Self-supplied consumables:
Microplate reader or visible spectrophotometer (able to measure absorbance at 550 nm)
Thermostat, ice machine, low temperature centrifuge
96-well plate or micro-glass cuvette, adjustable pipetting gun and tip
Deionized water
Homogenizer or mortar (if it is a tissue sample)
Reagent preparation:
ReagentⅠ : ready-to-use; Before use, balance to room temperature; The samples were stored at 4 ° C.
ReagentⅡ : ready-to-use; Before use, balance to room temperature; The samples were stored at 4 ° C.
ReagentⅢ : ready-to-use; Before use, balance to room temperature; They were stored at 4 ° C in the dark.
Preparation of working solution: Before use, ReagentⅤ and ReagentⅥ were transferred to ReagentⅣ in turn, mixed and dissolved. If the test sample is of mammalian origin, please incubate at 37℃ for 5 min; If the samples were other species, they were incubated at 25 ° C for 5 min. The inexhaustible reagents were packaged and stored at -20℃ in the dark for 1 week, and repeated freezing and thawing were prohibited.
Sample preparation:
Note: it is recommended to use fresh samples to ensure that the enzyme activity.
Extraction of mitochondrial respiratory chain complex Ⅳ :
1. Accurately weigh 0.1 g of tissue or collect 5 million cells, add 1 mL ReagentⅠ and 10 µL ReagentⅢ, and homogenize with a homogenizer or mortar ice bath.
2. The homogenate was centrifuged at 600 g for 5 min at 4 ° C, and the supernatant was collected into a new centrifuge tube and the precipitate was discarded.
3. The supernatant was centrifuged again at 11,000 g for 10 min at 4 ° C, and the precipitate was the extracted mitochondria, which was used as the fifth step.
4. (optional) is the cytoplasm supernatant fluid extract, can be used as samples for determination of mitochondria from leakage of mitochondrial respiratory chain complexes Ⅳ, used to determine mitochondrial extraction effect.
5. 200 µL ReagentⅡ and 2 µL ReagentⅢ were added to the precipitate, and the precipitate was fully resuspended for further detection of mitochondrial respiratory chain complex Ⅳ enzyme activity.

Experimental procedures:
1, enzyme standard instrument and visible spectrophotometer preheating above 30 min, adjust the wavelength of 550 nm, visible spectrophotometer with deionized water to zero.
2, in 96 - well plates or trace color glass dish, in turn, to join in 200 (including L working liquid and 10 (including L samples, after thoroughly incorporated, immediately read at 550 nm 0 min initial absorbance value of A1 and 1 min after the absorbance value of A2, calculate Δ A = A1 and A2.

Note: In order to ensure the accuracy of the experimental results, it is necessary to take 1-2 samples for pre-experiment. If ΔA is too high (higher than 1.0), the sample can be diluted by ReagentⅡ before determination, and the results should be multiplied by the dilution factor. If Δ A small, can increase the numerical sample volume to improve detection is added, if Δ A negative, then samples do not contain complex Ⅳ or degradation.

The results were calculated as follows:
Note: We provide you with the calculation formula, including the derivation process calculation formula and the concise calculation formula. The two are exactly the same. The concise formula in bold is recommended as the final formula.
1, using the calculation formula determined in 96-well plates
(1) Calculated according to fresh weight of sample

Unit: the definition of each g group in the reaction system in the catalytic degradation of 1 nmol per minute with cytochrome C is defined as a unit of enzyme activity.

Mitochondrial respiratory chain complexes in supernatant Ⅳ dynamic calculation:

Supernatant of complex Ⅳ energy (U/g fresh weight) = [Δ A1 * V is the total present (epsilon (d) x 10 9] present sample (W present V extraction (V) present T = 2221 x Δ A1 present W

Precipitation in the complex Ⅳ dynamic calculation:

Precipitation in the complex Ⅳ energy (U/g fresh weight) = [Δ A2 * V is the total present (epsilon (d) x 10 9] present sample weight suspended x (W present V V) present T = 444 x Δ A2 present W

Calculation of total activity of complex IV:

The total complex Ⅳ activity was calculated as the sum of the activity of mitochondrial respiratory chain complex Ⅳ in the supernatant and in the precipitate.

Total complex Ⅳ activity (U/g fresh weight) = 2221 * Δ Δ A1 present W + 444 A2 present W

(2) calculated by cell density

Unit Definition: Catalytic degradation of 1 nmol of reduced cytochrome C per minute per 10 000 cells was defined as one unit of enzyme activity.

Mitochondrial respiratory chain complexes Ⅳ activity (U / 10 4 cells) = [Δ A * V is the total present (epsilon (d) x 10 9] present present V (V sample weight suspended x 500) present T = 0.888 x Δ A

The volume V reverse total: reaction system, 2.1 x 10-4 L; Epsilon: cytochrome C molar extinction coefficient, 19.1 x 10 3 mol/L/cm; D: 96 light orifice diameter, 0.5 cm; 10 9: unit conversion coefficient, 1 mol=10 9 nmol; V-like: added sample volume, 0.01 mL; T: the reaction time, 1 min; Δ A1: supernatant measurements; W: sample weight, g; V extraction: extraction system volume, 1.01 mL; ΔA2: precipitation measurement value; V heavy suspension: suspension precipitation volume, 0.202 mL; 500: total number of cells, 5 million.

2. Calculation formula for determination using micro-glass cuvette


The optical diameter d: 0.5 cm in the above calculation formula can be adjusted to d: 1 cm for calculation.

The results show that:

Experimental examples:

1. 0.1 g of mouse brain tissue was taken for sample processing, followed by the determination steps, and measured with 96-well plates:


ΔA1=A1-A2=0.4568-0.4446=0.0122, ΔA2=A3-A4=0.4373-0.4188=0.0185

2. Calculated by sample quality:


Mitochondrial respiratory chain complex Ⅳ activity (U/g fresh weight)=2,221×ΔA1÷W=2,221×0.0122÷0.1=270.962 U/g

Precipitation in the mitochondrial respiratory chain complexes Ⅳ activity (U/g fresh weight) = 444 x Δ A2 present W = 444 x 0.0185 present 0.1 = 82.14 U/g

The mitochondrial respiratory chain complexes Ⅳ fresh weight (U/g) = 2221 * Δ Δ A1 present W + 444 A2 present W = 270.962 + 82.14 = 353.102 U/g