NIPRO ENZYMESPYRUVATE KINASE (PK)[EC 2.7.1.40]
from Bacillus stearothermophilus
ATP + Pyruvate ↔ ADP + Phosphoenolpyruvate
SPECIFICATIONStateSpecific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Isoelectric point
Thermal stability
Michaelis constants: Lyophilized: more than 230 U/mg protein: (as PK activity = 100 %)Adenylate kinase
Lactate dehydrogenase: ca. 260,000: ca. 68,000: 7.0: 8.0 - 10.0
: 5.2
: No detectable decrease in activity up to 55 °C.
: (76 mM Imidazole-HCl buffer, pH 7.2, at 30 °C)
Phosphoenolpyruvate
ADPSTORAGE
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)
0.6 mM
0.9 mMNIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
ADP + PEPPK

ATP + Pyruvate

Pyruvate + NADH + H+LDH

Lactate + NAD+

Unit Definition
One unit of activity is defined as the amount of PK 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 (0.507 g ADP disodium salt∙2H2O/(9.0 mL distilled water + 1.0 mL 1 NNaOH))
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., Product Code: LDH-02)
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
SolutionⅤ
0.48 mL
SolutionⅡ 2.40 mL
SolutionⅥ
0.90 mL
SolutionⅢ 0.45 mL
SolutionⅦ
0.06 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 (cm 2/μmol)*Protein concentration ; determined by Bradford’s method
REFERENCE
1. Sakai, H., Suzuki, K., and Imahori, K.; J. Biochem., 99, 1157 (1986)

Remaining activity (%)

Relative activity (%)

NIPRO ENZYMES

100500100

500345678910 11456pH8910 11 12pH

Fig. 1 pH profile

Fig. 2 pH stability△ acetate, □ imidazole-HCl,● Tris-HCl

treated for 24 hr at 4 °C in thefollowing buffer solution (0.1 M);△ acetate, □ imidazole-HCl,● Tris-HCl, ▲ carbonate

100

Remaining activity (%)

Remaining activity (%)

7500100500203040506070Temperature (°C)
Fig. 3 Thermal stability
treated for 15 min in 0.1 MTris-HCl buffer, pH 8.5

800153045Time (min)60

Fig. 4 Thermal stability
treated in 0.1 M Tris-HCl buffer,
pH 8.5○ 55 °C, □ 60 °C, ● 65 °C