TOYOBO Ideas & Chemistry Biotechnology Operating
Department

SARCOSINE OXIDASE

PREPARATION and SPECIFICATION

Appearance Yellowish amorphous powder, lyophilized
Activity GradeⅢ　8.0 U/mg-solid or more
Contaminants Catalase ≤1.0 %
Stabilizers Potassium chloride

PROPERTIES

Stability Stable at −20 ℃ for at least one year （Fig.1）
Molecular weight approx.43,000 (by SDS-PAGE)
Isoelectric point 4.8±0.1
Michaelis constant 2.8×10-3 M
Inhibitors Cu2+, Ag＋, Hg2+, p-chloromercuribenzoate, N-ethylmaleimide, SDS
Optimum pH 7.5−8.5 （Fig.2）
Optimum temperature 55−60 ℃ （Fig.3）
pH Stability 6.0−9.5 (25 ℃, 20 hr) （Fig.4）
Thermal stability below 60 ℃ (pH 7.5, 30 min) （Fig.5）
Effect of various chemicals (Table.1)

APPLICATIONS

This enzyme is useful for enzymatic determination of creatinine, creatine, and sarcosine, in combination with creatinine amidohydrolase (CNH-211, CNH-311) and creatine amidinohydrolase (CRH-211, CRH221, CRH-229).

ASSAY

Principle

Principle

Unit definition

One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per minute under the conditions detailed below.

Method

Reagents

A. Sarcosine solution 0.2 M: Weigh out 1.78 g of sarcosine (MW ＝ 89.09), dissolve in 80 mL of 0.125 M TrisHCl buffer, pH 8.0, containing 0.125 % Triton X-100, and, after adjusting to pH 8.0 at 25 ℃ with 1.0 N NaOH or 1.0 N HCl, make up to 100 mL with H2O (stable for 1 week if stored at 0−5 ℃).
B. 4-AA solution 0.1 %: 100 mg of 4-aminoantipyrine / 100 mL of H20 (store at 4 ℃ in a brownish bottle).
C. Phenol solution 0.1 %: 100 mg of phenol / 100 mL of H2O (store at 4 ℃ in a brownish bottle).
D. Peroxidase solution 0.025 %: 25 mg of peroxidase (110 purpurogallin units / mg) / 100 mL of H2O (should be freshly prepared).
E. SDS solution 0.25 %: 1.25 g of sodium dodecyl sulfate / 500 mL of H2O
F. Enzyme diluent 20 mM Tris-HCl buffer, pH 8.0 containing 2.0 mM EDTA

Procedure

1.Prepare the following working solution(for 100 tests) in a brownish bottle and store on ice.

50 mL Sarcosine solution (A)
10 mL 4-AA solution (B)
20 mL Phenol solution (C)
20 mL Peroxidase solution (D)
Concentration in assay mixture
Tris-HCl buffer 48 mM
Sarcosine 95 mM
4-Aminoantipyrine 0.47 mM
Phenol 2.0 mM
Triton X-100 0.045%
POD Approx. 5.2 U/mL

2.Pipette 1.0 mL of working soluton into a test tube and equilibrate at 37 ℃ for approximately 5 minutes.

3.Add 0.05 mL of the enzyme solution* and mix.

4.After exactly 10 minutes at 37 ℃, add 2.0 mL of SDS solution (E) to stop the reaction, and measure the optical density at 500 nm against water (OD test).
At the same time, prepare the blank using the same method as for the test except that the enzyme diluent is used instead of the enzyme solution (OD blank).

*Dissolve the enzyme preparation in ice-cold enzyme diluent (F) and dilute to 0.07−0.17 U/mL with the same buffer, immediately before the assay.

Calculation

Activity can be calculated by using the following formula :

  • Volume activity (U/mL) =

  • ΔOD (OD test−OD blank)×Vt×df

    13.3×1/2×1.0×t×Vs

  • ＝ ΔOD×0.917×df

Weight activity (U/mg) ＝ (U/mL)×1/C

Vt : Total volume (3.05 mL)
Vs : Sample volume (0.05 mL)
13.3 : Millimolar extinction coefficient of quinoneimine dye under the assay condition (cm2/micromole)
1/2 : Factor based on the fact that one mole of H2O2 produced half a mole of quinoneimine dye
t : Reaction time (10 minutes)
1.0 : Light path length (cm)
C : Enzyme concentration in dissolution (c mg/mL)

REFERENCES

1) N.Mori, M.Sato, Y.Tani and Y.Yamada; Agric.Biol.Chem., 44, 1391 (1980).

2) M.Suzuki; J. Biochem., 89, 599 (1981).

3) M.Suzuki and M.Yoshida; Proceedings of the Symposium on Chemical Physiolosy and Pathology (Kyoto), Vol16, p.220 (1976).

4) T.Kinoshita and Y.Hiraga; Chem.Pharm.Bull., 28, 3501 (1980).

5) Y.Nishiya, S.Zuihara and T.Imanaka; APPLIED AND ENVIROMENTAL MICROBIOLOGY., 61, 367 (1995).

Table 1. Effect of Various Chemicals on Sarcosine oxidase

[The enzyme dissolved in 50 mM K-phosphate buffer, pH 7.5 (10 U/mL) was incubated at 30 ℃ for 30 minutes.］

  • Chemical Concn.(mM) Residual
    activity(%)
    None - 100
    Metal salt 2.0
    MgCl2 99
    CaCl2 99
    Ba(OAc)2 98
    FeCl3 99
    CoCl2 98
    MnCl2 99
    Zn(OAc)2 98
    Cd(OAc)2 99
    NiCl2 96
    CuSO4 43
    Pb(OAc)2 98
    AgNO3 0.4
    HgCl2 0.4
    PCMB 2.0 36
    MIA 2.0 101
  • Chemical Concn.(mM) Residual
    activity(%)
    NAF 2.0 99
    NaN3 20.0 90
    EDTA 5.0 97
    o-Phenanthroline 2.0 99
    α,α′-Dipyridyl 2.0 97
    Borate 50 98
    IAA 2.0 97
    NEM 2.0 74
    Hydroxylamine 2.0 97
    Triton X-100 0.10% 99
    Brij 35 0.10 % 99
    Tween 20 0.10 % 97
    Span 20 0.10 % 101
    Na-cholate 0.10 % 99
    SDS 0.05 % 68
    DAC 0.05 % 97

Ac, CH3CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate;
IAA, Iodoacetate; 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. pH-Activity

    Fig.2. pH-Activity

    37 ℃ in 0.1 M buffer solution : pH 6.5-7.5 K-phosphate,:pH 7.5-8.5 Tris-HCl:pH 8.5-9.5 Glycine-NaOH

  • Fig.3. Temperature activity

    Fig.3. Temperature activity

    (in 0.1 M Tris-HCI buffer : pH 8.0)

  • Fig.4. pH-Stability

    Fig.4. pH-Stability

    25 ℃,24hr-treatment with 100 mM buffer solution:pH 5-6 Acetate buffer, :pH 6-8 K-phosphate,:pH 8-9 Tris-HCl, :pH 8.5-9.5 Glycine-NaOH

  • Fig.5. Thermal stability

    Fig.5. Thermal stability

    30 min-treatment with 50 mM K-phosphate pH 7.5 (contg. 100 mM NaCl) enzyme concn. 5 U/mL

活性測定法（Japanese）

1. 原理

原理

4-AminoantipyrineとPhenolの酸化縮合物である 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

  • 0.2Mサルコシン溶液〔1.78gのサルコシン(MW＝ 89.09)を80mlの0.125%Triton X-100を含む 0.125M Tris-HCl緩衝液, pH8.0に溶解後，1.0N のNaOHあるいはHClでpHを8.0に調整(25℃)し, 蒸留水で100mlとする〕(0～5℃保存で1週間は使用可能)8
  • 0.1%4-AA水溶液(100mgの4−アミノアンチピリンを100mlの蒸留水に溶解する)(褐色瓶中で4℃ 保存)
  • 0.1%フェノール水溶液(100mgのフェノールを100ml の蒸留水に溶解する) (褐色瓶中で4℃保存)
  • POD溶液〔25mgのペルオキシダーゼ(POD)(110 プルプロガリン単位/mg)を蒸留水100mlに溶解する〕(用時調製)
  • 0.25%SDS水溶液〔1.25gのsodium dodecyl sulfate(SDS)を500mlの蒸留水に溶解する〕

酵素溶液：酵素標品を予め氷冷した2.0mM EDTAを含む20mM Tris-HCl緩衝液, pH8.0で溶解し, 分析直前に同緩衝液で0.07〜0.17U/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)。

50ml サルコシン溶液 (A)
10ml 4-AA水溶液 (B)
20ml フェノール水溶液 (C)
20ml POD水溶液 (D)

2.反応混液1.0mlを試験管に採り,37℃で約5分間予備加温する。

3.酵素溶液0.05mlを加え,反応を開始する。

4.37℃で正確に10分間反応させた後,SDS水溶液 (E)2.0mlを加えて反応を停止させる。この液につき 500nmにおける吸光度を測定する(OD test)。

5.盲検は酵素溶液の代わりに酵素希釈液(2.0mM EDTAを含む20mM Tris-HCl緩衝液, pH8.0)を用い, 上記同様に操作を行って吸光度を測定する(OD blank)。

5.計算式

  • U/ml ＝

  • ΔOD (OD test−OD blank)×3.05(ml)×希釈倍率

    13.3×1/2×1.0×10(分)×0.05(ml)

＝ ΔOD×0.917×希釈倍率
U/mg ＝ U/ml×1/C
13.3 : Quinoneimine色素の上記測定条件下でのミリモル分子吸光係(cm2/micromole)
1/2 : 酵素反応で生成したH2O2の1分子から形成する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)