Solarbio Pyrophosphate: Fructose 6-phosphate-1 Phosphotransferase Assay Kit

Components:

Extract solution: 55mL × 1, stored at 4 °C.

Reagent 1: 40mL × 1, stored at 4 °C and protected from light.

Reagent 2: Powder × 1, stored at -20 °C and protected from light. Just before use, add 6 mL of distilled water to fully dissolve. Unused reagents are stored at -20 °C for 1 week after dispensing to avoid repeated freeze-thaw cycles.

Reagent 3: powder × 1, stored at -20 °C and protected from light. Just before use, add 6 mL of distilled water to fully dissolve. Unused reagents are stored at -20 °C after dispensing. Prohibition of repeated freeze-thaw cycles.

Reagent 4: 91µL × 2, stored at 4 °C and protected from light. Just before use, add 0.209 mL of distilled water to fully dissolve. Stored at 4 °C for 1 week.

Reagent 5: Powder × 2, stored at -20 °C and protected from light. Just before use, add 0.3 mL of distilled water to fully dissolve. Unused reagents are stored at -20 °C for 1 week after dispensing.

Reagent 6: 60 µL × 1, stored at 4 °C and protected from light. Just before use, add 0.6 mL of distilled water to fully dissolve. Stored at 4 °C for 1 week.

Product Description:

Pyrophosphate: Fructose-6-phosphate-1-phosphotransferase (PFP, EC2.7.1.90) is a cytosolic enzyme that is widely present in plant tissues and catalyzes the phosphorylation of fructose-6-phosphate like phosphofructokinase. As a result, the single PEP catalytic reaction is a reversible reaction, and pyrophosphate is used instead of ATP, which plays an important role in carbon metabolism of photosynthesis.

PFP catalyzes the conversion of fructose 6-phosphate to fructose 1,6-diphosphate, which is converted to dihydroxyacetone phosphate by the action of aldolase and triose phosphate isomerase, and then catalyzed by α-phosphate glycerol dehydrogenase and NADH to from Glycerol 3-diphosphate and NAD. The change in absorbance at 340 nm reflects the level of PFP activity.

Required material

Low temperature centrifuge, spectrophotometer, water bath/constant temperature incubator, mortar/homogenizer, 1 mL quartz cuvette, transfer pipette, ice and distilled water, EP tube.

Procedure:

I. Sample Extraction:

1. 1. Tissue sample:

According to the mass of the tissue (g): the volume of the extract solution (mL) is 1: 5 ~ 10.  Suggested 0.1g of tissue with 1mL of extract solution. Fully grind on ice, centrifugated at 20000g and 4℃ for 15 min. Supernatant is placed on ice for test.

2. Bacteria or cells:

According to the number of cells (104): the volume of the extract solution (mL) is 500 ~ 1000: 1. Recommend 5 million with 1mL of Extract Solution. Use ultrasonication to split bacteria or cells (power 300W, work time 3s, interval 7s, total time 3 min). Centrifugated at, 20000g and 4℃ for 15 min. Supernatant is placed on ice for test.

3. Liquids: direct detection.

II. Determination procedure:

• Preheat the spectrophotometer 30 min, adjust wavelength to 340 nm, set zero with distilled
• Add reagents with the following list:

III. Calculation of PFP activity:

1 Calculated by micro quartz cuvette

• Calculated by protein concentration:

Unit definition: One unit of enzyme activity is defined as that 1 mg of tissue protein per minute consumes 1 nmol of NADH.

PFP activity (U/mg prot) =ΔA÷（ε×d）×VRT×109÷(VS×Cpr) ÷T=53.59×ΔA÷Cpr

• Calculated by sample weight

Unit definition: One unit of enzyme activity is defined as that 1g of tissue per minute consumes 1  nmol of NADH.

PFP activity (U/g fresh weight) =ΔA÷（ε×d）×VRT×109÷(W ×VS÷VE) ÷T=53.59×ΔA÷W

• Calculated by bacteria or cell amount:

Unit definition: One unit of enzyme activity is defined as that 10 thousand bacteria or cells per minute consumes 1 nmol of NADH.

PFP activity (U/104 cell) =ΔA÷（ε×d）×VRT×109÷(VS×N÷VE) ÷T=53.59×ΔA÷N（104）

• Calculated by serum and other liquids:

Unit definition: One unit of enzyme activity is defined as that 1 mL of liquids per minute consumes 1 nmol of NADH.

PFP activity (U/mL) =ΔA÷（ε×d）×VRT×109÷VS÷T=53.59×ΔA

VRT: total volume of reaction system, 0.001 L;

ε: NADH molar extinction coefficient, 6.22 × 103 L/mol/cm; d: cuvette light path, 1 cm;

VS: added sample volume, 0.1 mL;

VE: volume of extract solution added, 1 mL; T: reaction time, 30 min;

Cpr: sample protein concentration, mg/mL; W: sample mass, 0.1 g;

109:conversion factor, 1 mol = 109 nmol N: number of cell

2. Calculated by 96-well UV plate:

Modify d = 1 cm in the above formula to d-0.6 cm (the light path of a 96-well plate) for calculation.

Note:

1. The number of samples should not be too large to avoid delaying the enzymatic reaction time.

Experimental examples:

1. Take 0.1 g of bean sprouts and add 1 mL of Extract solution for sample processing.  After centrifugation to take the supernatant, proceed according to the determination procedure. CalculateΔA = (A1T-A2T)-(A1B-A2B)=  (1.041-0.963)-0=0.078. The enzyme activity is calculated according to the sample mass.

PFP activity (U/g fresh weight) =53.59×ΔA÷W=41.8 U/g fresh weight.

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