Origin

Bacillus licheniformis

Reaction

NADH+H⁺+O2 → NAD⁺+ H²O²

Appearance

White amorphous powder.

Activity

More than 50 units/mg protein.

Unit definition

One unit is the amount of enzyme required to oxidize 1 μmol of NADH per minute at pH7.0 at 30 ºC.

Storage

Stable for one year when stored below 5 ºC and also stable at room temperature for at least one week. For prolonged storage, keep at -20 ºC.

Contaminants

Sometimes, trace amount of catalase might be detected. Therefore, the addition of 10 mM NaN₃ into the reaction mixture is recommended when the complete elimination of catalase is needed

Properties

Molecular weight	Approx. 240,000 Da
Optimum pH	6.5-7.5
Optimum temperature	45℃
pH stability	7.0-8.5
Thermal stability	below 30 ºC (pH 7.5, 10 min)and below 40 ºC (in the coexistence of 0.1% bovine serum albumin,pH 7.5, 10 min)
Michaelis constant	3.2×10-5M（NADH) 6.7×10-6M（FAD)
Substrate specificity	In the absence of added FAD both NADH and NADPH are oxidized equally, but by the addition of FAD (about 30 μM) to reaction mixture the reaction velocity to NADH is accelerated about 20–30 times in contrast to 2–3 times of NADPH. Accordingly, the substrate specificity of NADH is about 10 times larger than that of NADPH in the presence of added FAD.

Assay method

(Spectrophotometric method)

Reagents

1. Potassium phosphate buffer, pH 7.0 : 250 mM
2. Cofactor solution : 1 mM FAD
3. Substrate solution : 2 mM NADH
4. Enzyme solution : Prepare approximately 5 units/ml solution of NADH oxidase in 30 mM potassium phosphate buffer, pH 7.5. This solution, stored at 5 ºC, is stable for at least one week. For assay of activity dilute this enzyme solution to approximately 0.2 unit/ml with enzyme diluent.
5. Enzyme diluent : 30 mM Potassium phosphate buffer (pH 7.5) containing 0.1% bovine serum albumin.

Procedure

1. Prepare the following reaction mixture in a cuvette (d =1.0 cm) and equilibrate at 30 ºC for about 10 minutes.
   1.9 ml H₂O
   0.6 ml Potassium phosphate buffer (A)
   0.3 ml FAD solution (B)
   0.1 ml NADH solution (C)
2. Add 0.1 ml enzyme solution (approximately 0.2 unit/cuvette) (D) and mix.
3. Read the decrease of optical density at 340 nm against the reaction mixture omitted NADH and enzyme as reference for several minutes at timed interval in a spectrophotometer thermostated at 30 ºC, and calculate the DA340 per minute from the initial linear portion of the decrease of optical density (usually, DA340 per minute is calculated from the decrease between 30 and 90 seconds after the addition of enzyme solution).

Calculation

unit / cuvette	= DA340 x 3 ÷ 6.22
where 3	= volume (ml) of solution in a cuvette
6.22	= millimolar extinction coefficient of NADH

Package

5 units, 25 units