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Assay Procedure for N-Acetylneuraminic Acid Aldolase

Principle

The disappearance of NADH is measured at 340 nm by spectrophotometry.

Unit definition

One unit causes the oxidation of one micromole of NADH per minute under the conditions described below.

Method

Reagents

 

A. NANA solution
:50mM [Dissolve 309mg of N-acetylneuraminic acid (MW=309) in ca. 15ml of 50mM K-phosphate buffer, pH 7.5 and, after adjusting the pH to 7.5 with 1N KOH, fill up to 20ml with the same buffer.] (Stable for at least one week if stored at 0- 5℃)
B. LDH solution :ca. 50U/ml [Dilute pig heart lactate dehydrogenase to a concentration of ca. 50U/ml with ice-cold 50mM Kphosphate buffer, pH 7.5] (Should be freshly prepared)
C. NADH solution :1.0mM [Dissolve 7.6mg of NADH・Na2・3H2O (MW=763) in 10ml of 50mM Kphosphate buffer, pH 7.5] (Should be freshly prepared)
D. Buffer solution :50mM K-phosphate buffer, pH 7.5
E. Enzyme diluent :50mM K-phosphate buffer, pH 7.5 containing 0.2% BSA

Procedure

  1. Prepare the following reaction mixture in a cuvette (d=1.0cm and equilibrate at 37℃ for about 5 minutes.

    1.0ml Substrate solution (A)
    0.5ml LDH solution          (B)
    0.5ml NADH solution       (C)
    0.4ml Buffer solution        (D)
Concentration in assay mixture
K-Phosphate buffer 50 mM
NANA 20 mM
NADH 0.2mM
LDH ca.10 U/ml
  1. Add 0.1ml of the enzyme solution* and mix by gentle inversion.
  2. Record the decrease in optical density at 340nm against water for 3 to 4 minutes in spectrophotometer thermostated at 37℃, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).

At the same time, measure the blank rate (ΔOD blank) using the same method the test except that the enzyme diluent (E) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (E) and dilute to 0.1-0.3U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula:

 

Weight activity (U/mg)=(U/ml)×1/C

 

Vt :Total volume (2.5ml)
Vs :Sample volume (0.1ml)
6.22 :Millimolar extinction coefficient of NADH (F/micromole)
1.0 :Light path length (cm)
df :Dilution factor
C :Enzyme concentration in dissolution (c mg/ml)

 

Materials

     

This procedure is for informational purposes. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.

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