DIAPHORASE from microorganism.

DAD-311

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

PREPARATION and SPECIFICATION
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%

PROPERTIES
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)

APPLICATIONS

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.

ASSAY

Principle:

diaphorase

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.

Method:

Reagents
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

Procedure

Concentration in assay mixture
Tris buffer	27 mM
NADH	1.2 mM
DCPIP	80 µM
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.

Calculation

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

20.9×1.0×Vs

Weight activity (U/mg)=(U/ml)×1/C

Vt: : Total volume (3.0ml)
Vs: : Sample volume (0.1ml)
20.9: : Millimolar extinction coefficient of DCPIP under the assay conditions (㎠/micromole)
1.0: : Light path length (cm)
df: : Dilution factor
C: : 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 activity(%)	Chemical	Concn.(mM)	Residual activity(%)
None	−	100	NaN₃	2.0	104
Metal salt	2.0		EDTA	5.0	105
MgCl₂		102	o-Phenanthroline	2.0	105
CaCl₂		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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