TOYOBO Ideas & Chemistry バイオ事業総括部

URICASE

PREPARATION and SPECIFICATION

Appearance White amorphous powder, lyophilized
Activity GradeⅡ　4.0U/mg-solid or more
(containing approx.20% of stabilizers)
Contaminants Catalase ≤1.0%
Stabilizers Borate, EDTA, nonionic detergents

PROPERTIES

Stability Stable at −20℃ for at least one year （Fig.1）
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℃, 20hr)（Fig.6）
Thermal stability below 50℃ (pH 8.5, 10min)（Fig.7）
Effect of various chemicals (Table.1)

APPLICATIONS

This enzyme is useful for enzymatic determination of uric acid in clinical analysis.

ASSAY

Principle

Principle

The disappearance of uric acid is measured at 290nm by spectrophotometry.

Unit definition

One unit causes the oxidation of one micromole of uric acid per minute under the conditions described below.

Method

Reagents

A. Uric acid solution0.001%［Dilute the stock solution (0.01%) to 10-fold volume with 50mM borate buffer containing 0.001% Triton X-100 and 1.0mM EDTA, pH 8.5］(Should be prepared fresh) stock solution : 10mg uric and/100ml of above buffer (store at 0−5℃)
B. KOH solution20%
C. Enzyme diluent50mM borate buffer containing 0.001% Triton X-100 and 1.0mM EDTA, pH 8.5

Procedure

1.Prepare the following reaction mixture in a test tube and equilibrate at 25℃ for about 5 minutes.

2.0ml Uric acid solution (A)
0.5ml Distilled water
Concentration in assay mixture
Borate buffer 42 mM
Uric acid 40 μM
EDTA 0.83 mM
Triton X-100 0.00083%

2.Add 0.5ml of the enzyme solution* and mix by gentle inversion.

3.After exactly 5 minutes at 25℃, add 0.2ml of 20% KOH solution (B) to stop the reaction and measure the optical density at 290nm against water (OD test).
At the same time, prepare the blank by first mixing the reaction mixture with 0.2ml of KOH solution after 5min-incubation at 25℃, followed by the addition of the enzyme solution (OD blank).

*Dissolve the enzyme preparation in ice-cold enzyme diluent (C) and dilute to 0.01−0.02U/ml with the same buffer and store on ice.

Calculation

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 (3.2ml)
Vs : Sample volume (0.5ml)
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)

REFERENCES

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)

Table 1 Effect of Various Chemicals on Uricase

[The enzyme dissolved in 50mM borate buffer, pH 8.5 containing 0.001% Triton X-100 and 1.0mM EDTA (10U/ml) was incubated with each chemical at 25℃ for 1hr.］

  • Chemical Concn.(mM) Residual
    activity(%)
    None - 100
    Metal salt2.0
    MgCl295
    CaCl291
    Ba(OAc)295
    FeCl388
    CoCl288
    MnCl284
    ZnCl269
    CdCl264
    NiCl279
    CuSO49.8
    Pb(OAc)274
    AgNO30
    HgCl20
    2-Mercaptoethanol2.0101
    PCMB1.034
  • Chemical Concn.(mM) Residual
    activity(%)
    MIA2.070
    NEM2.041
    IAA2.094
    Hydroxylamine2.095
    EDTA5.090
    o-Phenanthroline2.098
    α,α′-Dipyridyl1.099
    Borate5090
    Naf2.087
    NaN32.096
    Triton X-1000.10%111
    Brij 350.10%94
    Tween 200.10%85
    Span 200.10%91
    Na-cholate0.10%96
    SDS0.05%91
    DAC0.05%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)

    Fig.1. Stability (Powder form)

    (kept under dry conditions)

  • Fig.2. Stability (Powder form)

    Fig.2. Stability (Powder form)

    (kept under dry conditions)

  • Fig.3. Stability (Liquid form)

    Fig.3. Stability (Liquid form)

    enzyme concentration : 3.0U/ml buffer composition : 0.2M borate buffer contg. 1mM EDTA,pH8.5

  • Fig.4. pH-Activity

    Fig.4. pH-Activity

    (25℃,in 67mM borate buffer)

  • Fig.5. Temperature activity

    Fig.5. Temperature activity

    5min-reaction in 50mM borate buffer, pH8.5

  • Fig.6. pH-Stability

    Fig.6. pH-Stability

    25℃, 20hr-treatment with the following buffer solution: ○̶○ 67mM borate ; ●̶● 50mM phosphate

  • Fig.7. Thermal-stability

    Fig.7. Thermal-stability

    10min-treatment with 67mM borate buffer, pH8.5.

活性測定法（Japanese）

1. 原理

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

  • 0.0 01%　尿酸溶液〔保存溶液(0.01%)を 0.001% Triton X-100及び1.0mM EDTAを含む 50mMホウ酸緩衝液,pH8.5で10倍希釈する〕(用時調製)保存溶液は10.0㎎の尿酸を同上緩衝液 100mlに溶解して調製する(0～5℃で保存)
  • 20%KOH溶液

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。

2.0ml 尿酸溶液 (A)
0.5ml 蒸留水

2.酵素溶液0.5ml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)0.2ml 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)0.2mlを加えて混和し,次いで酵素溶液0.5ml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)