DIAPHORASE from Clostridium sp.

DAD-301

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:	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

PROPERTIES
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.

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⁺+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
below.

Method:

Reagents
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・2H₂O/10ml of H₂O](Should be prepared fresh)
D. Enzyme diluent:	Buffer solution(A) containing 0.1% of bovine serum albumin.

Procedure

Concentration in assay mixture
Tris buffer	27 mM
NADH	0.20mM
DCPIP	40 µM
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 H₂O
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.

Calculation

Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test−ΔOD blank ) × df

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

1.0×Vs

Weight activity (U/mg) = (U/ml) × 1/C

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

REFERENCES

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 activity(%)	Chemical	Concn.(mM)	Residual activity(%)
None	−	100	NaF	2.0	102
Metal salt	2.0		NaN₃	2.0	100
MgCl₂		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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