Enzymatic Assay of Ferredoxin NADP Reductase (EC 1.18.12)

1. Objective

To standardize a procedure for the enzymatic assay of ferredoxin NADP reductase (EC.1.18.12).

2. Scope

This scope of this procedure applies to all products that have a specification for ferredoxin NADP reductase.

3. Definitions

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

3.2 Unit Definition - One unit of enzyme will reduce 1.0 millimole of cytochrome C per minute at 25 °C at pH 7.5 in the presence of spinach ferredoxin and NADP⁺.

3.3 NADPH = b-Nicotinamide adenine dinucleotide phosphate, reduced

3.4 NADP⁺ = b-Nicotinamide adenine dinucleotide phosphate, oxidized

3.5 CYT-OX = Cytochrome C-Oxidized

3.6 CYT-RD = Cytochrome C-Reduced

4. Discussion

CYT-OX + Ferrodoxin-NADPH ^{Ferredoxin NADP Reductase} > CYT-RD + Ferredoxin-NADP⁺

5. Responsibilities

Analytical services personnel should follow this protocol as written.

6. Safety

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

7. Procedure

7.1 CONDITIONS:
T = 25 °C, pH = 7.5, A_550nm, Light path = 1 cm

7.2 METHOD:
Continuous Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1 500 mM Tris-HCl, Buffer, pH 7.8 at 25 °C (Buffer A)

7.3.1.1 Prepare 100 mL in purified water using Trizma Base (T1503).

7.3.1.2 Adjust to pH 7.8 at 25 °C with 5 N HCl.

7.3.2 150 mM Tris-HCl, Buffer, pH 7.3 at 25 °C (Buffer B)

7.3.2.1 Prepare 100 mL in purified water using Trizma Base (T1503).

7.3.2.2 Adjust to pH 7.3 at 25 °C with 1 N HCl.

7.3.3 100 mM Tris-HCl, Buffer, pH 8.5 at 25 °C (Buffer C).

7.3.3.1 Prepare 100 mL in purified water using Trizma Base (T1503).

7.3.3.2 Adjust to pH 8.5 at 25 °C with 1 N HCl.

7.3.4 1.5 mM Cytochrome C (CYT-OX)

7.3.4.1 Immediately before use, prepare 2 mL in cold purified water using Cytochrome C from bovine heart (C3131).

7.3.4.2 Prepare in an amber vial.

7.3.4.3 Correct the concentration for percent water and percent reduced.

7.3.5 100 uM Ferredoxin Solution (Ferredoxin)

7.3.5.1 Immediately before use, prepare at 1.1 mg Protein / mL in cold Reagent 7.3.2 (Buffer B) ferredoxin from Spinacia oleracea, Product No.F3013.Prepare fresh.

7.3.5.2 The ferredoxin (F5875) comes as a frozen solution. It should be thawed once.

7.3.5.3 If F3013 is not available, dilute ferredoxin from Spinacia oleracea using Product No. F5875., to 1.1 mg protein / mL with cold 7.3.2 (Buffer B). One vial should only be used once and disposed of after use.

7.3.6 10 mM b-Nicotinamide adenine dinucleotide phosphate, reduced (NADPH)

7.3.6.1 Immediately before use, prepare a minimum of 2 mL in cold 7.3.3 (Buffer C) using b-nicotinamide adenine dinucleotide phosphate, reduced, tetrasodium salt (N1630).

7.3.6.2 Prepare fresh and correct concentration for percent water, Percent solvent, and percent purity by high-pressure liquid chromatography.

7.3.7 Ferredoxin NADP Reductase (Enzyme)

7.3.7.1 Immediately prior to pipetting into cuvette, prepare a solution at 1 mg / mL in cold purified water. Place on ice.

7.3.7.2 This solution is stable for a maximum of thirty minutes.

7.3.7.3 For each aliquot and immediately before pipetting into cuvette dilute to 0.02 to 0.08 units of ferredoxin NADP reductase /mL.

7.3.7.4 If unit vials, dilute by adding 0.100 mL, 0.250 mL, and 0.500 mL of cold purified water to a 1 unit vial, 5 unit vial, and 10 unit vial, respectively. For each aliquot and immediately before pipetting into the cuvette, dilute to 0.02 to 0.08 units of ferredoxin NADP reductase /mL.

7.4 TEST METHOD

7.4.1 Reagent Suitability:

7.4.1.1 Pipette (in milliliters) the following reagents in the following sequence into appropriate cuvettes :

7.4.1.2 Mix by inversion and equilibrate to 25 °C using a suitable spectrophotometer. Measure and record the A_340nm , A_420nm, A_530nm, and A_550nm versus purified water.

7.4.1.3 The A_340nm for Reagent 7.3.6.1 (NADPH) must be ≥ 1.25.

7.4.1.4 The A_530nm for Reagent 7.3.4.1 (CYT-OX) must be > A_550nm and the A_550nm 1.0 to 1.2.

7.4.1.5 The A_420nm for Reagent 7.3.5.1 (Ferredoxin) must be > 0.75.

7.4.1.6 If the criteria of 7.4.1.3 to 7.4.1.5 are met, proceed with enymatic assay. If not, reorder the non-conforming reagent or prepare the appropriate reagent again.

7.4.2 Enzymatic Assay

7.4.2.1 Pipette (in milliliters) the following reagents in the following sequence into appropriate cuvettes :

7.4.2.2 Immediately mix thoroughly by swirling or vortexing and incubate at room temperature for 30 min.

7.4.2.3 Mix by inversion and equilibrate to 25 ^oC for one minute. Then add:

7.4.2.4 Immediately mix by inversion and record the increase A_550nm for a minimum total reaction time of 2 minutes. Read the A_550nm at a maximum of every 4 seconds. Obtain the ΔA_550nm /minute using the maximum linear rate for all test and blanks over a time interval of thirty seconds.

7.4.2.5 For all enzymatic rates, the net Δ_A550nm / minute must be in the range of 0.06 to 0.25.

7.5 CALCULATIONS

7.5.1 Net ΔA_550nm / minute Test_{t-1 to t-3} = ΔA_550nm / minute (Test_{t-1 to t-3}) - ΔA_550nm / minute (Blank_{Blank–1 to Blank-3})

where,
df = dilution factor of enzyme solution
1.0 = volume (in milliliters) of total enzyme reaction mixture
0.10= volume (in milliliters) of enzyme used
28.0= Millimolar extinction coefficient of Cytochrome C from bovine heart,(reduced)

7.6 FINAL ASSAY CONTENTRATION:
In a 1.00 mL enzymatic reaction mix, the final concentrations are 67 mM Trizma, 75 mM cytochrome C, 10 mM ferredoxin, 0.2 mM b-nicotinamide adenine dinucleotide phosphate, reduced, and 0.001 to 0.008 units of ferredoxin NADP reductase.