Enzymatic Assay of Neuraminidase

(EC 3.2.1.18) N-Acetylneuramin-Lactose as Substrate

1. Objective

To standardize a procedure for the enzymatic determination of neuraminidase.

2. Scope

2.1. This procedure applies to products that have a specification for neuraminidase content by enzymatic determination.

2.2. Do not use this assay for neuraminidase from Newcastle disease virus, Sigma-Aldrich Product Number N5146, and neuraminidase from Streptococcus sp., Sigma-Aldrich Product Number N5271.

3. Definitions

3.1. Unit Definition - One unit will liberate 1.0 μmole of n-acetylneuaminic acid per minute at pH 5.0 at 37oC using NANA-Lactose as substrate.

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

3.3. NANA - N-Acetylneuraminic Acid

4. Discussion

N-Acetylneuramin-Lactose + H2O    Neuraminidase   > NANA + Lactose

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

7.1.1   T = 37ºC

7.1.2   pH 5.0

7.1.3   A550nm

7.1.4   Light path = 1 cm

7.2 Method
Colorimetric

7.3. Reagents

7.3.1   100 mM Sodium Acetate Buffer with 2 mM Calcium Chloride, pH 5.0 at 37oC (Buffer)

7.3.1.1   Prepare 100 ml in purified water using Sodium Acetate, Anhydrous, Sigma-Aldrich Product Number S8750 and Calcium Chloride, Dihydrate, Sigma-Aldrich Product Number C3881.

7.3.1.2   Adjust to pH 5.0 at 37oC with 1 M HCl.

7.3.1.3   Only neuraminidase from Vibrio cholera requires calcium ions. However, the presence of calcium ions does not interfere with the activity of neuraminidase from other sources.

7.3.2   0.085% (w/v) N-Acetylneuramin-Lactose (2→3) isomer Solution (NAN-Lactose)

7.3.2.1   Prepare a 0.85 mg/ml solution in Reagent 7.3.1 using N-Acetylneuramin-lactose, Sigma-Aldrich Product Number A3307.

7.3.2.2   This assay system is operating at less than Km value for the substrate. It is critical that the concentration of the 2→3 isomer is 0.085% (w/v). Please correct for water and isomer content.

7.3.3   0.2% (w/v) Bovine Serum Albumin Solution (BSA)
Prepare a 2 mg/ml solution in purified water using Albumin, Bovine, Sigma-Aldrich Product Number A6003.

7.3.4   5% (w/v) Phosphotungstic Acid Solution (PT)
Prepare 50 mg/mL in 2.5 M HCl using Phosphotungstic Acid, Free Acid, Sigma-Aldrich Product Number P4006.

7.3.5   Neuraminidase Enzyme Solution (Enzyme)
Immediately before use, prepare a solution containing approximately 0.03 – 0.09 unit/ml of Neuraminidase in cold Reagent 7.3.3.

7.3.6   9 M Phosphoric Acid Solution (PAS)
Prepare 20 mL in purified water using Phosphoric acid, Aldrich Product Number 215104.

7.3.7   200 mM Sodium m-Periodate Solution (Per)
Prepare 10 mL in Reagent 7.3.6 using Sodium m-Periodate, Sigma-Aldrich Product Number S1878.

7.3.8   10% (w/v) Sodium m-Arsenite with 50 mM Sulfuric Acid and 500 mM Sodium Sulfate Solution (Ars)
Prepare 50 mL in purified water using sodium m-Arsenite, Sigma-Aldrich Product Number S7400, Sulfuric Acid, Aldrich Product Number 258105, and Sodium Sulfate, Anhydrous, Sigma-Aldrich Product Number S9627.

7.3.9   0.6% (w/v) Thiobarbituric Acid and 500mM Sodium Sulfate Solution (TBA)
Prepare 200 mL in purified water using 2-Thiobarbituric Acid, Sigma-Aldrich Product Number T5500, and Sodium Sulfate, Anhydrous, Sigma-Aldrich Product Number S9627.

7.3.10   5% (v/v) HCl in Butanol Solution (But)
Prepare by combining 5 mL of concentrated HCl, Aldrich Product Number 258148 and 95 mL of n-Butanol, Sigma-Aldrich Product Number B0878.

7.3.11   0.20 mM N-Acetylneuraminic Acid Standard Solution (NANA)
Prepare 100 mL in purified water using N-Acetylneuraminic Acid, Sigma-Aldrich Product Number A2388

7.4 Enzyme Incubation Test Method

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

    Test
  Test Blank
Reagent 7.3.1 (Buffer) 0.20 0.20
Reagent 7.3.2 (NAN-Lactose) 0.20 0.20

7.4.2   Mix by inversion and equilibrate to 37oC. Then add:

Reagent 7.3.5 (Enzyme) 0.10 ------
Reagent 7.3.3 (BSA) ------ 0.10

7.4.3   Immediately mix by inversion and incubate at 37oC for exactly 10 minutes. Then add:

Reagent 7.3.4 (PT) 0.50 0.50

7.4.4   Immediately, mix by inversion and centrifuge for 3 minutes at low speed.

7.5 Standard Curve

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

              Std
  Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Blank
Reagent 7.3.11 (NANA) 0.05 0.10 0.20 0.30 0.40 0.50 -----
Purified water 0.45 0.40 0.30 0.20 0.10 ----- 0.50
Reagent 7.3.4 (PT) 0.50 0.50 0.50 0.50 0.50 0.50 0.50

7.5.2   Immediately, mix by inversion and centrifuge for 3 minutes at low speed.

7.6 Color Development

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

    Test             Std
  Test Blank Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Blank
Test supernatant 0.50 ------- ------- ------- ------- ------- ------- ------ ------
Test Blank supernatant ------- 0.50 ------- ------- ------- ------- ------- ------ ------
Std 1 supernatant ------- ------- 0.50 ------- ------- ------- ------- ------ ------
Std 2 supernatant ------- ------- ------- 0.50 ------- ------- ------- ------ ------
Std 3 supernatant ------- ------- ------- ------- 0.50 ------- ------- ------ ------
Std 4 supernatant ------- ------- ------- ------- ------- 0.50 ------- ------ ------
Std 5 supernatant ------- ------- ------- ------- ------- ------- 0.50 ------ ------
Std 6 supernatant ------- ------- ------- ------- ------- ------- ------- 0.50 ------
Std Blank supernatant ------- ------- ------- ------- ------- ------- ------- ------ 0.50
Reagent 7.3.7 (Per) 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10

7.6.2   Mix by inversion and incubate for 20 minutes at 25°C. Then add:

Reagent 7.3.8 (Ars) 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00   1.00

7.6.3   Vortex until the yellow-brown color disappears. Then add:

Reagent 7.3.9 (TBA) 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00

7.6.4   Transfer to a boiling water bath and incubate for 15 minutes. Allow to cool to room temperature. Then add:

Reagent 7.3.10 (But) 4.60 4.60 4.60 4.60 4.60 4.60 4.60 4.60 4.60

7.6.5   Shake or vortex the solution until most of the pink color has been extracted from the lower layer.

7.6.6   Centrifuge for 2-3 minutes at low speed, ~1000 rpm.

7.6.7   Transfer the upper layer to suitable cuvettes and immediately record the A550nm for Test, Test Blank, Standard, and Standard Blank.

7.7 Calculations

7.7.1   Standard Curve:

7.7.1.1   ΔA550nm Standard = A550nm Standard – A550nm Standard Blank

7.7.1.2   Plot the ΔA550nm Standard vs. μmoles N-Acetylneuraminic Acid. Obtain the slope, y-intercept and R2 for the standard curve.

7.7.2   Sample Determination:

7.7.2.1   Determine the μmoles of N-Acetylneuraminic Acid liberated using the Standard Curve.

7.7.2.2. Units/mL enzyme = (μmoles of NANA liberated)(df)
    (0.10)(10)


df = Dilution factor of enzyme
0.10 = volume (in milliliters) of enzyme used
10 = time (in minutes) of the assay per the unit definition

7.8 Final Assay Concentration
In a 0.50 mL reaction mix, the final concentrations are 80 mM sodium acetate, 1.6 mM calcium chloride, 0.034% (w/v) n-acetylneuramin-lactose (2→3 isomer), 0.04% (w/v) Bovine Serum Albumin, and 0.003 – 0.009 unit neuraminidase.

8. References & Attachments

8.1 Schneir, M.L. and Rafelson, M.E., Jr. (1996) Biochim. Biophys. Acta. 130, 1-11.

8.2 Cassidy, J.T., Jourdia, G.W., and Roseman, S. (1965) J. Biol. Chem. 240, 3501-3506.

8.3 Warren, L. (1959) J Biol. Chem. 234, 1971-1975.

9. Approval

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Materials

     
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