Enzymatic Assay of Fructose-6-Phosphate Kinase Pyrophosphate Dependent

1. Objective

To standardize a procedure for the enzymatic assay of Fructose -6- Phosphate Kinase (Pyrophosphate Dependent) from Propionibacterium Freudenreichii (Shermanii).

2. Scope

This procedure applies to Sigma-Aldrich Product Number F8381, which has a specification for Fructose -6- Phosphate Kinase (Pyrophosphate Dependent).

3. Definitions

3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC

3.2 Unit Definition - One unit will convert 1 μmole of pyrophosphate and fructose 6-phosphate to fructose 1,6-diphosphate and inorganic phosphate per minute, at pH 7.4 and 30ºC.

3.3 PPi - Pyrophosphate

3.4 F-6-P - Fructose 6-Phosphate

3.5 F-1-6-DP - Fructose 1,6-Diphosphate

3.6 GAP - Glyceraldehyde Phosphate

3.7 DHAP - Dihydroxy Acetonephosphate

3.8 NAD+ - Nicotinamide Adenine Dinucleotide, Oxidized Form

3.9 NADH - Nicotinanmide Adenine Dinucleotide, Reduced Form

3.10 GDH - Glycerophosphate Dehydrogenase

3.11 TPI -Triosephosphate Isomerase

4. Discussion

PPi + F-6-P    PPi-PFK   > F-1-6-DP + Pi
F-1-6-DP    Aldolase   > Glyceraldehyde Phosphate (GAP)+Dihydroxy Acetonephosphate
(DHAP)
GAP    TPI   > DHAP
2H+ +2NADH +2DHAP    GDH   > 2NAD+ +2 Glycerol -3-phosphate

5. Responsibilities

It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. Safety

Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:
T = 30°C, pH = 7.4, A340nm, Light path = 1 cm

7.2 METHOD:
Continuous Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1    150 mM Imidazole with 1 mM Magnesium Chloride, pH 7.4 at 30°C (Buffer) Prepare 100 ml by making a 10.2 mg/ml solution of Imidazole, Sigma-Aldrich Product Number I0250, and 0.001 ml/ml solution of Magnesium Chloride, 1.0 M Solution, Sigma-Aldrich Product Number M1028.

7.3.1.1    Dissolve the Imidazole in 90 ml of purified water and adjust the pH to 7.4 at 30°C with 1 N HCl/NaOH.

7.3.1.2    Add 0.10 ml of Magnesium Chloride to Reagent 7.3.1.1 and dilute to 100 ml with purified water.

7.3.2    90 mM Fructose-6-Phosphate (F-6-P) Prepare a 30 mg/ml solution in Reagent 7.3.1(Buffer) using Fructose-6-Phosphate, Sigma-Aldrich Product Number F1502. Correct for water and solvent content.

7.3.3    7.5 mM Nicotinanmide Adenine Dinucleotide, Reduced Form (NADH) Prepare a 5.0 mg/ml solution in Reagent 7.3.1(Buffer), using Nicotinamide Adenine Dinucleotide, Reduced Form, Sigma-Aldrich Product Number N8129. Correct for water and solvent content.

7.3.4    50 un/ml solution of Aldolase (Ald) Prepare a 50 unit/ml solution in cold Reagent 7.3.1(Buffer) by using Aldolase, Sigma-Aldrich Product Number A2714.

7.3.5    25 units/ml solution of Glycerophosphate Dehydrogenase-Triosephosphate Isomerase (α-GDH-TPI) Prepare a 25 units/ml solution in cold Reagent 7.3.1 (Buffer) by using Glycerophosphate Dehydrogenase-Triosephosphate Isomerase, Sigma-Aldrich Product Number G6755.

7.3.6    25 units/ml solution of Glycerophosphate Dehydrogenase-Triosephosphate Isomerase (α-GDH-TPI) Prepare a 25 units/ml solution in cold Reagent 7.3.1 (Buffer) by using Glycerophosphate Dehydrogenase-Triosephosphate Isomerase, Sigma-Aldrich Product Number G6755.

7.3.7    Fructose-6-Phosphate Kinase, Pyrophosphate Dependent enzyme solution (Enzyme) Immediately before use prepare a 0.1-0.5 units/ml solution of Fructose-6- Phosphate Kinase, Pyrophosphate Dependent enzyme solution in cold Reagent 7.3.1 (Buffer)

7.4 TEST METHOD

7.4.1    Pipette (in milliliters) the following reagents into suitable containers:

 

  Test Blank
Reagent 7.3.1 (Buffer) 2.50 2.55
Reagent 7.3.2 (F-6-P) 0.10 0.10
Reagent 7.3.3 (NADH) 0.10 0.10
Reagent 7.3.4 (Ald) 0.10 0.10
Reagent 7.3.5 (α-GDH-TPI) 0.10 0.10
Reagent 7.3.6 (PPi) 0.05 0.05

 

7.4.2    Mix by inversion, and using a suitable thermostatted spectrophotometer, equilibrate to 30°C for five minutes. Then add:

Reagent 7.3.7 (Enzyme) 0.05 -----

 

7.4.3    Immediately mix by inversion and record the decrease in A340nm for approximately 10 minutes.

7.4.4    Obtain the ΔA340nm using the maximum linear rate over a one minute period, and at least four data points. Obtain maximum linear rate for both Test and Blank.

7.5 CALCULATIONS

7.5.1 Units/ml enzyme = (A340nm Test - A340nm BLank)(3.0)(df)
(6.22)(0.05)(2.0)

 

 


    3.0 = Total volume (in milliliters) of solution
    df = Dilution Factor
    6.22 = Millilmolar extinction coefficient of β-NADH at 340nm
    0.05= (Volume in milliliters) of enzyme used
    2.0= 2 moles of NADH oxidized per 1 mole PPi consumed

7.5.2 Units/mg solid = Units/mL enzyme
mg solid/mL enzyme

 

7.5.3 Units/mg protein = Units/mL enzyme
mg protein/mL enzyme

 

7.6 FINAL ASSAY CONCENTRATION :
In a 3.00 ml reaction mix the final concentrations are 148 mM Imidazole, 0.98 mM Magnesium Chloride, 3.0 mM Fructose-6-Phosphate, 0.25 mM Nicotinamide Adenine Dinucleotide (NADH), 1.67 units of Aldolase, 0.83 units of Glycerophosphate Dehydrogenase-Trisosephosphate Isomerase (α-GDH -TPI), 1.67 mM Inorganic Pyrophosphate (PPi) and 0.0016-0.0083 units of Fructose–6-Phosphate Kinase, Pyrophosphate Dependent (Enzyme).

8. References & Attachments

8.1 Determination of pyrophosphate. O’Brien W., et al: J Biol Chem 250-8690,1975

8.2 Continuous spectrophotometric assays based on pyrophosphate formation. O’Brien W: Anal Biochem 76:423,1976

8.3 Replaces SPFRUC10.

9. Approval

Review, approvals and signatures for this document will be generated electronically using the EDMS. Print a “For Use” copy if hardcopy with signature verification is required.

Materials

     
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