activity Assay

Measurement of NADH:Q reductase (Complex I) activity 

A. Preparation of stock solutions:

Tris–HCl buffer pH 7.2                           100 mM

NaCl                                                      100 mM

NADH or deamino-NADH                    10 mM

potassium ferricyanide K₃[Fe(CN)₆]     100 mM

B.  Procedure:

1. Mix the stock solutions listed in A to the final reaction concentrations: 50 mM Tris–HCl, pH 7.2, 50 mM NaCl, 0.2 mM NADH or deamino-NADH, and 1 mM K₃[Fe(CN)₆]. (see the table  below)
2. The reaction is initiated by adding mitochondrial sample.
3. The absorbance at 420 nm using spectrophotometry is measured over time.

Stock solution                  volume           Final concentration

------------------------------------------------------------------------------------------------         

1 M Tris-HCl                      50 µl             50 mM

1 M NaCl                           50 µl             50 mM

10 mM NADH                   20 µl             0.2 mM  

50 mM K₃[Fe(CN)₆]          20 µl             1 mM

H₂O                                  850µl

------------------------------------------------------------------------------------------------

Mitochondria                   10-20 µg

Note 1:  deamino-NADH (Nicotinamide hypoxanthine dinucleotide, reduced form, sodium salt; SIGMA CAT# N6756).

Note 2:  The extinction coefficient of K₃[Fe(CN)₆] at 420 nm is 1.03 mM^-1 cm^-1.

Note 3: Amount of mitochondria used is dependent on enzymatic activity to get a good reading.

Category