(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-05REACH compliant product

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

Substrate	Relative activity (%)
Choline	100
Betaine aldehyde	46
Diethanolamine	1
Triethanolamine	3
N, N-Dimethylaminoethanol	5
N-Methylethanolamine	0

Table 2. Effect of various chemicals on COD activity

Additives	Consentration	Relative activity (%)
None	–	100
Triton X-100	0.1%	96
Adekatol SO-120	0.1%	106
Sodium laurylsulfate	0.1%	94
Deoxycholate	0.1%	94
Sodium laurylbenzene sulfate	0.1%	91
CaCl₂	5mM	101
MgCl₂	5mM	100
FeCl₃	5mM	0
ZnCl₂	5mM	8
MnCl₂	5mM	98
CoCl₂	5mM	31
MoCl₂	5mM	58
KCl	5mM	93
NaCl	5mM	97
NH₄Cl	5mM	100
LiCl	5mM	97
BaCl₂	5mM	103

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

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

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

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.
Enzyme dilution buffer
10 mM Tris–HCl buffer (pH 8.) containing 2 mM
EDTA: Ethylenediaminetetraacetic acid
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

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 1.90 ml of reaction mixture into a small test tube and preincubate at 37℃.
After 5 min, add 50 μl of enzyme solution and mix to start the reaction at 37℃.
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

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)

1/2 :	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

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

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

COD 活性測定法 (Japanese)

試薬液

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。
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
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

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3.0ml 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50 μl を正確に加えて混和し、37℃で反応を開始する。
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)

CHOLINE OXIDASE [COD] (T-05)