DIAPHORASE from microorganism.


NAD(P)H:(acceptor)oxidoreductase(EC 1.6.99.-)
NAD(P)H+ H⁺+ Acceptor(ox)                        NAD(P) ⁺+ Acceptor(red)

Appearance Yellowish amorphous powder, lyophilized
Activity Grade III 500 U/mg-solid or more
Contaminants Myokinase ≤5.0×10⁻¹%
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
Isoelectric point 5.0
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.




NADH +H⁺+DCPIP                                               NAD + DCPIP(red)

The reduction of DCPIP(2,6-dichlorophenol-indophenol) is measured at 600 nm by spectrophotometry.

Unit definition:

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
NADH 1.2 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.

2.4ml H₂O
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) =                                                                      = ΔOD/min× 1.43× df


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)


Table 1. Effect of Various Chemicals on Diaphorase
[The enzyme dissolved in 0.1M HEPES buffer, pH7.5 (5U/ml) was incubated with each chemical at 25oC for 1 hr.]
Chemical Conc.(mM) Residual
Chemical Concn.(mM) Residual
None 100 NaN 2.0 104
Metal salt 2.0   EDTA 5.0 105
MgCl₂   102 o-Phenanthroline 2.0 105
  99 α,α′-Dipyridyl 1.0 102
Ba(OAc)₂   100
Borate 5.0 104
FeCl₃   4.4
IAA 2.0 105
CoCl₂   94 NEM 2.0 106
MnCl₂   55 Hydroxylamine 2.0 107
ZnCl₂   84 Triton X-100 0.10% 109
Cd(OAc)₂   101 Brij 35 0.10% 109
NiCl₂   101 Tween 20 0.10% 116
CuSO₄   23 Span 20 0.10% 113
Pb(OAc)₂   46 Na-cholate 0.10% 110
AgNO₃   94 SDS 0.05% 91
MIA 1.0 104
DAC 0.05% 110
NaF 2.0 105      

MIA, Monoiodoacetate; EDTA, ethylenediaminetetraacetate; IAA, iodoacetamide; NEM, N-Ethylmaleimide; SDS, sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride

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