DIAPHORASE from microorganism.
NAD(P)H+ H⁺+ Acceptor(ox)
► NAD(P) ⁺+ Acceptor(red)
|Appearance:||Yellowish amorphous powder, lyophilized|
|Activity:||Grade III 500 U/mg-solid or more|
NADH oxidasee ≤1.0×10⁻¹%
|Stability:||Stable at -20°C (Fig.1)|
|Molecular weight :||approx. 48,000|
|Michaelis constant:||2.2 × 10-⁴M (NADH), 2.9 × 10-²M(NADPH)|
|Inhibitors :||Fe³⁺, Mn²⁺, Cu²⁺, Pb²⁺
|Optimum pH :||8.0(Fig.3)|
|Optimum temperature :||60°C(Fig.4)|
|pH Stability :||5.0–10.0 (25°C, 20hr)(Fig.5)|
|Thermal stability :||below 70°C (pH7.5, 15min)(Fig.6)|
|Substrate specificity:||Either NADH or NADPH can be used as a reductant.|
|Effect of various chemicals :||(Table 1)|
This enzyme is useful for colorimetric determination of NAD(P)H and many dehydrogenases when coupled with various dyes which act as hydrogen acceptors from NAD(P)H.
The reduction of DCPIP(2,6-dichlorophenol-indophenol) is measured at 600 nm by spectrophotometry.
One unit causes the decrease of one micromole of DCPIP per minute under the conditions described below.
|A. Buffer solution :||0.2 M Tris-HCl, pH 8.0|
|B. NADH solution:||36 mM (Prepare freshly and store on ice)|
|C. DCPIP solution :||2.4 mM [7.8mg DCPIP(Mw:326.11)/10ml of H₂O](Should be prepared fresh)|
|D. Enzyme diluent:||Buffer solution (A) containing 0.5% of Tween20|
|Concentration in assay mixture|
|Tris buffer||27 mM|
|Tween20||ca. 167 µg/ml|
1. Prepare the following reaction mixture in a cuvette
(d=1.0cm) and equilibrate at 37oC for about 4 minutes.
0.3ml Buffer solution (A)
0.1ml NADH solution (B)
2. Add 0.1 ml of the enzyme solution* and mix by gentle pipetting and equilibrate at 37oC for another 1 min.
3. Add 0.1 ml of DCPIP solution (C) and mix by rapid inversion.
4. Record the decrease of optical density at 600 nm against water for 3 to 4 min in a spectrophotometer
thermostated at 37oC, and calculate the ΔOD per minute from the initial linear portion of the curve (OD test).
At the same time , measure the blank rate (OD blank) by the same method as test except that the enzyme
diluent is added instead of the enzyme solution.
* Dissolve the enzyme preparation in ice-cold buffer solution (A) (approx. 1.0% solution), dilute to 0.10−0.25U/ml with ice-cold enzyme diluent (D) and store on ice.
Activity can be calculated by using the following formula :
ΔOD/min(OD test–OD blank)× Vt× df
Volume activity (U/ml) =
Weight activity (U/mg)=(U/ml)×1/C
- : Total volume (3.0ml)
- : Sample volume (0.1ml)
- : Millimolar extinction coefficient of DCPIP under the assay conditions (㎠/micromole)
- : Light path length (cm)
- : Dilution factor
- : Enzyme concentration in dissolution (C mg/ml)
MIA, Monoiodoacetate; EDTA, ethylenediaminetetraacetate; IAA, iodoacetamide; NEM, N-Ethylmaleimide; SDS, sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride