CHOLINE OXIDASE from Alcaligenes sp.
Appearance | Yellowish amorphous powder, lyophilized | |
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Activity | GradeⅢ 10U/mg-solid or more (containing approx. 20% of stabilizers) |
|
Contaminant | Catalase | ≤1.0×102% |
Stabilizers | EDTA, BSA, amino acids (glycine, sodium glutamate, etc.) |
Stability | Stable at −20℃ for at least one year (Fig.1) |
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Molecular weight | approx. 95,000 |
Isoelectric point | 4.1±0.1 |
Michaelis constants | 2.84×10-3M (Choline), 5.33×10-3M (Betaine aldehyde) |
Structure | One mol of FAD is covalently bound to mol of the enzyme 8) |
Inhibitors | p-Chloromercuribenzoate, Cu++,Co++,Hg++,Ag+ |
Optimum pH | 8.0−8.5(Fig.4) |
Optimum temperature | 40−45℃(Fig.5) |
pH Stability | pH 7.0−9.0 (30℃, 2hr)(Fig.6) |
Thermal stability | below 37℃ (pH 7.5, 10min)(Fig.7) |
Effect of various chemicals | (Table 1) |
This enzyme is useful for enzymatic determination of phospholipids when coupled with phospholipase D and for choline esterase-activity in clinical analysis.9〜11)
The appearance of quinoneimine dye is measured at 500nm by spectrophotometry.
One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per minute under the conditions described below.
A. Choline chloride solution | 2.1%[2.1g choline chloride/100ml of Tris-HCl buffer (D)](Should be prepared fresh) |
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B. 4-AA solution | 1.0% (1.0g 4-aminoantipyrine/100ml of H2O)(Store at 4℃ in a brownish bottle) |
C. Phenol solution | 1.0% (1.0g phenol/100ml of H2O)(Store at 4℃ in a brownish bottle) |
D. Tris-HCl buffer | 0.1M Tris-HCl buffer, pH 8.0[Dissolve 12.1g of Tris (MW=121.14) in ca.800ml of H2O and, after adjusting the pH to 8.0 at 25℃ with 2.0 N HCl, fill up to 1,000ml with H2O.] |
E. Enzyme diluent | 10mM Tris-HCl buffer, pH 8.0 contg. 2mM EDTA and 1.0% KCl. |
1.Prepare the following working solution (100ml) in a brownish before use and store on ice in a brownish bottle.
97ml | Substrate solution | (A) |
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1.0ml | 4-AA solution | (B) |
2.0ml | Phenol solution | (C) |
5.0mg | Peroxidase from horseradish (110 purpurogallin units/mg) (Toyobo GradeⅢ) |
Concentration in assay mixture | |
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Tris buffer | 97 mM |
Choline chloride | 0.14 M |
EDTA | 33 μM |
KCI | 2.2 mM |
4-Aminoantipyrine | 0.48 mM |
Phenol | 2.1 mM |
POD | ca.4.92U/ml |
2.Pipette 3.0ml of working solution into a cuvette (d=1.0cm) and equilibrate at 37℃ for about 5 minutes.
3.Add 0.05ml of the enzyme solution* and mix by gentle inversion.
4.Record the increase in optical density at 500nm against the working solution for 3 to 4 minutes in a spectrophotometer thermostated at 37℃, and calculate theΔOD per minute from the initial linear portion of the curve.
*Dissolve the enzyme preparation in ice-cold Tris-HCl buffer (D) and dilute to 0.1−0.5U/ml with enzyme diluent (E).
Activity can be calculated by using the following formula :
Volume activity (U/ml) =
ΔOD/min×Vt×df
12.0×1/2×1.0×Vs
= ΔOD/min×10.17×df
Weight activity (U/mg) = (U/ml)×1/C
Vt | : Total volume (3.05ml) |
Vs | : Sample volume (0.05ml) |
12.0 | : Millimolar extinction coefficient of quinoneimine dye under the assay conditions(cm2/micromole) |
1/2 | : Factor based on the fact that one mole of H2O2 produces half a mole of quinoneimine dye. |
1.0 | : Light path length (cm) |
df | : Dilution factor |
C | : Enzyme concentration in dissolution (c mg/ml) |
1)P.J.G Mann and J.H.Quastel; Biochem.J.,31, 869 (1937).
2)P.J.G Mann et al; Biochem.J.,32, 1024 (1938).
3)H.S.Shieh; Can.J.Microbiol., 10, 837 (1964).
4)H.S.Shieh; Ibid.,11, 375 (1965).
5)G.J.J.Kortstee; Arch.Mikirobiol., 71, 235 (1970).
6)S.Ikuta, K.Matuura, S.Imamura, H.Misaki, Y.Horiuti; J.Biochem., 82, 157 (1977).
7)S.Ikuta, S.Imamura, H.Misaki and Y.Horiuti ; J.Biochem., 82, 1741 (1977).
8)M.Ohta-Fukuyama, Y.Miyake, S.Emi and T.Yamano; J.Biochem., 88, 197 (1980).
9)M.Takayama et al; Clin. Chim. Acta, 79, 93 (1977).
10)K.Sugawara and A.Kihara; Eisei Kensa, 27(1), 106 (1978).
11)H.Okabe et al ; Clin.Chim.Acta, 80, 87 (1977).
[The enzyme dissolved in 10mM Tris-HCl buffer, pH 8.0 contg. 2mM EDTA and 1.0% KCl (5U/ml) was incubated with each chemical at 25℃ for 1hr.]
Chemical | Concn.(mM) | Residual activity(%) |
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None | ー | 100 |
Metal salt | 2.0 | |
MgCl2 | 87 | |
CaCl2 | 92 | |
Ba(OAc)2 | 89 | |
FeCl3 | 87 | |
CoCl2 | 89 | |
MnCl2 | 91 | |
ZnCl2 | 88 | |
CdCl2 | 92 | |
NiCl2 | 91 | |
CuSO4 | 92 | |
Pb(OAc)2 | 87 | |
AgNO3 | 80 | |
HgCl2 | 48 | |
2-Mercaptoethanol | 2.0 | 90 |
PCMB | 1.0 | 13 |
Chemical | Concn.(mM) | Residual activity(%) |
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MIA | 2.0 | 87 |
NEM | 2.0 | 100 |
IAA | 2.0 | 95 |
Hydroxylamine | 2.0 | 77 |
EDTA | 5.0 | 92 |
o-Phenanthroline | 2.0 | 90 |
α,α′-Dipyridyl | 1.0 | 91 |
Borate | 50 | 94 |
NaF | 2.0 | 92 |
NaN3 | 2.0 | 92 |
Triton X-100 | 0.10% | 96 |
Brij 35 | 0.10% | 92 |
Tween 20 | 0.10% | 95 |
Span 20 | 0.10% | 94 |
Na-cholate | 0.10% | 96 |
SDS | 0.05% | 95 |
DAC | 0.05% | 91 |
Fig.1. Stability (Powder form)
(kept under dry conditions)
Fig.2. Stability (Powder form)
(kept under dry conditions)
Fig.3. Stability (Liquid form at 37℃)
enzyme concentration: 1.0mg/ml buffer composition: 0.1M K-phosphate buffer, pH7.5
Fig.4. pH-Activity
(37℃, in 50mM K-phosphate buffer)
Fig.5. Temperature activity
(in 50mM K-phosphate buffer,pH7.5)
Fig.6. pH-Stability
30℃, 2hr-treatment with 50mM buffer solution:pH6.0-9.0, K-phosphate; pH9.0-10.0, glycine-NaCl-NaOH.
Fig.7. Thermal stability
15min-treatment with 50mM K-phosphate buffer, pH7.5
1. 原理
4-AminoantipyrineとPhenolの酸化縮合生成物であるQuinoneimine色素を500nmで測定し,上記反応で生成したH2O2量を定量する。
2.定義
下記条件下で1分間に1マイクロモルのH2O2を生成する酵素量を1単位 (U)とする。
3.試薬
酵素溶液:酵素標品を予め氷冷した0.1M Tris-HCl緩衝液,pH8.0で溶解し,2.0mM EDTAと1.0%のKClを含む10mM Tris-HCl緩衝液,pH8.0で0.1〜0.5 U/mlに希釈する。
4.手順
1.下記反応混液を調製する(褐色瓶にて氷冷保存)。
97.0ml | 基質溶液 | (A) |
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1.0ml | 4-AA水溶液液 | (B) |
2.0ml | フェノール水溶液 | (C) |
5.0ml | peroxidase(110プルプロガリン単位/mg) |
2.反応混液3.0mlをキュベット(d=1.0cm)にとり,37℃で約5分間予備加温する。
3.酵素溶液0.05mlを添加し,ゆるやかに混和し,反応混液を対照に37℃に制御された分光光度計で500nmの吸光度変化を3〜4分間記録し,その初期直線部分から1分間あたりの吸光度変化を求める(ΔOD/min)。
5.計算式
U/ml =
ΔOD/min×3.05(ml)
12.0×1/2×1.0×0.05(ml)
×希釈倍率
= ΔOD/min×10.17×希釈倍率 | |
U/mg | =U/ml×1/C |
12.0 | : Quinoneimine色素の上記測定条件下でのミリモル分子吸光係数(cm2/micromole) |
1/2 | : 酸素反応で生成したH2O2の1分子のから形成するQuinoneimine色素は1/2分子である事による係数。 |
1.0 | : 光路長(cm) |
C | : 溶解時の酵素濃度(c mg/ml) |
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