NIPRO ENZYMESDIAPHORASE 3 (DI-3)[EC 1. 6. 99. - ]from recombinant E. coli
NAD(P)H + Acceptor(ox.) + H+ ↔ NAD(P) + + Acceptor(red.)
SPECIFICATIONStateSpecific activity
Contaminants

PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Isoelectric point
Thermal stability
Michaelis constants: Lyophilized: more than 1,000 U/mg protein: (as Diaphorase activity = 100 %)Adenylate kinase
NADH oxidase: ca. 20,000(SDS-electrophoresis): 8.0: 7.5 - 9.5: 4.7: No detectable decrease in activity up to 60 °C.
: See Table 1< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)

STORAGE
Stable at -20 to 5 °C for one year
APPLICATIONThe enzyme is useful for the measurement of various dehydrogenase reactions in visible spectral
range.

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 600 nm according to the following reaction.
NAD(P)H + DCIP(ox.) + H+DI-3

NAD(P)+ + DCIP(red.)

Unit Definition
One unit of activity is defined as the amount of DI-3 that reduces 1 μmol of DCIP per minute at30 °C.
SolutionsⅠ Buffer solution ; 500 mM Tris-HCI, pH 8.5
Ⅱ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O/10 mL distilled water)
Ⅲ 2,6-Dichlorophenolindophenol (DCIP) solution ; 1.2 mM (2.0 mg DCIP sodium salt∙2H2O/5mL
distilled water) (prepare freshly)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 1.0 to 2.0 U/mL with 50 mM
potassium phosphate buffer, pH 7.5.
Procedure
1. Prepare the following reaction mixture and pipette 2.28 mL of reaction mixture and 0.12 mL ofSolution Ⅲ into a cuvette.
SolutionⅠ 3.00 mL
SolutionⅡ 2.28 mL
23.22 mL
H2O
2. Incubate at 30 °C for about 3 minutes.
3. Add 0.008 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 600 nm per minute (∆Abs(test)) in linear portion of curve. Repeat theProcedure 3 using distilled water in place of enzyme solution, and ∆Abs(blank) is obtained.
Calculation
Volume activity (U/mL) =(∆Abs (test) - ∆Abs (blank)) X (2.40 + 0.008)

Specific activity (U/mg protein) =19 X 0.008

X d.f.

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

d.f. ; dilution factor
19 ; millimolar extinction coefficient of DCIP (cm2/μmol)*Protein concentration ; determined by Bradford’s method
REFERENCE
1. Mains, I., Power, D.M., Thomas, E.W. and Buswell J. A.; Biochem. J., 191, 457 (1980)

NIPRO ENZYMESRemaining activity (%)

Relative activity (%)

10050045678910050010 11 1245Fig. 1 pH profile● Tris-HCl,

910 11 12treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M);○ phosphate,△ acetate,● Tris-HCl

100

Remaining activity (%)

Remaining activity (%)

8Fig. 2 pH stability

1005007pHpH○ phosphate,■ Gly-KCl-KOH

650020 30 40 50 60 70 80 90 100
Temperature (℃)
Fig. 3 Thermal stability
treated for 15 min in 0.1 M
potassium phosphate buffer,
pH 7.5010 20 30 40 50 60 70Time (min)Fig. 4 Thermal stability
treated in 0.1 M potassium
phosphate buffer, pH 7.5○ 50 ℃, □ 60 ℃, ● 70 ℃

NIPRO ENZYMESTable 1. SUBSTRATE SPECIFICITY OF DIAPHORASEAcceptor

DCIP*1

NTB*2

MTT*3

Km Acceptor

(mM)

0.02

0.15

0.9

Km

NADH

(mM)

0.37

0.01

0.05

Km NADPH

(mM)

32.7

0.31

2.0

8.0

108.0

Optimum pH
Assay Mixture

Tris-HCI(pH 8.5)
NAD(P)HDCIP

Wavelength for
Measurement(nm)

Extinction Coefficient(cm2/μmol)

50 mM
1 mM
0.06 mM

TEA(pH 7.0)
50 mM
NAD(P)H
1 mM
NBT
0.5 mM
Triton X-100 0.1 %TEA(pH 7.0)
NAD(P)H
MTT
Triton X-100

600

550565

1912.42050 mM
1 mM
0.5 mM
0.5 %*1 2,6-Dichlorophenolindophenol*2 Nitrotetrazolium Blue
*3 Thiazolyl Blue Tetrazolium Bromide

pH profiles of DI-3 (Acceptor; NTB or MTT)

100

Relative activity (%)

Relative activity (%)

100505006789101112pH
Fig. 5 pH profile (NTB)□ triethanolamine, ○ phosphate,● Tris-HCl,■ Gly-KCl-KOH

067891011pH
Fig. 6 pH profile (MTT)□ triethanolamine, ○ phosphate,● Tris-HCl,■ Gly-KCl-KOH

12