NIPRO ENZYMESDIAPHORASE 22 (Di-22)[EC 1. 8. 1. 4]from recombinant E.coli
NADH + Acceptor(ox.) + H+ ↔ NAD+ + Acceptor(red.)
SPECIFICATIONStateSpecific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants

Substrate specificity: Lyophilized: more than 150 U/mg protein: (as Diaphorase activity = 100 %)Adenylate kinase
NADH oxidase< 0.01 %< 0.20 %: ca. 110,000: ca. 50,000: 8.0(Fig,1): 6.0 – 9.0(Fig.2): No detectable decrease inactivity up to 70 °C.
(Fig. 3, 4): (50 mM HEPES buffer, pH 7.0, at 30 °C)
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT)
0.345 mM
NADH
0.033 mM
(Table 1): NADH
100 %
NADPH
1%
MTT
100 %
Lipoate103 %(Table 1)

STORAGEStore at -20℃
APPLICATIONThe enzyme is useful for measurement of various dehydrogenase reactions in the visible spectral range.

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 565 nm according to the following reaction.
NADH + MTT(ox.) + H+Di-22

NAD+ + MTT(red.)

Unit Definition
One unit of activity is defined as the amount of Diaphorase that forms 1 μmol of NAD+ per minute at30 °CSolutions
Ⅰ Buffer solution ; 100 mM HEPES, pH 7.0
Ⅱ 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution; 10 mM (20 mg MTT disodium salt･2H2O/5 mL distilled water)
Ⅲ NADH solution ; 13.1 mM (0.100 g NADH disodium salt・3H2O /10 mL distilled water)
Ⅳ Triton solution ; 10 % (1 mL TritonX-100 dilute with distilled water up to10 mL)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 1 to 5 U/mL with 50 mM potassium
phosphate buffer, pH 7.5, 1 mg/mL BSA.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 15.00 mL
SolutionⅣ
1.50 mL
10.80 mL
SolutionⅡ 1.50 mL
H2O
SolutionⅢ 1.20 mL
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 565 nm per minute (∆Abs565 ) in the linear portion of curve.
Calculation(∆Abs565) X (3.00 + 0.01)
Volume activity (U/mL) =20.0 X 0.01

Specific activity (U/mg protein) =X d.f.

Volume activity (U/mL)
Protein concentration (mg/mL)*

d.f. ; dilution factor
20.0 ; millimolar extinction coefficient of MTT (cm2/μmol)*Protein concentration ; determined by Bradford's method
REFERENCE
1. Packman, L.C., and Perham. R.N.; FEBS Lett., 139, 155 (1982)

NIPRO ENZYMESTable 1. SUBSTRATE SPECIFICITY OF DIAPHORASE 22Acceptor

MTT

LipoateKm Acceptor

(mM)

0.345

2.0

Km NADH

(mM)

0.033

0.01

100

103

Relative Activity
Assay Mixture

Wavelength for
Measuremen
Extinction
coefficient

HEPESI (pH 7.0)
NADH
MTT
Triton X-100(nm)(cm2/μmol)

50 mM
0.5 mM
0.5 mM
0.5 %Potassium Phosphate(pH 6.5)
70.5 mM
NADH
0.2 mM
NAD
0.3 mM
Lipoate10.2 mM
EDTA
0.81 mM
BSA
0.7 mg/mL

565

340

20

6.22

NIPRO ENZYMES
200

Remaining activity (%)

Relative activity (%)

100.0150

100

50045678910110.01256789pH

Fig. 1 pH profile

Fig. 2 pH stability○ Bicine,▲ HEPES

10treated for 24 hr at 4 ℃ in the following
buffer solution (0.1 M), :△ Gly-KOH,○ Bicine,● phosphate, ▲ HEPES,□ MES,■ Citrate

100100

Remaining activity (%)

Remaining activity (%)

4pH△ Gly-KOH,● phosphate,

50050.02030

4050607080

Temperature (℃)

90500015304560

Time (min)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 0.1M
potassium phosphate buffer,
pH 7.5treated for in 0.1M potassium
phosphate buffer, pH 7.5○ 60 ℃, □ 70 ℃, ● 80 ℃

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