Sialidase

UNIT DEFINITION

One unit is defined as the amount of enzyme which hydrolyze 1 μmol of sialic-acid per minute under the conditions described below.

REAGENTS PREPARATION

Reagent I: 50 mM pH 7.5 potassium phosphate buffer.

Reagent II: 8.0 mM N-Acetylneuraminosyl-D-lactose (Dissolve 52.5mg of 3’-sialylactose in 10 mL of precooled reagent I in ice bath for standby).

Reagent III: 100U/mL NAL: N-acetylneuraminic acid aldolase was diluted with precooled reagent I to 100U/ ml, use ice bath for standby, and store at 4 ℃.

Reagent IV: 100U/ml LDH: Dilute L-LDH (L-lactate dehydrogenase) to 100U/mL with precooled reagent I, use ice bath for standby, and store at 4 ℃.

Reagent V: 2.5 mM NADH: 17.8mg NADH dissolve in 10 mL deionized water and store at -20 ℃ after sub packaging, Thaw before use and use ice bath for standby.

Reagent VI: Enzyme diluent: Dissolve 500 mg BSA in 100 mL reagent I until the final concentration is 0.5 % (W/V). Use ice bath for standby and store at 4 ℃.

Sample: dilute the enzyme with enzyme diluent to 0.1- 0.25 U/mL.

PROCEDURE

1. Pipette Reagent I 125μL, Reagent II 300μL, Reagent V 50μL, Reagent III 62.5μL and Reagent IV 62.5μL fully mix into a cuvette and equilibrate at 37 ℃ for about 2 minutes.

2. Add 25 μL of the enzyme solution to the cuvette and mix at 37 ℃ for 10 min.

3. Record the ΔAs at 340 nm in 7-10 minute in a spectrophotometer thermostated at 37 ℃.

* At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.

∆A = ∆As – ∆Ab