PYRUVATE KINASE - Toyobo

PYK-301
TOYOBO ENZYMES
(Diagnostic Reagent Grade)
PYRUVATE KINASE
from Rabbit muscle
ATP : pyruvate 2-O-phosphotransferase (EC 2.7.1.40)
COOH
ATPCO
CH 3
Pyruvate
COOH
ADPCO P
CH 2
Phosphoenolpyruvate
PREPARATION and SPECIFICATION
Appearance
: White amorphous powder, lyophilized
Activity
: Grade100U/mg-solid or more
Contaminants : Lactate dehydrogenase ≤1.010 2 %
Myokinase ≤1.010 2 %
NADH oxidase ≤1.010 2 %
PROPERTIES
Stability : Stable at 20 for at least 6 months Fig.1
Molecular weight 1 : 237,000
Isoelectric point 2 : 5.98
Michaelis constants 3 : 8.610 4 M (ATP), 1.010 2 M (Pyruvate),
3.010 4 M (ADP), 7.010 5 M (PEP)
Structure
: 4 subunits per mole of enzyme
Inhibitors : p-Chloromercuribenzoate, N-ethylmaleimide, SDS, DAC, Zn ,Cd ,
Cu ,Ag ,Hg
Activators
: Mg ,K
Optimum pH : 7.0 Fig.2
Optimum temperature : 40 Fig.3
pH Stability : pH 5.38.0 (25, 20hr) Fig.4
Thermal stability : below 45 (pH 7.6, 10min) Fig.5
Substrate specificity 4 : The enzyme transfers phosphate moiety of phosphoenolpyruvate to
ADP, GDP, UDP or CDP with activity decreasing in this order.
Effect of various chemicals : (Table 1)
APPLICATIONS 3,4
This enzyme is useful for enzymatic determination of ADP, ATP, pyruvate or phsphoenolpyruvate(PEP)
when coupled with the related enzymes.

PYK-301
ASSAY
Principle:
PEPADP
pyruvate kinase
PyruvateNADHH
PyruvateATP
lactate dehydrogenase
LactateNAD
The disappearance of NADH is measured at 340nm by spectrophotometry.
Unit definition:
One unit causes the oxidation of one micromole of NADH per minute under the conditions described below.
Method:
Reagents
A. Triethanolamine buffer, pH 7.60.1M containing 11.5mM KCl and 3.0mM MgCl 2
B. ADP solution
140mMDissolve 264mg of ADPNa 2 (MW471.2) in ca.2ml of H 2 O, adjust pH
to ca.7.0 with 1N KOH and fill up to 4.0ml with H 2 O(Should be prepared fresh)
C. PEP solution
16mMDissolve 3.3mg of PEPK (MW206.2) in 1.0ml of H 2 O(Should be
prepared fresh)
D. NADH solution
6.0mMDissolve 4.25mg of NADHNa 2 (MW709.4) in 1.0ml of H 2 O
(Should be prepared fresh)
E. LDH solution
ca.45U/mlDilute lactate dehydrogenase (ammonium sulfate suspension,
Grade from Toyobo) to ca.45U/ml with 0.1M triethanolamine buffer, pH 7.6
containing 0.3% of BSA
F. Enzyme diluent
0.1M Triethanolamine buffer, pH 7.6 containing 0.3% of BSA
Procedure
1. Prepare the following reaction mixture in a cuvette (d1.0cm)
Concentration in assay mixture
and equilibrate at 25 for about 5 minutes.
Triethanolamine buffer 90 mM
2.60ml Buffer solution (A) MgCl 2
2.6 mM
0.10ml ADP solution (B) KCl
9.8 mM
ADP
4.6 mM
0.10ml PEP solution (C)
PEP
0.52mM
0.10ml NADH solution (D)
NADH
0.20mM
0.10ml LDH solution (E)
LDH
ca.1.5 U/ml
2. Add 0.05ml of the enzyme solution and mix by gentle inversion.
3. Record the decrease in optical density at 340nm against water for 3 to 4 minutes in a spectrophotometer
thermostated at 25, and calculate the OD per minute from the initial linear portion of the curve (OD
test).
At the same time, measure the blank rate (OD blank) by using the same method as the test except that
the enzyme diluent (F) is added instead of the enzyme solution.
Dissolve the enzyme preparation in ice-cold enzyme diluent (F) and dilute to 0.251.0U/ml with the same
buffer and store on ice.
Calculation
Activity can be calculated by using the following formula
OD/min (OD testOD blank)Vtdf
Volume activity (U/ml)
6.221.0Vs
Weight activity (U/mg)(U/ml)1/C
OD/min9.807df
Vt
Vs
Total volume (3.05ml)
Sample volume (0.05ml)
6.22 Millimolar extinction coefficient of NADH at 340nm (F/micromole)
1.0 Light path length (cm)
df
C
Dilution factor
Enzyme concentration in dissolution (c mg/ml)
REFERENCES
1) G.L.Cotton, P.F.Hollenberg and M.J.Coon; J.Biol.Chem,244, 1481 (1969)
2) A.Morawiecki; Arch.Immunl.Terap.Doswiadczalnej, 8, 243 (1960)
3) J.T.McQuate and M.F.Utter; J.Biol.Chem.,234, 2151 (1959)
4) F.J.Kayne; The Enzyme, A, 3rd Ed.,p353,Academic Press,New York (1973)

PYK-301
Table 1. Effect of Various Chemicals on Pyruvate kinase
[The enzyme dissolved in 0.1M triethanolamine buffer, pH 7.6 (25U/ml) containing 0.3% of BSA was incubated
with each chemical at 25 for 1hr.
Chemical
Concn.(mM)
Residual
activity
None 100%
Metal salt 2.0
CaCl 2 97
Ba(OAc) 2 98
FeCl 2 87
CoCl 2 73
MnCl 2 100
ZnSO 4
0.8
Cd(OAc) 2 0
NiCl 2 90
CuSO 4
Pb(OAc) 2
0.4
27
AgNO 3 0
HgCl 2 0
2-Mercaptoethanol 2.0 94
PCMB 0.1 12
MIA 2.0 56
Chemical
Concn.(mM)
Residual
activity
NEM 2.0 0.2
IAA 2.0 24
Hydroxylamine 2.0 94
EDTA 5.0 100
,-Dipyridyl 2.0 89
o-Phenanthroline 2.0 94
Borate 50 102
NaF 2.0 100
NaN 3 20 95
TritonX-100 0.10% 97
Brij 35 0.10% 95
Tween 20 0.10% 95
Span 20 0.10% 95
Na-cholate 0.10% 90
SDS 0.05%
0.08
DAC 0.05% 8
Ac, CH3CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA,
Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethyl-benzyl-alkyl-ammonium
chloride.
100
100 100
Residual Activity,%
50
-20
4
25
35
Relative Activity
50
Residual Activity,%
50
0 1 2
3
6
0
5
6
7
8
9
0
4
5
6
7
8 9
(weeks) Period(months)
Fig.1. Stability (Powder form)
kept under dry conditions
pH
Fig.2. pH-Activity
25 in the following buffer solution:
,Britton-Robinson buffer;
,0.1M triethanolamine buffrer
pH
Fig.4. pH-Stability
25 20hr-treatment with the follwing
buffer solution:,Britton-
Robinson buffer;,0.1M
triethanolamine buffrer
100
100
Relative Activity
50
Residual Activity, %
50
0
20
30
40
50
60
0
20
30
40
50
60
Temperature,
Fig.3. Temperature activity
in 0.1M triethanolamine buffer,
pH7.6
Temperature,
Fig.5. Thermal stability
10min-treatment with 0.1M triethanolamine
buffer, pH7.6

PYK-301
pyruvate kinase
lactate dehydrogenase
2
2
P
P
P
2
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P