CODE No.:B1D211

DIAPHORASE II (Di-2)

[EC 1.8.1.4]
from Bacillus stearothermophilus

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 150U/mg protein
Contaminants	:	(as Diaphorase activity = 100%)
		Adenylate kinase	<0.01%
		NADH oxidase	< 0.10%

PROPERTIES

Molecular weight	:	ca. 110,000
Subunit molecular weight	:	ca. 50,000
Optimum pH	:	6.5	(Fig.1)
pH stability	:	6.5 - 8.0	(Fig.2)
Isoelectric point	:	4.8
Thermal stability	:	No detectable decrease in activity up to 60°C.	(Fig.3, 4)
Michaelis constants	:	(70mM Potassium phosphate buffer, pH 6.5, at 30°C)
		Lipoate	2.0mM
		NADH	0.01 mM
Substrate specificity	:	Lipoate	100%
		Lipoamide	43%
		2,6-Dichlorophenolindophenol (DCIP)	20%
		Nitro blue tetrazolium (NBT)	110%
		NADH	100%
		NADPH	1%
Effectors	:	cations and anions	(Fig.5,6)

STORAGE

stable at -20°C for at least one year

APPLICATION

The enzyme is useful for measurement of various dehydrogenase reactions in the visible spectral range.

ASSAY

Principle

The change in absorbance is measured at 340 nm according to the following reaction.

Unit Definition

One unit of activity is defined as the amount of Di-2 that forms 1μmol of NAD⁺ per minute at 30°C.

Solutions

1. Buffer solution ; 100mM Potassium phosphate, pH 6.5
2. EDTA solution ; 27 mM (0.100 g EDTA disodium salt·2H₂O/10mL distilled water)
3. Lipoate solution ; 60mM (0.124 g DL-α-lipoate/10mL 0.1 M K₂HPO₄ solution)
4. BSA solution ; (100 mg BSA/10mL distilled water)
5. NADH solution ; 13.1 mM (0.100 g NADH disodium salt·3H₂O/10mL distilled water)
6. NAD⁺ solution ; 30mM (0.215 g NAD⁺·3H₂O/10mL distilled water)

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50mM potassium phosphate buffer, pH 7.5.

Procedure

1. Prepare the following reaction mixture and pipette 3.00mL of reaction mixture into a cuvette.

   Solution I	21.15mL	Solution IV	2.10mL
   Solution II	0.90mL	Solution V	0.45mL
   Solution III	5.10mL	Solution VI	0.30mL

2. Incubate at 30°C for about 3 minutes.
3. Add 0.01 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 340 nm per minute (ΔAbs₃₄₀) in the linear portion of curve.

Calculation

• d.f. ; dilution factor
• 6.22 ; millimolar extinction coefficient of NADH (cm²/μmol)
• *Protein concentration ; determined by Bradford's method

REFERENCE

1. Packman, L.C., and Perham. R.N.; FEBS Lett., 139, 155 (1982)

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