CHOLINE OXIDASE [COD] (T-05)

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(Diagnostic Reagent Grade) Nagase Diagnostics ENZYMES T-05REACH適合品

CHOLINE OXIDASE [COD]

from Arthrobacter globiformis
(Choline: oxygen 1-oxidoreductase, EC 1.1.3.17)

Choline + O2 → Betaine aldehyde + H2O2
Betaine aldehyde + O2 + H2O → Betaine + H2O2

Preparation and Specification

Appearance
: Yellowish amorphous powder, lyophilized
Specific activity
: More than 8 U/mg solid
Contaminants
: CatalaseLess than 10.0 % (U/U)
 
: Glucose oxidaseLess than 0.01 % (U/U)

Properties

Substrate specificity
: See Table 1
Molecular weight
: 83 kDa (Sephadex G–150)
Isoelectric point
: pH 4.5
Michaelis constants
: Choline 1.2 × 10-3M
Betaine aldehyde 8.7 × 10-3M
Optimum pH
: 7.5–8.0Figure 1
pH stability
: 7.5–9.0 (37℃, 10 min) Figure 2
Thermal stability
: Stable at 40℃ and below (pH 7.5, 10 min) Figure3
Storage stability
: At least one year at –20℃Figure4
Effect of various
chemicals
: See Table 2

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of phospholipids coupled with phospholipase D (T–07) .

 PLD
Phosphatidylcholine + H2O→Choline + Phosphatidic acid
 COD
Choline + 2 O2 + H2O→Betaine + 2 H2O2
 POD
2 H2O2 + 4-AA + Phenol→Quinoneimine dye + 4 H2O

 

Table 1. Substrate specificity

SubstrateRelative activity
(%)
Choline100
Betaine aldehyde46
Diethanolamine1
Triethanolamine3
N, N-Dimethylaminoethanol5
N-Methylethanolamine0

 

Table 2. Effect of various chemicals on COD activity

AdditivesConsentrationRelative activity
(%)
None–100
Triton X-1000.1%96
Adekatol SO-1200.1%106
Sodium laurylsulfate0.1%94
Deoxycholate0.1%94
Sodium laurylbenzene sulfate0.1%91
CaCl25mM101
MgCl25mM100
FeCl35mM0
ZnCl25mM8
MnCl25mM98
CoCl25mM31
MoCl25mM58
KCl5mM93
NaCl5mM97
NH4Cl5mM100
LiCl5mM97
BaCl25mM103

Fig.1 pH Optimum


□: Dimethylglutarate-NaOH buffer
■: Phosphate buffer
〇: Glycylglycine buffer
●: Glycine-NaOH buffer

Fig.2 pH Stability


37℃, 10 min.
△: Acetate buffer
▲: Dimethylglutarate-NaOH buffer
〇:Tris-HCI buffer
●: Glycine-NaOH buffer

Fig.3 Thermal Stability


pH 7.5,10 min

Fig.4 Storage (lyophilized powder)


〇: －20℃
□: 5℃
△: 30℃

Assay

Principle

  1. The assay is based on the increase in absorbance at 500 nm as the formation of quinoneimine dye proceeds in the following reactions:

 COD
Choline+O2→Betaine aldehyde+H2O2
 COD
Betaine aldehyde+O2+H2O→Betaine+H2O2
 POD
2 H2O2+4-AA+Phenol→Quinoneimine dye+4 H2O
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
    1.211 g of Tris (hydroxymethyl) amino methane, 2.1 g of choline chloride and 2 ml of 1 % (W/V) phenol are dissolved with 1 N HCl and adjusted to pH 8.0 (25℃) . Then, 1 ml of 1 % (W/V) 4–AA and 3 ml of 100 PPU/ ml POD are added to make a total of 100 ml.
  2. Enzyme dilution buffer
    10 mM Tris–HCl buffer (pH 8.) containing 2 mM
    EDTA: Ethylenediaminetetraacetic acid
  3. Reagents
    Choline chloride:
    FUJIFILM Wako Pure Chemical Corporation
    1st Grade #033–09812
    4-AA: NACALAI TESQUE, INC. Special grade #01907–52
    POD: Sigma Chemical Co. Type Ⅱ #P–8250
    EDTA (2 Na・2H2O) : KISHIDA CHEMICAL Co., Ltd.
    #060–29133
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 1.90 ml of reaction mixture into a small test tube and preincubate at 37℃.
  2. After 5 min, add 50 μl of enzyme solution and mix to start the reaction at 37℃.
  3. After starting the reaction, measure the rate of increase per minute in absorbance at 500 nm. The rate must be measured within the linear portion of the absorbance curve.
Calculation
  1. Activity (U/mg of powder) = {(△ A/min)/(12.0×1/2)} × 3.05/0.05 × 1/x
    12.0 :millimolar extinction coefficient of quinoneimine dye at 500 nm (cm2/μmole)
  1. 1/2 :multiplier derived from the fact that 2 mole of H2O2 produce 1 mole of quinoneimine dye.
    3.05 :final volume (ml)
    0.05 :volume of enzyme solution (ml)
    X :concentration of the sample in enzyme solution
    ( mg/ml)
Storage

  1. Storage at –20℃ in the presence of a desiccant is recommended. Enzyme activity will be retained for at least one year under this condition (Figure 4)

References
  1. Ikuta, S., Matsuura, K., Imamura, S., Misaki, H. and Horiuchi, Y. (1977) J. Biochem., 82, 157–163.
  2. Ikuta, S., Imamura, S., Misaki, H. and Horiuchi, Y. (1977) ibid, 82, 1741–1749.
  3. Ohta–Fukuyama, M., Miyake, Y., Emi, S. and Yamano, T. (1989) J. Biochem., 88, 197–203.
  4. Takayama, M. et al. (1977) Clin. Chim. Acta, 79, 93.
  5. Sugawara, K. and Kihara, A. (1978) Eisei Kensa, 27 (1) , 106–111.
  6. Okabe, H. et al. (1977) Clin. Chim. Acta, 80, 87.

COD 活性測定法 (Japanese)

試薬液
  1. 反応試薬混合液
    トリス (ヒドロキシメチル) アミノメタン1.211gと塩化コリン2.1g 及び1％ (W/V) フェノール液2ml を精製水に溶解した後、1N HCl でpH8.0 (25℃) に調整し、さらに1% (W/V) 4–AA 溶液1ml と100PPU/ml POD 溶液3ml を加えて溶かし、全容100ml とする。
  2. 酵素溶解希釈用液
    2mM EDTA と1％ (W/V) KCl を含む10mM トリス－ HCl 緩衝液pH8.0 溶液
    酵素溶解希釈用液
    50mM トリス−HCl 緩衝液 pH9.0
  3. 試薬
    塩化コリン：富士フイルム和光純薬製　一級
    #033–09812
    4-AA：ナカライテスク製　特級　#01907–52
    POD：シグマ製　Type Ⅱ　#P–8250
    EDTA (エチレンジアミン四酢酸・2Na・2H2O) ：
    キシダ化学製 #060–29133
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3.0ml 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50 μl を正確に加えて混和し、37℃で反応を開始する。
  3. 反応開始後、500nm における吸光度を測定して直線的に反応している1 分間当たりの吸光度変化を求める。
    ΔA/min ≦ 0.040 Abs/min
計算
活性 (U/mg) = {(△ A/min)/(12.0×1/2)} × 3.05/0.05 × 1/x
12.0 :キノンイミン色素の500nm におけるミリモル分子吸光係数 (cm2/ μmole)
1/2 :H2O2 2 モルからキノンイミン色素1 モルが生成することによる係数
3.05 :反応総液量 (ml)
0.05 :反応に供した酵素試料液量 (ml)
X :酵素試料液の検品濃度 (mg/ml)
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