G3D-301 - Toyobo

G3D-301
●TOYOBO ENZYMES●
(Diagnostic Reagent Grade )
GLYCEROL-3-PHOSPHATE DEHYDROGENASE
from Rabbit Muscle
sn-Glycerol-3-phosphate : NAD+ 2-oxidoreductase (EC 1. 1. 1. 8)
CH2OH
HO
C
CH2OH
＋
H
NAD
+
P
CH2O
sn-Glycerol-3-phosphate
C
O
＋
NADH
＋
H
+
P
CH2O
Dihydroxyacetone 3-phosphate
PREPARATION and SPECIFICATION
Appearance
: White amorphous powder, lyophilized
Activity
: GradeⅢ 15U/mg-solid or more
Contaminants
: Lactate dehydrogenase
≤3.0×10−1%
Pyruvate kinase
≤3.0×10−1%
Triose phosphate isomerase
≤1.0×10 %
Aldolase
≤5.0×10−2%
Glycerol kinase
≤1.0×10−2%
＋
: NAD , lactose
Stabilizers
PROPERTIES
: Stable at −20℃ for at least 6 months
Stability
Molecular weight
1,2）
Michaelis constants
（Fig.1）
: approx. 78,000
: 7.7×10−4M (Glycerol-3-P),1.6×10−4M (NAD＋),
4.5×10−4M (DAP),1.3×10−5M (NADH)
Inhibitors
3）
: SH reagents, DAP, fructose 1,6-diphosphate
Optimum pH
: 9.0
（Fig.2）
Optimum temperature
: 45−50℃
（Fig.3）
pH Stability
: pH 6.0−9.0 (25℃, 21hr)
（Fig.4）
Thermal stability
: below 40℃ (pH 8.5, 15min)
（Fig.5）
Substrate specificity
: The enzyme is specific for L-glycerol-3-P and dihydroxyacetone
phosphate. The reaction velocity with NADPH is one-tenth of that with
NADH.
APPLICATIONS
This enzyme is useful for enzymatic determination of glycerol when coupled with glycerol kinase (GYK301, GYK-311) in clinical analysis.
G3D-301
ASSAY
Principle:
glycerol-3-phosphate dehydrogenase
Glycerol-3-phosphate＋NAD＋
Dihydroxyacetone phosphate＋NADH＋H＋
The appearance of NADH is measured at 340nm by spectrophotometry.
Unit definition:
One unit causes the formation of one micromole of NADH per minute under the conditions described below.
Method:
Reagents
A. BICINE buffer, pH 9.0
：0.1M［Dissolve 1.63g of N, N-Bis (2-hydroxyethyl) glycine (MW＝163.16) in ca.
80ml of H2O and after adjusting the pH to 9.0 with 5N NaOH, fill up to 100ml with
H2O］
＋
B. NAD solution
：51mM［Dissolve 183mg of β-NAD＋・3H2O (MW＝717.4) in 5ml of H2O］(Should
C. G-3-P solution
：0.87M G-3-P［Dissolve 1.77g of glycerol-3-phosphate (dicyclohexylammoniumsalt・
be prepared fresh)
2H2O, MW＝406.5) in 5ml of H2O］(Should be prepared fresh)
D. Enzyme diluent
：50mM BICINE buffer, pH 9.0 contg. 0.1% BSA (Should be prepared fresh)
Procedure
1.
Prepare the following reaction mixture in a cuvette (d＝1.0cm)
and equilibrate at 37℃ for about 5minutes.
2.7ml
BICINE buffer, pH 9.0
(A)
0.1ml
NAD solution
(B)
0.1ml
G-3-P solution
(C)
＋
Concentration in assay mixture
BICINE buffer
90 mM
NAD＋
1.7 mM
G-3-P
29 mM
BSA
33μg/ml
2.
Add 0.1ml of the enzyme soluion* and mix by gentle inversion.
3.
Record the increase in optical density at 340nm against water for 4 to 5 minutes in a spectrophotometer
thermostated at 37℃, and calculate theΔOD 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 (D) is added instead of enzyme solution.
*
Dissolve the enzyme preparation in ice-cold enzyme diluent (D) and dilute to 0.06−0.3U/ml with the same
buffer, immediately before assay.
Calculation
Activity can be calculated by using the following formula：
ΔOD/min (ΔOD test−ΔOD blank)×Vt×df
Volume activity (U/ml) ＝
＝ΔOD/min×4.82×df
6.22×1.0×Vs
Weight activity (U/mg)＝(U/ml)×1/C
Vt
：Total volume (3.0ml)
Vs
：Sample volume (0.1ml)
6.22 ：Millimolar extinction coefficient of NADH (F/micromole)
1.0
：Light path length (cm)
df
：Dilution factor
C
：Enzyme concentration in dissolution (c mg/ml)
REFERENCES
1) J.van Eys, B.J.Nuenke and M.K.Patterson; j.Biol.Chem., 234, 2308 (1959).
2) J.Otto, A.Raggi, W.Machleidt and T.Bucher; Hoppe-Seyler's Z.Phsyol.Chem., 353, 332 (1972).
3) B.Borrebeak, S.Abraham and I.L.Chaikoff; Biochim.Biophys.Acta., 96, 237 (1965).
G3D-301
Relative Activity
Residual Activity,%
50
100
Residual Activity,%
100
100
50
50
-20℃
0 7
8
9
10
11
0 4
6
pH
2
3
4
5
6
Period (months)
Fig.1. Stability (Powder form)
kept under dry conditions
Fig.2. pH-Activity
12
25℃,21hr-treatment with 0.1M buffer
solution:pH4.0-5.5,acetate buffer;
pH5.5-7.5,MES;pH7.5-9.0,BICINE;
pH9.0-10.0, CHES;pH10.0-11.0
CAPS.enzyme concn.:17U/ml
100
100
50
0
10
Fig.4. pH-Stability
37℃,in 0.1M buffer solution:pH7.07.5,TES;pH7.5-9.0,BICINE;pH9.09.7,CHES;pH9.8-10.5,CAPS
Residual Activity,%
1
Relative Activity
0
8
pH
20
30
40
50
60
Temperature, ℃
Fig.3. Temperature activity
in 0.1M BICINE buffer, pH9.0
50
0 20
30
40
50
60
Temperature, ℃
Fig.5. Thermal stability
15min-treatment with 50mM BICINE
buffer ,pH8.5,enzyme concn.:40U/ml
G3D-301
活性測定法（Japanese）
1.原理
Glycerol-3-phosphate＋NAD＋ glycerol-3-phosphate dehydrogenase
Dihydroxyacetone phosphate＋NADH＋H＋
NADHの生成量を340nmの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マイクロモルのNADHを生成す
る酵素量を1単位(U)とする。
3.試薬
A.
0.1M BICINE緩衝液, pH 9.0〔1.63gの N,N-ビ
ス(2-ヒドロキシエチル)グリシン(分子量＝163.16)
を約80Pの蒸留水に溶解し, 5N-NaOHでpH9.0
に調整した後,蒸留水で100Pとする。
〕
B.
51mM NAD ＋水溶液〔183㎎のβ-NAD・3H 2O
(分子量＝717.47)を蒸留水に溶解し５Pとする〕
(用時調製)。
C.
0.87M G-3-P水溶液〔1.77gのグリセロール-3-リン
酸 ( 分子量＝406.5 )を蒸留水に溶解し,5 Pとする〕
(用時調製)。
酵素溶液：酵素標品を予め氷冷した50mM BICINE緩
衝液, pH9.0で溶解し,分析直前に0.1%
BSAを含む同緩衝液で0.06∼0.3U/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)
2.7P
BICINE緩衝液, pH9.0
＋
(B)
0.1P
NAD 水溶液
(C)
0.1P
基質溶液
②酵素溶液0.1P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340nm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(ΔOD test)。
③盲検は酵素溶液の代りに酵素希釈液を0.1P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(ΔOD blank)。
5.計算式
U/P
U/mg
6.22
1.0
C
＝
ΔOD/min (ΔOD test−ΔOD blank)×3.0(P)×希釈倍率
6.22×1.0×0.1(P)
＝ ΔOD/min×4.82×希釈倍率
＝ U/P×1 / C
: NADHの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)

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