Cytochrome Oxidase Activity Colorimetric Assay Kit rev. 04/13
(Catalog # K287-100; 100 assays; Store at -20°C)
I. Introduction:
Cytochrome c Oxidase (EC 1.9.3.1) or Complex IV is the fourth complex of the Electron Transport Chain located in the mitochondrial (or
bacterial) membrane. It provides energy to the cell by coupling electron transport through the Cytochrome c chain with the process of
oxidative phosphorylation. Complex IV contains 13 different subunits encoded by both mitochondrial DNA and nuclear DNA. It receives
an electron from each of the four Cytochrome c molecules, and transfers it to one oxygen molecule, converting it into two molecules of
water. In this process, it also binds to four proton molecules and translocates them across the membrane to establish electrochemical
gradient, which is utilized for the synthesis of ATP.
Cytochrome Oxidase Activity Assay Kit is simple, fast and high-throughput adaptable. This assay kit can be used for purified
mitochondria or tissue extracts containing mitochondria. The activity of the enzyme is determined colorimetrically by following the
oxidation of reduced Cytochrome c as an absorbance decrease at 550 nm. The overall reaction is as follows:
II. Application:
• Fast and simple measurement of Cytochrome Oxidase enzymatic activity in 96-well plate format.
• Mitochondrial respiration studies, assembly of the complexes, Mitochondria outer membrane integrity.
III. Sample Type:
• Purified mitochondria
• Cells/Tissue extracts
IV. Kit Contents:
V. User Supplied Reagents and Equipment:
• Multi-well spectrophotometer capable of reading absorbance.
• Multi-channel pipet
VI. Storage and Handling:
Store kit at -20°C, protected from light. Warm Assay Buffer to room temperature before use. Keep Enzyme Dilution Buffer on ice. Read
the entire protocol before performing the assay.
VII. Reagent Preparation and Storage Conditions:
• DTT: Aliquot and store at -20°C. Thaw just before use.
• Cytochrome c: Reconstitute each vial with 1 ml of Cytochrome Oxidase Assay Buffer. Mix by vortexing to dissolve compley. Add 5
<l of DTT solution. Mix well and wait for 15 min. at room temperature. Keep this working solution at room temperature. After assay is
completed, aliquot and save rest of the Cytochrome c solution at -20°C. This is now reduced form of Cytochrome c.
VIII. Complex IV Activity Assay Protocol:
1. Efficiency of Reduction of Cytochrome c: In a 96-well plate, mix 20 <l of reduced Cytochrome c with 100 <l of Cytochrome Oxidase
Assay Buffer. Prepare a parallel well as blank with only Assay Buffer. Read OD at 550 nm. The OD at 550 nm of reduced Cytochrome
c is between 0.2-0.6. If not, add 5 <l of DTT/ml of reconstituted Cytochrome c and wait for 15 min. to read again the OD.
2. Sample Preparation: Isolate mitochondria from cultured cells, yeast or tissues by using Mitochondria/Cytosol Fractionation Kit (BV
cat. # K256) or Yeast Mitochondria Isolation Kit (K259-50) or use cell or tissue lysate (BV cat. # 1067). The recommended range of
purified mitochondria is 0.5- 5 <g and tissue extract is 1-60 <g per reaction. Dilute the test samples, if needed by Enzyme Dilution
Buffer.
3. Cytochrome c preparation: Prepare 1:6 dilution of Cytochrome c by using pre-warmed Cytochrome Oxidase Assay Buffer (one part
of Cytochrome c to 5 parts of buffer) in a separate tube depending on the number of assay samples and controls. Prepare 120 <l of
diluted Cytochrome c per reaction.
4. Complex IV Activity Assay: Before the reaction, set the spectrophotometer at 550 nm on kinetic program for 30-45 minutes at 30 sec
interval. Add the test samples (approx. volume 5-10 <l) to each well of a 96-well plate. For negative control (Blank), add equal volume
of Enzyme Dilution Buffer. Add 120 <l of the diluted Cytochrome c from Step 3 to each sample and control using a multichannel
pipette. Shake and immediay read and record decrease in OD over a period of 30-45 min.
Note: The rate of the reaction is relative to a control or normal sample. The rate is calculated in linear range.
5. Calculations: Calculate rate of the reaction by calculating change in OD: OD/min by using the maximum linear rate. The oxidation