Components:

Extract solution: 80ml×1. Storage at 4℃.

Reagent 1: 40ml×1. Storage at 4℃.

Reagent 2: Powder×1. Storage at -20℃, dissolve with 1ml of acetone. The dissolved reagent 2 can be stored at -20℃ after dispensing. Dilute 100 times when used.

Reagent 3: Powder×1. Storage at 4℃, dissolve with 3ml of acetone before use. Reagent 4: 5ml×1. Storage at 4℃.

Product Description:

Mitochondrial complex II is the same as succinate-Co-enzyme Q reductase, which exists widely in mitochondria of animal, plant, microorganisms and cultured cells. It catalyzes succinic acid to form fumaric acid, reduce FAD to form FADH2. The FADH2 reduce oxidized CoQ to form reduced CoQ, which is a branch of respiratory electron transport chain.

CoQ that a catalytic product of complex II could reduce 2,6-dichloroindophenol, which has absorbance at 605 nm, the activity of enzyme can be calculated by detecting the decrease rate of 2, 6-dichlorindolepheno.

Reagents and Equipment Required but Not Provided:

Spectrophotometer, water bath, desk centrifuge, transfer pipette, 1ml glass cuvette, mortar/ homogenizer, acetone, ice and distilled water.

1. Complex II extraction:

• Collecting 0.1g of tissue or 5 million cells, add 1ml extract solution and grind on ice with mortar/homogenizer;
• centrifuge at 600g and 4℃for 10 min. Discard the precipitate and transfer supernatant to another tube, centrifuge at, 11000g and 4℃ for 15 min;
• The supernatant, i.e., cytoplasmic extract, can be used to determine the complex II leaking from mitochondria, this step can show the effect of mitochondrial extraction;
• Dodati 400 μL extraction solution to sediment, splitting with ultrasonication (power 20%, work time 5s, interval 10s, repeat 15 times), used to detect Complex Ⅱ activity and protein content.

Determining step

1. Preheat spectrophotometer for 30 min, adjust the wavelength to 605 nm, set the counter to zero with distilled
2. Sample determination

• Making working solution: mix reagent 2 and reagent 3 according to 1:1 before use. Prepare when used. Prepared when the solution will be used.
• Preheat reagent1 at 37℃ (mammal cell) or 25℃(other species) za 15
• Add the following reagents in 1ml glass cuvette:

Calculation:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1nmol of 2, 6-dichlorindolepheno per mg of tissue protein in every minute.

Complex Ⅱ Activity (U/mg prot)=[ΔA×Vrv÷(ε×d)×109]÷(Vs×Cpr)÷T =476.2×ΔA÷Cpr

ε: 2, 6-dichlorindolepheno molar extinction coefficient, 2.1×104L/mol/cm; d: light path of cuvette, 1 cm;

Vrv: total reaction volume,1mL; Vs: sample volume (mL), 0.05 mL;

Cpr: sample protein concentration (mg/mL); T: reaction time (min), 2 min;

Note:

1. Take two or three different samples for prediction before test to ensure the accuracy of experimental results. Dilute supernatant with distilled water if absorbance is higher than 1.5. Dilute sample with distilled water if ΔA>0.4, multiply dilute times in the formula. Increase sample volume if ΔA is slow.
2. Detect sample protein concentrate by yourself, you can use Solarbio (PC0020 BCA Protein Komplet za analizu). Because protein is contained in the extract, the protein content of the extract itself should be subtracted when determining the protein concentration of the sample.
3. It is recommended to use the sample protein concentration to calculate the enzyme activity. If the sample fresh weight is used to calculate, the enzyme activity of cytoplasmic extract needs to be measured, and the sum of supernatant and precipitation enzyme activity is the total enzyme activity.
4. It’s enough for 50 tube reactions.
5. Prilog: Sample weight(50T/24S)

• Supernatant:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1nmol of 2, 6-dichlorindolepheno in 1 min every gram of tissue weight.

Complex Ⅱ Activity(U/g)=[ΔA1×Vrv÷(ε×d)×109]÷(W÷Ve×Vs)÷T =476.2×ΔA1÷W ΔA1: supernatant absorbance;

Vrv: total reaction volume,1mL;

ε: 2, 6-dichlorindolepheno molar extinction coefficient, 2.1×104L/mol/cm; d: light path of cuvette, 1 cm;

Ve: extract solution volume,1mL; Vs: sample volume (mL), 0.05 mL; T: reaction time (min), 2 min;

W: sample weight, g.

• Sediment:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1nmol of 2, 6-dichlorindolepheno in 1 min every gram of tissue weight.

Complex Ⅱ Activity(U/g)= [ΔA2×Vrv÷(ε×d)×109]÷(W÷Ve×Vs)÷T =190.5×ΔA2÷W ΔA2: sediment absorbance;

Vrv: total reaction volume,1mL;

ε: 2, 6-dichlorindolepheno molar extinction coefficient, 2.1×104L/mol/cm; d: light path of cuvette, 1 cm;

Ve: sediment resuspended volume,0.4 mL; Vs: sample volume (mL), 0.05 mL;

T: reaction time (min), 2 min; W: sample weight, g.

• Total activity is the sum of ComplexⅡactivity in supernatant and sediment. Complex Ⅱ(U/g) =476.2×ΔA1÷W+190.5×ΔA2÷W.

Experimental example:

1. Take 0.1g of rabbit liver sample, add 1 mL of Extract solution, grind and centrifuge the homogenate, and operate according to the determination steps. ΔA1 = A1-A2 = 1.134-1.054 = 0.08 in the supernatant, and ΔA2 =A1-A2 = 1.371-1.347 = 0.024 in the precipitation.

The activity of complex II in the supernatant (U/g mass) = 476.2×ΔA1÷W = 476.2 × 0.08÷0.1 = 380.96 U/g mass

The activity of complex II in the precipitation (U/g mass) = 190.5×ΔA2÷ W = 190.5×0.024÷0.1 = 45.72

U/g mass

Complex II (U/g mass) = 476.2× ΔA1÷W + 190.5×ΔA2÷W = 476.2×0.08÷0.1 + 190.5×0.024 ÷ 0.1 =426.8U/g mass.

References：

[1] Mühling J, Tiefenbach M, López-Barneo J, et al. Mitochondrial complex II participates in normoxic and hypoxic regulation of α-keto acids in the murine heart[J]. Journal of molecular and cellular cardiology, 2010, 49(6): 950-961.

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