DIAPHORASE from Clostridium sp.


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 GradeⅢ 30U/mg-solid or more
(containing approx. 15% of stabilizers)
Contaminants Myokinase ≤5.0×10⁻¹%
NAD(P)H oxidase ≤5.0×10⁻¹%
Stabilizers FMN, NAD(P)H
Stability Stable at -20°C for at least Two years (Fig.1)
Molecular weight 24,000 ¹ ⁾
Michaelis constants 2.0×10⁻⁵M (NADH), 6.0×10⁻⁶M (NADPH)
Structure One mol of FMN per mol of enzyme ¹ ⁾
Inhibitor N-Ethylmaleimide
Optimum pH 8.5(Fig.3)
Optimum temperature: 50℃(Fig.4)
pH Stability pH 7.5 (30℃, 3hr)(Fig.5)
Thermal stability  below 30℃ (pH 7.5, 30min)(Fig.6)
Substrate specificty Either NADH or NADPH can be used as a reductant. The catalytic
ratio (NADPH/NADH) is 0.6 in the assay method. Neither oxygen nor cytochrome C can be utilized as a hydrogen acceptor.


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⁺+Leucodye

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

Unit definition:

One unit causes the decrease of one unit absorbance (1.0) of DCPIP per minute under the condeitions described


A. Buffer solution 0.2M Tris-HCl, pH 7.5
B. NADH solution 6.0mM (Prepare freshly and store on ice)
C. DCPIP solution 1.2mM [3.9mg DCPIP・2HO/10ml of HO](Should be prepared fresh)
D. Enzyme diluent Buffer solution(A) containing 0.1% of bovine serum albumin.


Concentration in assay mixture
Tris buffer 27 mM
NADH 0.20mM
BSA ca.33µg/ml

1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 25℃ for about 5 minutes.

2.4 ml HO
0.3ml Buffer solution (A)
0.1ml NADH solution (B)

2. Add 0.1ml each of the enzyme solution* and DCPIP solution (C) in this order and mix by rapid inversion.

3. Record the decrease of optical density at 600nm against water for 2 to 3 minutes in a spectrophotometer thermostated at 25°C, and calculate theΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by using the same method as the 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 0.4− 0.8U/ml with ice-cold enzyme diluent (D) and store on ice.


Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test−ΔOD blank ) × df

Volume activity (U/ml) =                                                               =ΔOD/min×10×df



Weight activity (U/mg) = (U/ml) × 1/C

: Sample volume (0.1ml)
: Unit absorbance at 600nm due to unit definition
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)


  1. F.Kaplan, P.Setlow and N.O.Kaplan; Arch,Biochem.Biophys., 132, 91 (1969).

Table 1. Effect of Various Chemicals on Diaphorase
[The enzyme dissolved in 0.2M Tris-HCl buffer, pH 7.5 (40U/ml) was incubated with each chemical at 25℃ for 1hr.]
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 NaF 2.0 102
Metal salt 2.0   NaN₃ 2.0 100
  99 EDTA 5.0 99
CaCl₂   102 o-Phenanthroline 2.0 99
Ba(OAc)₂ 100 α,α′-Dipyridyl 1.0 101
FeCl₂   90 Borate
5.0 100
CoCl₂   101 IAA 2.0 99
MnCl₂   96 NEM 2.0 100
ZnCl₂   100 Hydroxylamine 2.0 101
Cd(OAc)₂   100 TritonX-100 0.10% 106
NiCl₂   99 Brij 35 0.10% 104
CuSO₄   87 Tween 20
0.10% 107
Pb(OAc)₂   88 Span 20 0.10% 101
AgNO₃   103 Na-Cholate 0.10% 99
HgCl₂   103 SDS 0.05% 32
PCMB 2.0 90 DAC 0.05% 32
MIA 1.0 100      

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