CATALASE from Microorganism
Appearance | Olive green solution |
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Activity | GradeⅤ 150,000 U/ml or more |
Stability | Stable at 4℃(Fig.1) |
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Molecular weight | approx. 53,000 (by mass spectrometry) |
Inhibitors | NaN3 |
Optimum pH | 7.2-9.0(Fig.2) |
Optimum temperature | 35-40℃(Fig.3) |
pH Stability | pH 6.2-8.9 (25℃, 16hr)(Fig.4) |
Thermal stability | below 35℃ (pH 7.0, 30min)(Fig.5) |
Effect of various chemicals | (Table 1) |
This enzyme is useful for eliminating the interference of hydrogen-peroxide in clinical analysis.
The disappearance of hydrogen peroxide is measured by the Titanium color method 1).
One unit causes the hydrolysis of one micromole of hydrogen peroxide per minute under the conditions described below.
A. 10mM Phosphate buffer, pH 7.0 (at 25℃) | |
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B. H2O2 solution | 16mM[0.182ml of 30% (W/V)H2O2/100ml of buffer A](Should be prepare fresh and store on ice.) |
C. Titanium reagent | (Nacalai tesque) |
D. Enzyme diluent | Buffer A |
1.Prepare 0.25ml of the substrate solution (B) into a test tube and equilibrate at 25℃ for about 5 minutes
2.Add 0.25ml of the enzyme solution* and mix.
3.After exactly 5 minutes at 25℃, add 2.5ml of Titanium reagent (C) to stop the reaction and measure the optical density at 410nm against water (OD test)
At the same time, prepare the blank by first mixing the substrate solution with 2.5ml of Titanium reagent after 5 min-incubation at 25℃, follow by the addition of enzyme solution (OD blank)
*Dilute the enzyme preparation to 0.35-1.35U/ml with ice-cold enzyme diluent (D).
Activity can be calculated by using the following formula :
Volume activity (U/ml) =
ΔOD(OD blank-OD test)×Vt×df
F×t×1.0×Vs
= ΔOD/min×2.4×1/F×df
Weight activity (U/mg) = (U/ml)×1/C
Vt | : Total volume (3.0ml) |
Vs | : Sample volume (0.25ml) |
F | : Extinction coefficient of Titanium color product developed by the presence of 1.0mM hydrogen peroxide (F should be determined in each lot of Titanium reagent by using a known concentration of hydrogen peroxide. F is usually around 0.7.) |
t | : Reaction time (5 minutes) |
1.0 | : Light path length (cm) |
df | : Dilution factor |
C | : Enzyme concentration in dissolution (c mg/ml) |
1)F.Patti and P.B.-Maury; Bull.Soc.Chem.Biol.,35, 1177 (1953)
[The enzyme dissolved in 10mM K-phosphate buffer, pH 7.0 (200U/ml) was incubated with each chemical at 25℃ for 1hr.]
Chemical | Concn.(mM) | Residual activity(%) |
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None | - | 100 |
Metal salt | 2.0 | |
AgNO3 | 76.6 | |
BaCl2 | 99.6 | |
CaCl2 | 62.4 | |
CoCl2 | 101.2 | |
CuSO4 | 99.7 | |
FeSO4 | 98.2 | |
MgSO4 | 99.2 | |
MnCl2 | 28.7 | |
NiCl2 | 53.6 | |
ZnCl2 | 96.1 |
Chemical | Concn.(mM) | Residual activity(%) |
---|---|---|
NaF | 2.0 | 89.3 |
NaN3 | 2.0 | 3.8 |
EDTA | 5.0 | 96.6 |
IAA | 2.0 | 99.4 |
Borate | 20 | 98.8 |
SDS | 0.05% | 96.1 |
Triton X-100 | 0.10% | 95.7 |
Brij 35 | 0.10% | 97.8 |
Span 20 | 0.10% | 94.5 |
Na-cholate | 0.10% | 95.3 |
Fig.1. Stability (Liquid form)
Fig.2. pH-Activity
in 10mM buffer solution: pH 5-8,K-phosphate; pH 7.5-10, Tris-HCl
Fig.3. Temperature activity
(in 10mM K-phosphate buffer, pH 7.0)
Fig.4. pH-Stability
in 10mM buffer solution: pH 5-8,K-phosphate; pH 7.5-10, Tris-HCl.Enzyme concentration: 1U/ml
Fig.5. Thermal stability
30min-treatment with 10mM K-phosphate buffer, pH 7.0.Enzyme concentration: 200U/ml
1. 原理
過酸化水素の減少量をチタン呈色法で測定する。
2.定義
下記条件下で1分間に1マイクロモルの過酸化水素を分解する酵素量を1単位(U)とする。
3.試薬
4.手順
1.試験管に0.25mlの基質溶液(B)を採り,25℃で約5分間予備加温する。
2.酵素溶液0.25mlを添加し,緩やかに混和する。
3.25℃で正確に5分間反応させた後,チタン試薬(C)2.5mlを加えて反応を停止させ,水を対照にして410nmの吸光度を測定する(OD test)。
4.盲検は5分間の反応の後, 最初に基質溶液(B)0.25㎖をチタン試薬(C)2.5mlに加えて混和し,次いで酵素溶液を添加する(OD blank)。
5.計算式
U/ml =
ΔOD (OD blank-OD test)×3.0(ml)×希釈倍率
F×5(分)×1.0×0.25(ml)
= ΔOD/min×2.4×1/F×df | |
U/mg | = U/ml×1 / C |
F | 1.0mM過酸化水素によるチタン呈色生成物の吸光係数 (Fは濃度の分かっている過酸化水素を用いて各ロット毎に決定する。通常は0.7前後である) |
1.0 | 光路長(cm) |
C | 溶解時の酵素濃度(c mg/ml) |
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