Recycling Micro-Assay of β-NAD and β-NADH

1. OBJECTIVE

To standardize a procedure for the recycling micro-assay of β-NAD and β-NADH.

2. SCOPE

This procedure applies to products that have a specification for the recycling micro-assay of β-NAD and β-NADH.

3. DEFINITIONS

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

3.2. β-NADH = β-Nicotinamide Adenine Dinucleotide, Reduced Form

3.3. β-NAD = β-Nicotinamide Adenine Dinucleotide, Oxidized Form

3.4. MTT = (3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium Bromide)

3.5. PES = Phenazine Ethosiulfate

4. DISCUSSION

β - NAD + ethanol ^{AlcoholDehydrogenase} > β-NADH + Acetaldehyde
β-NADH + MTT + PES → colored dye + β - NAD

5. RESPONSIBILITIES

It is the responsibility of all Analytical Services personnel to 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: PH = 7.8, T = 25 °C, A570nm, Path Length = 1 cm

7.2. METHOD: Continuous Spectrophotometric Rate Determination

7.3. REAGENTS

7.3.1. 1 M Bicine
Prepare a 163.2 mg/ml solution using Bicine, Sigma-Aldrich product number B3876, in purified water. May vortex to aid in dissolution. Prepare fresh.

7.3.2. 200 proof Ethyl Alcohol
Sigma-Aldrich Product Number 459828 (ETOH)

7.3.3. 0.1 M Ethylenediaminetetraacetic Acid (EDTA)
Prepare a 41.6 mg/mL solution of Ethylenediaminetetraacetic Acid, Tetrasodium Salt, Hydrate, Sigma-Aldrich Product Number ED4SS, in purified water. May vortex to aid in dissolution.

7.3.4. 20 mM Phenazine Thosulfate (PES)
Prepare a 6.7 mg/mL solution of Phenazine Thosulfate, Sigma-Aldrich Product Number P4544, in purified water. May vortex to aid in dissolution. Prepare fresh and keep on ice. This reagent degrades quickly so use within two hours of dissolution.

7.3.5. 10 mM (3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium Bromide (MTT)
Prepare a 4.1 mg/mL solution of (3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium Bromide, Sigma-Aldrich Product Number M2128, in purified water. May need to vortex and/or sonicate to aid in dissolution. Be sure to protect reagent from light and keep at room temperature throughout assay.

7.3.6. 0.045 mM β-Nicotinamide Adenine Dinucleotide, Oxidized Form (β-NAD)
Prepare a 0.045 mM solution of β-Nicotinamide Adenine Dinucleotide, Oxidized Form, Sigma-Aldrich Product Number N1636, in cold purified water. Correct for water content of the specific lot. Use class A volumetric glassware. Sonication may be required to aid in dissolution. This reagent should be prepared just before use.

7.3.7. Test Solution (Test)
Prepare a test solution in cold purified water that gives a Δ A_570nm/minute over a 5-minute window of ≤ 0.1. Primarily this assay is performed on Alcohol Dehydrogenase (ADH). For ADH, the concentration is usually in the range of 1-10 mg protein/mL.

7.3.8. 120 mM Bicine, 3.7% ETOH, 5 mM EDTA (RC)
Prepare a reaction cocktail in class A volumetric glassware, by combining (in milliliters) the following:

	(RC)
Reagent 7.3.1	12.0
Reagent 7.3.2 (ETOH)	3.7
Reagent 7.3.3 (EDTA)	5.0
Purified Water	79.3

Adjust the pH to 7.8 at 25 °C using 1 M Sodium Hydroxide, Sigma-Aldrich Product Number S2567 or 1 M Hydrochloric Acid, Sigma-Aldrich Product Number H3162.

7.4. PROCEDURE

7.4.1. Pipette the following (in milliliters) into suitable cuvettes using a suitably thermostatted spectrophotometer at 25 °C in the exact order as listed below. Reagent 7.3.4, reagent 7.3.5 and reagent 7.3.6 should be added as quickly as possible after each other. More than two test levels may be performed:

Blank	Test 1	Test 2	Std.1	Std.2	Std.3
RC (Reagent 7.3.8)	2.50	2.50	2.50	2.50	2.50
Purified Water	0.10	------	------	------	------
Test (Reagent 7.3.7)	------	0.10	0.10	0.10	0.10
PES (Reagent 7.3.4)	0.25	0.25	0.25	0.25	0.25
MTT (Reagent 7.3.5)	0.13	0.13	0.13	0.13	0.13
β-NAD (Reagent 7.3.6)	------	------	------	0.005	0.010

Immediately mix by inversion and record the increase in A_570nm for 6 minutes. Obtain the Δ A_570nm/minute for a 5-minute window for all levels.

7.5. CALCULATIONS

7.5.1. Standard Curve:
Corrected A_570nm/minute Standards = (ΔA_570nm/minute Standard - ΔA_570nm/minute Blank - ΔA_570nm/minute Test)

Where: ΔA_570nm/minute Test is the average of all ΔA_570nm/minute corrected tests as determined in 7.5.2.
Plot corrected Δ A_570nm/minute of standards versus nanomoles of β-NAD/reaction mixture of standards and determine the slope, y-intercept and r-square value of the line. The r-square value should be 0.99 or better.

7.5.2. Test Determination:
Corrected ΔA_570nm/minute Test = (ΔA_570nm/minute Test - ΔA_570nm/minute Blank)
Determine the nmoles β-NAD/Test using the standard curve and average all test levels within 90% agreement.

nmoles ADH	=	(0.100)(10.0)(mg Protein/mg solid)(1,000,000)
Test		Molecular Weight

moles β-NAD	=	nmoles of β-NAD/Test
moles ADH		nmoles of ADH/Test

0.100 = Volume (in milliliters) of Reagent 7.3.7 (Test) used in the reaction mixture.
10.0 = Concentration (in mg solid/mL) of Reagent 7.3.7 (Test)
mg Protein/mg Solid = Protein results based on E1% at 280nm.
1,000,000 = Conversion from milligrams to nanograms
Molecular Weight = Molecular weight for Reagent 7.3.7 (Test). For alcohol dehydrogenase, the molecular weight equals 160,000 from equine liver and 141,000 from baker’s yeast.

7.5.3. For product A3263 the specification is suitable for recycling micro-assay of β-NAD and β-NADH. The suitability criterion has been established as follows: Since A7011 has bound β-NAD and β-NADH and A3263 is supposed to have very small amounts of bound β-NAD and β-NADH, then A3263 is deemed suitable if the mole/mole ratio of β-NAD/ADH is 10 times less than the average mole/mole ratio of β-NAD/ADH of A7011.

8. APPROVAL

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