URICASE from Candida sp.
Appearance | White amorphous powder, lyophilized | |
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Activity | GradeⅡ 4.0 U/mg-solid or more (containing approx.20 % of stabilizers) |
|
Contaminants | Catalase | ≤ 1.0 % |
Stabilizers | Borate, EDTA, nonionic detergents |
Stability | Stable at −20 ℃ for at least one year (Fig.1) |
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Molecular weight | approx. 120,000 |
Structure | 4 subunits per enzyme molecule (Reactive SH groups are present in the enzyme molecule) |
Isoelectric point | 5.4 |
Michaelis constant | 2.5×10-5 M (Uric acid) |
Inhibitors | Heavy metal ions, cyanide,various urate analogs |
Optimum pH | 8.5 (Fig.4) |
Optimum temperature | 40 ℃ (Fig.5) |
pH Stability | pH 7.0−11.0 (25 ℃, 20 hr)(Fig.6) |
Thermal stability | below 50 ℃ (pH 8.5, 10 min)(Fig.7) |
Effect of various chemicals | (Table.1) |
This enzyme is useful for enzymatic determination of uric acid in clinical analysis.
The elimination of uric acid is measured at 290 nm by spectrophotometry.
One unit causes the oxidation of one micromole of uric acid per minute under the conditions detailed below.
A. Uric acid solution | 0.001 %: Dilute the stock solution (0.01 %) to 10-fold volume with 50 mM borate buffer containing 0.001 % Triton X-100 and 1.0 mM EDTA, pH 8.5 (should be prepared fresh) stock solution : 10 mg uric and/100 mL of above buffer (store at 0−5 ℃) |
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B. KOH solution | 20 % |
C. Enzyme diluent | 50 mM borate buffer containing 0.001 % Triton X-100 and 1.0 mM EDTA, pH 8.5 |
1.Prepare the following reaction mixture in a test tube and equilibrate at 25 ℃ for approximately 5 minutes.
2.0 mL | Uric acid solution | (A) |
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0.5 mL | Distilled water |
Concentration in assay mixture | |
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Borate buffer | 42 mM |
Uric acid | 40 μM |
EDTA | 0.83 mM |
Triton X-100 | 0.00083 % |
2.Add 0.5 mL of the enzyme solution* and mix by gentle inversion.
3.After exactly 5 minutes at 25 ℃, add 0.2 mL of 20 % KOH solution (B) to stop the reaction and measure the
optical density at 290 nm against water (OD test).
At the same time, prepare the blank by mixing the reaction mixture with 0.2 mL of KOH solution after
incubation for 5 minutes at 25 ℃, and then adding the enzyme solution (OD blank).
*Dissolve the enzyme preparation in ice-cold enzyme diluent (C) and dilute to 0.01−0.02 U/mL with the same buffer and store on ice.
Activity can be calculated by using the following formula :
Volume activity (U/mL) =
ΔOD (OD blank−OD test)×Vt×df
12.2×1.0×t×Vs
= ΔOD×0.105×df
Weight activity (U/mg) = (U/mL)×1/C
Vt | : Total volume (3.2 mL) |
Vs | : Sample volume (0.5 mL) |
12.2 | : Millimolar extinction coefficient of uric acid (cm2/micromole) |
t | : Reaction time (5 minutes) |
1.0 | : Light path length (cm) |
df | : Dilution factor |
C | : Enzyme concentration in dissolution (c mg/mL) |
1) K.Itaya,T.Yamamoto and J.Fukumoto; Agric.Biol.Chem., 31, 1256 (1967)
2) Y.Nakagiri and T.Yamamoto; Eisei Kensa(Japanese), 20, 751 (1971)
3) N.Kageyama et al.; Eisei Kensa(Japanese), 19, 338 (1969)
4) N.Kageyama et al.; Eisei Kensa(Japanese), 18, 59 (1969)
5) N.Kageyama; Rirsho Kensa(Japanese), 16, 891 (1972)
6) N.Kageyama; Clin.Chim.Acta, 31, 421 (1971)
7) T.Kawashima et al.; Nihon Kagakukaishi(Japanese), 10, 1542 (1980)
8) H.Nishimura et al.; J.Biochem., 91, 41-48 (1982)
[The enzyme dissolved in 50 mM borate buffer, pH 8.5 containing 0.001 % Triton X-100 and 1.0 mM EDTA (10 U/mL) was incubated with each chemical at 25 ℃ for 1 hr.]
Chemical | Concn.(mM) | Residual activity(%) |
---|---|---|
None | - | 100 |
Metal salt | 2.0 | |
MgCl2 | 95 | |
CaCl2 | 91 | |
Ba(OAc)2 | 95 | |
FeCl3 | 88 | |
CoCl2 | 88 | |
MnCl2 | 84 | |
ZnCl2 | 69 | |
CdCl2 | 64 | |
NiCl2 | 79 | |
CuSO4 | 9.8 | |
Pb(OAc)2 | 74 | |
AgNO3 | 0 | |
HgCl2 | 0 | |
2-Mercaptoethanol | 2.0 | 101 |
PCMB | 1.0 | 34 |
Chemical | Concn.(mM) | Residual activity(%) |
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MIA | 2.0 | 70 |
NEM | 2.0 | 41 |
IAA | 2.0 | 94 |
Hydroxylamine | 2.0 | 95 |
EDTA | 5.0 | 90 |
o-Phenanthroline | 2.0 | 98 |
α,α′-Dipyridyl | 1.0 | 99 |
Borate | 50 | 90 |
Naf | 2.0 | 87 |
NaN3 | 2.0 | 96 |
Triton X-100 | 0.10 % | 111 |
Brij 35 | 0.10 % | 94 |
Tween 20 | 0.10 % | 85 |
Span 20 | 0.10 % | 91 |
Na-cholate | 0.10 % | 96 |
SDS | 0.05 % | 91 |
DAC | 0.05 % | 89 |
Ac, CH3CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate;
IAA, Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.
Fig.1. Stability (Powder form)
(kept under dry conditions)
Fig.2. Stability (Powder form)
(kept under dry conditions)
Fig.3. Stability (Liquid form)
enzyme concentration : 3.0 U/mL buffer composition : 0.2 M borate buffer contg. 1 mM EDTA, pH 8.5
Fig.4. pH-Activity
(25 ℃,in 67 mM borate buffer)
Fig.5. Temperature activity
5 min-reaction in 50 mM borate buffer, pH 8.5
Fig.6. pH-Stability
25 ℃, 20 hr-treatment with the following buffer solution: ○̶○ 67 mM borate ; ●̶● 50 mM phosphate
Fig.7. Thermal-stability
10 min-treatment with 67 mM borate buffer, pH 8.5.
1. 原理
尿酸の消失量を290nmにおける吸光度の変化で測定する。
2.定義
下記条件下で1分間に1マイクロモルの尿酸を酸化する酵素量を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0.001 %Triton X100および1.0mM EDTAを含む50mMホウ酸緩衝液,pH8.5で溶解し,同緩衝液で0.01 ~0.02U/mLに希釈し氷冷保存する。
4.手順
1.試験管に下記反応混液を調製し,25℃で約5分間予備 加温する。
2.0 mL | 尿酸溶液 | (A) |
0.5 mL | 蒸留水 |
2.酵素溶液0.5 mLを加え,反応を開始する。
3.25℃で正確に5分間反応させた後,KOH溶液(B)0.2 mL を加えて反応を停止させる。この液につき290nmにおける吸光度を測定する(OD test)。
4.盲検は反応混液①を25℃で5分間放置後,KOH溶液 (B)0.2 mLを加えて混和し,次いで酵素溶液0.5 mLを加えて調製する。以下同様に吸光度を測定する(OD blank)。
5.計算式
U/mL =
ΔOD (OD blank−OD test)×3.2(mL)×希釈倍率
12.2×1.0×5(分)×0.5(mL)
= ΔOD×0.105×希釈倍率 | |
U/mg | = U/mL×1/C |
12.2 | : 尿酸のミリモル分子吸光係数 (cm2/micromole) |
1.0 | : 光路長(cm) |
C | : 溶解時の酵素濃度(c mg/mL) |
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