To standardize a procedure for the enzymatic assay of Fructose -6- Phosphate Kinase (Pyrophosphate Dependent) from Propionibacterium Freudenreichii (Shermanii).
This procedure applies to F8381, which has a specification for Fructose -6- Phosphate Kinase (Pyrophosphate Dependent).
3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC
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 30 ºC.
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
PPi + F-6-P PPi-PFK > F-1-6-DP + Pi
F-1-6-DP Aldolase > Glyceraldehyde Phosphate (GAP)+Dihydroxy Acetonephosphate
GAP TPI > DHAP
2H+ +2NADH +2DHAP GDH > 2NAD+ +2 Glycerol -3-phosphate
It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.
Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.
T = 30 °C, pH = 7.4, A340nm, Light path = 1 cm
Continuous Spectrophotometric Rate Determination
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, (I0250), and 0.001 mL/mL solution of Magnesium Chloride, 1.0 M Solution, M1028.
18.104.22.168 Dissolve the Imidazole in 90 mL of purified water and adjust the pH to 7.4 at 30 °C with 1 N HCl/NaOH.
22.214.171.124 Add 0.10 mL of Magnesium Chloride to Reagent 126.96.36.199 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, 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, 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, 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, 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, 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:
7.4.2 Mix by inversion, and using a suitable thermostatted spectrophotometer, equilibrate to 30°C for five minutes. Then add:
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.
|7.5.1||Units/ml enzyme =||(A340nm Test - A340nm BLank)(3.0)(df)
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 =||
mg solid/mL enzyme
|7.5.3||Units/mg protein =||
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).
8.1 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.
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