PYD-301
●TOYOBO ENZYMES●
(Diagnostic Reagent Grade )
PYRUVATE DEHYDROGENASE
from Microorganism
dehydrogenase
Pyruvate＋Pi＋Acceptor Pyruvate
Acetylphosphate＋CO2＋Reduced acceptor
FAD,TPP,Mg++
PREPARATION and SPECIFICATION
Appearance
: Yellowish amorphous powder, lyophilized
Activity
: GradeⅢ 2.0U/mg-solid or more
(containing approx.50% of stabilizers)
Stabilizers
: Mannitol, FAD
PROPERTIES
Stability
: Stable at −20℃
Molecular weight
: approx. 160,000
Isoelectric point
: 4.4±0.1
Michaelis constants
: 4.4×10−4M (Pyruvate)
（Fig.1）
1.4×10−2M (Phosphate)
Inhibitors
: Hg＋＋
Optimum pH
: 6.2−6.3
Optimum temperature
: 55℃
（Fig.3）
pH Stability
: pH 5.0−8.0 (25℃, 20hr)
（Fig.4）
（Fig.5）
Thermal stability
: below 50℃ (pH 6.3, 15min)
Substrate specificity
: (Table 1)
Effect of various chemicals
: (Table 2)
（Fig.2）
APPLICATIONS
This enzyme is useful for enzymatic determination of ADP, pyruvate, Pi, sialic acid, GOT and GPT when
coupled with the related enzymes.
PYD-301
ASSAY
Principle:
pyruvate dehydrogenase
Pyruvate＋Pi＋mPMS
TPP,FAD,Mg＋＋
2mPMS(red)＋NTB
Acetylphosphate＋CO2＋mPMS(red)
2mPMS＋Diformazan
The appearance of diformazan formed by the reduction of nitrotetrazorium blue (NTB) with 1-methoxy-5methylphenazinium methylsulfate (mPMS) is measured at 570nm by spectrophotometry.
Unit definition:
One unit causes the formation of half a micromole of diformazan per minute under the conditions described
below.
Method:
Reagents
A. Pyruvate solution
：300mM
B. Buffer solution
：0.15M K-Phosphate buffer, pH 6.3 containing 1.5% Triton X-100
C. FAD solution
：0.15mM
D. TPP solution
：3.0mM
E. EDTA・Na2 solution
：7.5mM
F. MgSO4 solution
：75mM
G. mPMS-NTB solution
：3.4mg of mPMS, 82mg of NTB／10ml of H2O containing 0.5% of Triton X-100 (store
H. Enzyme diluent
：50mM K-Phosphate buffer, pH 6.3
at 4℃ in a brownish bottle)
Procedure
1.
Prepare the following reaction mixture in a cuvette (d＝1.0cm)
and equilibrate at 37℃ for about 5 minutes.
0.50ml
Substrate solution
(A)
1.0 ml
Buffer solution
(B)
0.20ml
FAD solution
(C)
0.20ml
TPP solution
(D)
0.40ml
EDTA・Na2 solution
(E)
0.40ml
MgSO4 solution
(F)
0.30ml
mPMS-NTB solution
(G)
Concentration in assay mixture
Pyruvate
48.4 mM
Phosphate buffer
50.0 mM
FAD
9.67mM
TPP
0.19mM
EDTA
0.97mM
MgSO4
9.68mM
mPMS
97.7 μM
NTB
0.97mM
Triton X-100
0.53 %
2.
Add 0.10ml of the enzyme solution* and mix by gentle inversion.
3.
Record the increase in optical density at 570nm against water for 4 to 5 minutes in a spectrophotometer
thermostated at 37℃, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD
test).
At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that
the enzyme diluent (H) is added instead of the enzyme solution.
*
Dissolve the enzyme preparation in ice-cold enzyme diluent (H) and dilute to 0.10−0.5U/ml with the same
buffer and store on ice.
Calculation
Activity can be calculated by using the following formula：
ΔOD/min (ΔOD test−ΔOD blank)×Vt×df
Volume activity (U/ml) ＝
＝ΔOD/min×1.416×df
21.9×1.0×Vs
Weight activity (U/mg)＝(U/ml)×1/C
Vt
：Total volume (3.1ml)
Vs
：Sample volume (0.10ml)
21.9 ：Half a millimolar extinction coefficient of diformazan (F/micromole)
1.0
：Light path length (cm)
df
：Dilution factor
C
：Enzyme concentration in dissolution (c mg/ml)
PYD-301
Table 1. Substrate Specificity of Pyruvate dehydrogenase
Relative activity
Substrate(50mM)
Pyruvate
Acetoacetate
α-Ketoglutarate
Oxaloacetate
DL-Lactate
Substrate(50mM)
100
1.0
0
0
0
Relative activity
Acetate
Phosphoenol pyruvate
L-Alanine
L-Aspartate
0
0
0
0
Table 2. Effect of Various Chemicals on Pyruvate dehydrogenase
[The enzyme dissolved in 50mM K-phosphate buffer, pH 6.3 (10U/ml) was incubated with each chemical
at 25℃ for 1hr.]
Concn.(mM)
Residual
activity
None
―
100%
Metal salt
2.0
Chemical
Chemical
Concn.(mM)
Residual
activity
2.0
99.5
NaF
NaN3
20
EDTA
101
5.0
102
MgCl2
88.1
CaCl2
97.5
o-Phenanthroline
2.0
Ba(OAc)2
98.5
α,α′
-Dipyridyl
1.0
FeCl3
96.1
Borate
CoCl2
89.3
IAA
2.0
80.0
NEM
2.0
101
Hydroxylamine
2.0
101
0.10%
101
MnCl2
ZnSO4
100
98.5
100
50
99.0
98.5
Cd(OAc)2
98.0
Triton X-100
NiCl2
98.5
Brij 35
0.10%
94.1
99.4
Tween 20
0.10%
94.6
99.0
Span 20
0.10%
99.0
CuSO4
Pb(OAc)2
AgNO3
Na-cholate
0.10%
99.5
5.3
SDS
0.05%
99.0
95.9
DAC
0.05%
94.1
112
HgCl2
PCMB
1.0
MIA
2.0
98.5
Ac, CH3CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA,
Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethyl-benzyl-alkyl-ammonium chloride.
-20℃
50
0
0
1
2
3
4
5
6
7
8
9
10
100
Residual Activity,%
Relative Activity
100
50
0
4
5
Period (months)
6
7
8
50
0 4
5
6
pH
Fig.1. Stability (Power form)
8
9
Fig.4. pH-Stability
37℃ in 50mM K-phoshate buffer
25℃,20hr-treatment with 50mM buffer solution:
pH4.0-6.0,acetate;pH5.5-8.0 K-phosphate;
pH8.0-9.0,Tris-HCI;enzyme concn.:10U/ml
100
Residual Activity,%
100
50
0
7
pH
Fig.2. pH-Activity
kept under dry conditions
Relative Activity
Relative Activity,%
100
20
30
40
50
60
Temperature, ℃
Fig.3. Temperature activity
in 50mM K-phosphate buffer, pH6.3
50
0
20
30
40
50
60
Temperature, ℃
Fig.5. Thermal stability
15min-treatment with 50mM K-phosphate
buffer,pH6.3 enzyme concn.:10U/ml
PYD-301
活性測定法（Japanese）
1.原理
dehydrogenase
Pyruvate＋Pi＋mPMS pyruvate
AcetylphosTPP,FAD,Mg＋＋
phate＋CO2＋mPMS(red)
2mPMS(red)＋NTB
2mPMS＋Diformazan
酵素反応で生成した還元型mPMS ( 1-methoxy-5methyl phenazinium methylsulfate ) に よ り
NTB ( nitrotetrazorium blue ) を還 元し，
生 成した
diformazanの570nmにおける吸光度の変化で測定する。
2.定義
下記条件下で1分間に1/2マイクロモルのdiformazanを
生成する酵素量を1単位(U)とする。
3.試薬
A.
B.
0.3M
0.15M
ピルビン酸水溶液
K-リン酸緩衝液，pH6.3(1.5% Triton
X-100を含む)
C.
0.15mM FAD水溶液
D.
3.0mM TPP水溶液
E.
7.5mM EDTA・Na2水溶液
F.
75mM
MgSO4水溶液
G.
mPMS-NTB水溶液(3.4㎎ mPMS及び82㎎ NTB
を10Pの蒸留水に溶解する)(褐色瓶中で４℃保存)
酵素溶液：酵素標品を予め氷冷した50mM K-リン酸緩
衝液, pH6.3で溶解し，
分析直前に同緩衝液
で0.1∼0.5U/Pに希釈する。
4.手順
①下記反応混液をキュベット(d＝1.0cm)に調製し，
37℃
で約5分間予備加温する。
(A)
0.50P 基質溶液
(B)
1.00P K-リン酸緩衝液
(C)
0.20P FAD溶液
(D)
0.20P TPP溶液
(E)
0.40P EDTA・Na2水溶液
(F)
0.40P MgSO4水溶液
(G)
0.30P mPMS-NTB水溶液
②酵素溶液0.10Pを添加し，
ゆるやかに混和後，
水を対
照に37℃に制御された分光光度計で570nmの吸光
度変化を4∼5分間記録し，
その初期直線部分から1分
間当りの吸光度変化を求める(ΔODtest)。
③盲検は反応混液①に酵素溶液の代わりに酵素希釈
液(50mM K-リン酸緩衝液，pH6.3)を0.10P加え，
上
記同様に操作を行って，
1分間当りの吸光度変化を求
める(ΔODblank)。
5.計算式
U/P
U/mg
21.9
1.0
C
＝
ΔOD/min (ΔOD test−ΔOD blank)×3.1(P)×希釈倍率
21.9×1.0×0.10(P)
＝ ΔOD/min×1.416×希釈倍率
＝ U/P×1 / C
: Diformazan の1/2ミリモル分子吸光係数
(F/micromole)
: 光路長(cm)
: 溶解時の酵素濃度(c ㎎/P)