(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-107REACH適合品

L–α–GLYCEROPHOSPHATE OXIDASE [GPOM]

from Streptococcus sp.
(sn–Glycerol–3–phosphate: oxygen 2–oxidoreductase, EC 1. 1. 3. 21)

L–α–Glycerophosphate + O₂ → Dihydroxyacetone phosphate + H₂O₂

Preparation and Specification

Appearance: : Yellowish amorphous powder, lyophilized

Specific activity: : More than 15.0 U/mg solid

Properties

Substrate specificity: : See Table 1

Molecular weight: : 169 kDa (TSK gel G 3000 SW XL gel filtration)
65 kDa (SDS–PAGE)

Isoelectric point: : pH 4.4

Michaelis constants: : L–α–Glycerophosphate 0.64 mM (pH 7.5)

Optimum pH: : 8.5–9.0Figure 1

pH stability: : 6.0–8.0 (37℃, 30 min) Figure 2

Optimum temperature: : 37–42℃ (pH 6.5) Figure3

Thermal stability: : Stable at 40℃ and below (pH 6.5) Figure4

Effect of metal ions: : See Table 2

Effect of detergents: : See Table 3

tabilizers: : FAD

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of triglyceride.

	LP
TG + 3 H₂O	→	Glycerol + 3 Fatty acid
	GKZ
Glycerol + ATP	→	G-3-P + ADP
	GPOM
G-3-P + O₂	→	DHAP + H₂O₂
	POD
2 H₂O₂ + 4-AA + Phenol	→	Quinoneimine dye + 4 H₂O

TG: Triglyceride, DHAP: Dihydroxyacetone phosphate

Table 1. Substrate specificity

Substrate (300mM)	Relative activity (%)
L–α–Glycerophosphate	100
Glucose–1–phosphate	0
Glucose–6–phosphate	0
Glycerol	0
Glucose	0

Table 3. Effect of detergents on GPOM activity

Detergent (0.1%)	Relative activity (%)
None	100
EMULGEN 810	98
EMULGEN 911	98
RHEODOL TWL–106	99
RHEODOL 460	99
ADEKANOL NP–720	99
Triton X–100	98
Triton X–305	99
Tween 80	98

Table 2. Effect of metal ion on GPOM activity

Metal ion (2mM)	Relative activity (%)
None	100
MgCl₂	101
MgSO₄	102
ZnCl₂	102
ZnSO₄	102
NaCl	103
NH₄Cl	103
BaCl₂	103
Ba (CH₃COO) ₂	101
NiCl₂	103
CoCl₂	103
MnCl₂	114
LiCl	103
KCl	102
CaCl₂	103

Fig.1 Optimum pH

200 mM buffer
〇: MES buffer
●: PIPES buffer
□: Phosphate buffer
■: Tris buffer
△: DEA buffer
▲: TEA buffer
＋: Citrate buffer

Fig.2 pH Stability

37℃, 30 min.
200 mM buffer
〇: Citrate buffer
●: PIPES buffer
□: Phosphate buffer
■: Tris buffer
△: DEA buffer
▲: Glycine buffer
＋: MES buffer
－: Bis Tris buffer

Fig.3 Optimum Temperature

pH 6.5
200 mM PIPES buffer

Fig.4 Thermal Stability

pH 6.5, 10min.
50 mM PIPES buffer

Assay

Principle

The assay is based on the increase in absorbance at 600 nm as the formation of quinoneimine dye in the following reactions:

	GPOM
L–α–Glycerophosphate+O₂	→	Dihydroxyacetone phosphate+H₂O₂
	POD
2 H₂O₂+4–AA+DAOS	→	Quinoneimine dye+4H₂O

DAOS : [3,5–dimethoxy–N–ethyl–N– (2–hydroxy–3– sulphopropyl) aniline]

Unit definition
1. One unit is defined as the amount of enzyme which generates 1 μmole of H₂O₂ per minute at 37℃ under the conditions specified in the assay procedure.
Reagents
1. Reaction mixture
  Dissolve 6.05 g of PIPES and 9.45 g (purity calculation) of Disodium Glycerophosphate with 70 ml of distilled water and adjust pH to 6.5 with 4 N NaOH at 25℃. Add all reagents listed below and confirm pH is 6.5 at 25℃.
  Add distilled water to make a total of 100 ml.
  
  100 U/ml POD 1) solution 5.0 ml
  
  15 mM 4–AA solution 10.0 ml

100mM DAOS solution 1.0 ml

5% (W/V) Triton X–100 solution 1.0 ml

1) : 100 U/ml POD solution Dissolve 1,000 U (PPU) of POD with 10 ml of distilled water.
Reaction stopper
0.5% (W/V) SDS solution
SDS: Sodium dodecyl sulfate
Enzyme dilution buffer
10 mM PIPES–NaOH buffer pH 6.5
containing 0.1% (W/V) Triton X–100
Reagents
PIPES [Piperazine–1,4,–bis (2–ethanesulfonic acid) ]:
Dojindo Laboratories # 345–02225
DAOS (sodium salt) : Dojindo Laboratories #OC06
4–AA: NACALAI TESQUE, INC.
Special grade #01907–52
Triton X–100: The Dow Chemical Company
Disodium Glycerophosphate 5.5 Hydrate :
FUJIFILM Wako Pure Chemical Corporation
#192–02055
SDS (Sodium Dodecyl Sulfate) :
NACALAI TESQUE, INC. Extra pure　#31606–75
POD: Sigma Chemical Co.　Type Ⅱ　#P–8250

Enzyme solution

Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.

Procedure

Pipette accurately 1.0 ml of reaction mixture into a
small test tube and preincubate at 37℃.
After 5 min, add exactly 20 μl of enzyme solution and mix to start the reaction at 37℃.

※ In the case of a test blank, add 20 μl of enzyme dilution buffer in place of enzyme solution.
At 5 min after starting the reaction, add 2.0 ml of the

reaction stopper to stop the reaction.
Measure the absorbance at 600 nm.

Absorbance sample : As

blank : Ab

0.1 Abs ≦ △A (As−Ab) ≦ 0.2 Abs

Calculation

Activity (U/mg of powder) = {(△ A/5)/(16.8×1/2)} × 3.02/0.02 × 1/x

16.8 :	millimolar extinction coefficient of quinoneimine dye at 600 nm ( cm² /μmole)
1/2 :	a multiplier derived from the fact that 2 mole of H₂O₂ produces l mole of quinoneimine dye
5 :	reaction time (min)
3.02 :	final volume (ml)
0.02 :	volume of enzyme solution (ml)
X :	concentration of the sample in enzyme solution ( mg/ml)

Storage

Storage at −20℃ in the presence of a desiccant is recommended. Enzyme activity will be retained for at least one year under this condition.

References

Jacobs, N. J. and Van Demark, P. J. (1960) Arch.
Biochem. Biophys., 88, 250–255.
Koditschek, L. K. and Umbreit, W. W. (1969) J. Bacteriol., 98, 1063–1068.
Gancedo, C., Gancedo, J. M. and Sols, A. (1968) J. Biochem. ( Tokyo) , 5, 165–172.
Kistler, W. S., Hirsch, C. A., Cozzarelli, N. R. and Lin, E. C. C. (1969) J. Bacteriol., 100, 1133–1135.
Esders, T. W. and Michrina, C. A. (1979) J. Biol. Chem., 254, 2710–2715.

GPOM 活性測定法 (Japanese)

試薬液

反応試薬混合液
PIPES 6.05g とグリセロりん酸2Na 9.45g (純度換算) を精製水70ml に溶解した後、4N NaOH でpH6.5 (25℃) に調整し、その液に下記試薬を加えて混和し、pH6.5 (25℃) であることを確認した後、精製水で全容100ml とする。

100U/ml POD 溶液¹⁾ 5.0 ml

15mM 4–AA 溶液 10.0 ml

100mM DAOS 溶液 1.0 ml

5% (W/V) トリトンX–100 溶液 1.0 ml

1) ： 100U/ml POD 溶液
POD 1,000 単位 (PPU) を精製水10ml で溶解する。

反応停止液
0.5% (W/V) SDS 溶液
酵素溶解希釈用液
0 . 1 % ( W / V ) トリトン X – 1 0 0 を含む1 0 m M
PIPES–NaOH 緩衝液 pH6.5
試薬
PIPES［ピペラジン–1,4–ビス (2–エタンスルホン酸) ］：同仁化学製　#345–02225
DAOS［3,5–ジメトキシ–N–エチル–N– (2–ヒドロキ
シ–3–スルフォプロピル) アニリン］：
同仁化学製　#OC06
4–AA：ナカライテスク製　特級　#01907–52
トリトンX–100：Dow Chemical 製
グリセロりん酸二ナトリウム5.5 水和物：
富士フイルム和光純薬製　#192–02055
SDS (ドデシル硫酸ナトリウム) ：
ナカライテスク製　一級　#31606–75
POD：シグマ製　Type Ⅱ　#P–8250

酵素試料液

検品約20mg を精密に量り、酵素溶解希釈用液で溶解して全容20ml とする。
その液を酵素溶解希釈用液で適宜希釈する。

測定操作法

小試験管に反応試薬混合液1.0ml を正確に分注し、37℃で予備加温する。
5 分経過後、酵素試料液20 μl を正確に加えて混和し、37℃で反応を開始する。

※ 盲検は酵素試料液の代わりに酵素溶解希釈用液
20 μl を加える。
5 分経過後、反応停止液2.0ml を加えて混和し、反応を停止する。
600nm における吸光度を測定する。
求められた吸光度を試料液はAs、盲検液はAb とする。
0.1 Abs ≦ ΔA = (As−Ab) ≦ 0.2 Abs

計算

以下の計算式に従い、活性 (U/mg) を計算する。
活性 (U/mg) = {(△A/5)/(16.8×1/2)} × 3.02/0.02 × 1/x

16.8 :	キノンイミン色素の600nm におけるミリモル分子吸光係数 (cm² / μmole)
1/2 :	H₂O₂ 2 モルからキノン色素1 モルが生成することによる係数
5 :	反応時間 (min)
3.02 :	反応総液量 (ml)
0.02 :	反応に供した酵素試料液量 (ml)
X :	酵素試料液中の検品濃度 (mg/ml)

100U/ml POD 溶液¹⁾	5.0 ml
15mM 4–AA 溶液	10.0 ml
100mM DAOS 溶液	1.0 ml
5% (W/V) トリトンX–100 溶液	1.0 ml

Absorbance sample :	As
blank :	Ab

L–α–GLYCEROPHOSPHATE OXIDASE [GPOM] (T-107)