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

Lipase ActivityT-107 PDF download
(Diagnostic Reagent Grade) Nagase Diagnostics 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–Glycerophosphate100
Glucose–1–phosphate0
Glucose–6–phosphate0
Glycerol0
Glucose0

 

Table 3. Effect of detergents on GPOM activity

Detergent (0.1%)Relative activity
(%)
None100
EMULGEN 81098
EMULGEN 91198
RHEODOL TWL–10699
RHEODOL 46099
ADEKANOL NP–72099
Triton X–10098
Triton X–30599
Tween 8098

 

Table 2. Effect of metal ion on GPOM activity

Metal ion (2mM)Relative activity
(%)
None100
MgCl2101
MgSO4102
ZnCl2102
ZnSO4102
NaCl103
NH4Cl103
BaCl2103
Ba (CH3COO) 2101
NiCl2103
CoCl2103
MnCl2114
LiCl103
KCl102
CaCl2103

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

  1. 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]
  1. 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) solution5.0 ml
      15 mM 4–AA solution10.0 ml
  1. 100mM DAOS solution1.0 ml
    5% (W/V) Triton X–100 solution1.0 ml
    1) :100 U/ml POD solution Dissolve 1,000 U (PPU) of POD with 10 ml of distilled water.
    		 
  2. Reaction stopper
    0.5% (W/V) SDS solution
    SDS: Sodium dodecyl sulfate
  3. Enzyme dilution buffer
    10 mM PIPES–NaOH buffer pH 6.5
    containing 0.1% (W/V) Triton X–100
  4. 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
  1. 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
  1. Pipette accurately 1.0 ml of reaction mixture into a
    small test tube and preincubate at 37℃.
  2. 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.
  3. At 5 min after starting the reaction, add 2.0 ml of the
  1. reaction stopper to stop the reaction.
  2. 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
  1. Jacobs, N. J. and Van Demark, P. J. (1960) Arch.
    Biochem. Biophys., 88, 250–255.
  2. Koditschek, L. K. and Umbreit, W. W. (1969) J. Bacteriol., 98, 1063–1068.
  3. Gancedo, C., Gancedo, J. M. and Sols, A. (1968) J. Biochem. ( Tokyo) , 5, 165–172.
  4. Kistler, W. S., Hirsch, C. A., Cozzarelli, N. R. and Lin, E. C. C. (1969) J. Bacteriol., 100, 1133–1135.
  5. Esders, T. W. and Michrina, C. A. (1979) J. Biol. Chem., 254, 2710–2715.

GPOM 活性測定法 (Japanese)

試薬液
  1. 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。

  1. 反応停止液
    0.5% (W/V) SDS 溶液
  2. 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
  3. 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
  1. 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
  1. 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。
  2. 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。
  3. 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。
  4. 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
  1. 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
  1. 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)
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