Neuraminidase Assay

Method: The assay is based on the measurement of sialic acid (NANA) released from bovine submaxillary mucin (Worthington Code: MU). One unit causes the release of one micromole of sialic acid per minute at 37°C and pH 5.0, from bovine submaxillary mucin under the specified conditions. A coupled enzyme assay for determining neuraminidase is described by Ziegler and Hutchinson (1972).

Reagents

• 0.2 M Sodium metaperiodate in 9 M phosphoric acid
• 0.5 M Sodium sulfate
• 0.755 M Sodium arsenite in 0.5 M sodium sulfate with 0.1 N H2SO4. Prepare by dissolving 25 gm sodium arsenite (molecular weight 129.91) in 250 ml of 0.5 M sodium sulfate and add 5 ml 5 N H2SO4. Slight heating may be necessary in order to effect solution.
• 2.5 N HCl
• 5% Phosphotungstic acid in 2.5 N HCl
• 0.6% Twice crystallized thiobarbituric acid in 0.5 M Sodium sulfate. Prepare by dissoving 4.5 grams 2x crystallized thiobarbituric acid in 750 ml 0.5 M sodium sulfate. Heat to effect solution and allow to cool. A crystalline precipitate will form. Use only the supernatant for the assay.
• 0.1 M Acetic acid, pH 5.0
• 1% Mucin, pH 5.0. Prepare by dissolving 100 mgs Worthington Submaxillary Mucin (Code: MU) in 9 ml reagent grade water, adjust pH to 5.0 with acetic acid and dilute to a final volume of 10 ml. The solution will appear turbid at pH 5.0.
• Cyclohexanone. Caution: read product label for handling instructions.

Enzyme

Dissolve enzyme at one mg/ml in reagent grade water. Immediately prior to use prepare four dilutions ranging from 0.1 mg/ml to 0.005 mg/ml.

Procedure

Prepare a 37°C water bath. Adjust the spectrophotometer to 549 nm. Into numbered tubes pipette as follows:

Mucin	0.4 ml
0.1 M acetic acid	0.5 ml

At timed intervals, add 0.1 ml of the respective enzyme dilution. Include a blank of 0.1 ml of water in place of the enzyme. Incubate for 30 minutes in the 37°C water bath. Stop the reaction at timed intervals by adding 1 ml of 5% phosphotungstic acid to each tube. Centrifuge for 10 minutes. Remove a 0.5 ml aliquot of each supernatant to dry clean test tubes. To each, add 0.1 ml of 0.2 M sodium metaperiodate. Allow to stand for 20 minutes at room temperature, add 1.0 ml of 0.755 M sodium arsenite. Shake the tubes until the brown color disappears, then add 3.0 ml of the 0.6% thiobarbituric acid. Heat for 15 minutes in a boiling water bath, cool in an ice bath for 5 minutes and add 4.6 ml of cyclohexanone to each tube. Extract the color in cyclohexanone layer by vortexing. Centrifuge at high speed for 15 minutes. Remove colored cyclohexanone layer and read A₅₄₉ of the colored cyclohexanone layer versus a reagent grade water blank.

Note: The final A₅₄₉ of sample should not exceed 0.5 absorption unit.

Calculation