NIPRO ENZYMESPYRUVATE KINASE (PK2)[EC 2.7.1.40]
from Recombinant E. coli
ATP + Pyruvate ↔ ADP + Phosphoenolpyruvate
SPECIFICATIONStateSpecific activity
Contaminants

PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants: Lyophilized: more than 230 U/mg protein: (as PK activity = 100 %)Adenylate kinase
Lactate dehydrogenase: ca. 60,000: 6.0 - 6.5: 6.0 - 11.0
: No detectable decrease in activity up to 60 °C.
: (76 mM Imidazole-HCl buffer, pH 7.2, at 30 °C)
Phosphoenolpyruvate
ADP

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThe enzyme is useful for diagnostic reagent, for example, ADP determination.

< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
1.0 mM
1.5 mMNIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
ADP + PEP

PK2

ATP + Pyruvate

Pyruvate + NADH + H+

LDH

Lactate + NAD+

Unit Definition
One unit of activity is defined as the amount of PK2 that forms 1 μmol of pyruvate per minute at
30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Imidazole-HCl, pH 7.2
Ⅱ ADP solution ; 100 mM (0.45 g ADP sodium salt, Sigma-Aldrich A2754/(9.0 mL distilled water + 1.0mL 1 N NaOH))
Ⅲ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O/10 mL distilled water)
Ⅳ Phosphoenolpyruvate (PEP) solution ; 56 mM (0.150 g PEP MCA salt/10 mL distilled water)
Ⅴ MgCl2 solution ; 1 M (20.33 g MgCl2∙6H2O/100 mL distilled water)
Ⅵ KCI solution ; 2.5 M (18.64 g KCI/100 mL distilled water)
Ⅶ Lactate dehydrogenase (LDH) ; (from pig heart, Oriental Yeast Co. Ltd., LDH (P.H.)) ammonium
sulfate suspension, approx. 5,000 U/mL at 25 °C
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50 mM Tris-HClbuffer, pH 8.5.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 22.71 mL
SolutionⅤ0.48 mL
SolutionⅡ 2.40 mL
SolutionⅥ
0.90 mL
SolutionⅢ 0.45 mL
SolutionⅦ0.06 mL
SolutionⅣ 3.00 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 340 nm per minute (ΔAbs340) in the linear portion of curve.
Calculation
Volume activity (U/mL) =(∆Abs340) X (3.00 + 0.01)
6.22 X 0.01

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

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

d.f. ; dilution factor
6.22 ; millimolar extinction coefficient of NADH (cm2/μmol)*Protein concentration ; determined by Bradford’s method
REFERENCE
1. Sakai, H., Suzuki, K., and Imahori, K.; J. Biochem., 99, 1157 (1986)

NIPRO ENZYMES

100
Remaining activity (%)

Relative activity (%)

100

50034567pH

891050011345Fig. 1 pH profile

8pH

910 11 12

treated for 24 hr at 4 ℃ in thefollowing buffer solution (50 mM);
△ Acetate, □ Imidazole-HCl,● Tris-HCl, ■ Glycine-KOH

100
Remaining activity (%)

100
Remaining activity (%)

7Fig. 2 pH stability△ Acetate, □ Imidazole-HCl,● Tris-HCl, ■ Glycine-KOH

5006203040506070
Temperature (℃)

805000153045Time (min)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 50 mM
Tris-HCl buffer, pH 8.5

treated in 50 mM Tris-HClbuffer, pH 8.5○ 55 ℃, □ 60 ℃, ● 65 ℃60