TOYOBO Ideas & Chemistry バイオ事業総括部

PREPARATION and SPECIFICATION

Appearance Light brown amorphous powder, lyophilized
Activity GradeⅢ　2.0 U/mg-solid or more
　　　　　　(containing approx. 20% of stabilizers)
Stabilizer Na-Cholate

PROPERTIES

StabilityStable at −20℃ for at least one year（Fig.1,2）
Molecular weightapprox. 130,000
Isoelectric point4.1±0.1
Michaelis constants 3.9×10-5M (Linoleate),9.2×10-5M (Palmitate),
6.3×10-5M (Decylate),8.8×10-5M (Propionate)
InhibitorsHeavy metal ions (Hg＋＋, Ag＋, Fe＋＋＋)
Optimum pH4.8−8.0 (Cholesterol linoleate), 5.0 (serum)（Fig.5）
Optimum temperature55−60℃（Fig.6）
pH StabilitypH 2.5−7.5 (25℃, 20hr)（Fig.7）
Thermal stabilitybelow 55℃ (pH 5.5, 10min)（Fig.8）
Substrate specificity(Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of cholesterol when coupled with cholesterol oxidase (COO-311, COO-321, COO-331) in clinical analysis.

ASSAY

Principle

Principle

Principle

The appearance of indamine is measured at 590nm by spectrophotometry.

Unit definition

One unit causes the hydrolysis of one micromole of cholesterol ester per minute under the conditions described below.

Method

Reagents

A. COD-POD solution 14.4g Na2HPO4・12H2O, 31g boric acid, 15,000 U COD (GradeⅢ), 25,000 PUPOD, 290mg sodium cholate/1,000ml of H2O (adjusting the pH to 6.5) (stable for a month if stored at 0−5℃)
B. DMA-MBTH solution 400mg EDTA-Na2, 0.7ml DMA, 150mg MBTH/1,000ml of 0.5M acetate buffer, pH4.7 (stable for a week if stored at 0−5℃ in a brownish bottle)
C. Cholesterol linoleate solution 0.6mM［Dissolve 40mg of cholesterol linoleate in 8.0ml of absolute ethanol on a hot plate, mix 90ml of 1.0% (V/V) of Triton X-100 solution, keep at 70℃ in a hot water bath for 15 minutes and, after cooling down to room temperature in running water, fill up to 100ml with 1.0% (V/V) Triton X-100 (Should be prepared fresh)］
D. HCl solution 1.0 N
E. Enzyme diluent 10mM Phosphate buffer, pH 7.0.

Procedure

1.Prepare the following reaction mixture in a test tube and equilibrate at 37℃ for about 5 minutes.

1.0ml COD-POD solution (A)
1.0ml DMA-MBTH solution (B)
0.1ml enzyme solution*

2.Add 1.0ml of the substrate solution (C) and mix.

3.After exactly 10 minutes at 37℃, add 1.0ml of HCl solution (D) to stop the reaction and measure the optical density at 590 nm against water (OD test).
At the same time, prepare the blank by first mixing the reaction mixture (1.0ml of substrate solution is used instead of the enzyme solution) with 1.0ml of HCl solution (D) after 10 min-incubation at 37℃, followed by the addition of the enzyme of the enzyme solution (OD blank).

*Dissolve the enzyme preparation in ice-cold enzyme diluent and dilute to 0.02〜0.05U/ml with the same buffer.

Calculation

Activity can be calculated by using the following formula :

  • Volume activity (U/ml) =

  • ΔOD(OD test−OD blank)×Vt×df

    39.0×t×Vs

  • ＝ ΔOD×0.105×df

Weight activity (U/mg) ＝ (U/ml)×1/C

Vt : Total volume (4.1ml)
Vs : Sample volume (0.1ml)
39.0 : Millimolar extinction coefficient of indamine dye under the assay conditions (cm2/micromole)
t : Reaction time (10 minutes)
df : Dilution factor
C : Enzyme concentration in dissolution (c mg/ml)

REFERENCES

1)W.Richmond; Clin.Chem., 19, 1350 (1973).

2)H.M.Flegg; Ann.Clin.Biochem., 10, 79 (1973).

3)C.C.Allain et al.; Clin.Chem., 20, 470 (1974).

4)P.N.Tarbutton and C.R.Gunter; Clin.Chem., 20, 724 (1974).

5)S.Nomoto; Rinsho Kensa, 20, 688 (1976).

6)Y.Kameno et al.; Jap.J.Clin.Path., 24, 650 (1976).

Table 1. Substrate Specificity of Cholesterol esterase

［The reaction was carried out at 37℃ for 10min in 0.1M acetate buffer, pH 5.0, contg. 0.2mM each cholesterol ester and 0.33% Triton X-100.］

  • Cholesterol ester Relative activity(%)
    Linoleate(18 :2) 100
    Acetate(2 :0) 9
    Propionate(3 :0) 40
    Butyrate(4 :0) 38
    Crotonate(4 :1) 0
    Valerate(5 :0) 18
    Caproate(6 :0) 63
    Heptanoate(7 :0) 32
    Caprylate(8 :0) 94
    Nonanoate(9 :0) 120
    Decylate(10 :0) 143
    10-Undecenoate(11 :1) 141
  • Cholesterol ester Relative activity(%)
    Laurate(12 :0) 108
    Tridecanoate(13 :0) 59
    Myristate(14 :0) 57
    Pentadecanote(15 :0) 76
    Palmitate(16 :0) 111
    Heptadecanoate(17 :0) 99
    Stearate(18 :0) 39
    Oleate(18 :1) 59
    Lelaidate(18 :3) 43
    Linolenate(18 :3) 91
    Arachidonate(20 :4) 3
Number of carbon atoms and double bonds are given in parentheses.

  • Fig.1. Stability (COE-301)(Powder form)

    Fig.1. Stability (COE-301)(Powder form)

    (kept under dry conditions)

  • Fig.2. Stability (COE-302)(Powder form)

    Fig.2. Stability (COE-302)(Powder form)

    (kept under dry conditions)

  • Fig.3.Stability (Powder form)

    Fig.3.Stability (Powder form)

    (kept under dry conditions)

  • Fig.4. Stability (Liquid form at 40℃)

    Fig.4. Stability (Liquid form at 40℃)

    Enzyme concentration: 20mg/ml Buffer composition :0.2M boric acidborax contg. 0.1% sodium cholate and 0.6% Trinton X-100, pH5.5.

  • Fig.5.pH-Activity

    Fig.5.pH-Activity

    37℃, 10 min-reaction in 0.1M buffer solution: pH3.0-6.0,acetate;pH6.0-8.0,phosphate; pH8.0-10.0,boric acid-KCI-sodium carbonate

  • Fig.6. Temperature activity

    Fig.6. Temperature activity

    10min-reaction in 0.1M acetate buffer, pH5.5.

  • Fig.7. pH-Stability

    Fig.7. pH-Stability

    25℃,20hr-treatment with 50mM buffer solution:pH2.0-4.0,citrate; pH4.0-6.0, acetate;pH6.0-8.0, phosphate;pH8.0-9.0,boric acid-KCI-sodium carbonate

  • Fig.8. Thermal stability

    Fig.8. Thermal stability

    10min-treatment with 50mM acetate buffer,pH5.5 and 50mM phosphate buffer, pH7.0

活性測定法（Japanese）

1. 原理

原理

原理

MBTHとDMAの酸化縮合生成物であるIndamine色素を590nmで測定し,上記反応で生成したH2O2量(加水分解されたCholesterol esterの量)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cholesterol esterを加水分解する酵素活性を1単位 (U)とする。

3.試薬

  • COD-POD溶液〔Na2HPO4･12H2O 14.4g,ホウ酸31g,cholesterol oxidase (GradeⅢ)15,000単位,POD 25,000プリプロガリン単位及びsodium cholate 290mgを1,000㎖の蒸留水に溶解し,pH6.5に調整する。(0～5℃保存で1ヶ月使用可能)〕
  • DMA-MBTH溶液 〔EDTA-Na2 400mg, DMA0.7ml及びMBTH 150mgを1,000mlの0.5M酢酸緩衝液, pH4.7に溶解する。(0～5℃遮光保存で1週間使用可能)〕
  • 0.6mMコレステロールリノレート溶液〔40mgのコレステロールリノレートを精秤し,8.0mlの無水エタノールを加え,ヒーター上で加温溶解する。それを90mlの1％(V/V) トリトンX-100と混和して，ウォーターバス中で攪拌しながら70℃まで加温する。更に該温度で15分間保温した後，室温まで流水冷却する。次いで前記1％トリトンX-100で最終液量を100mlとする。(用時調製)〕
  • 1.0N HCl溶液

酵素溶液：酵素標品を予め氷冷した10mMリン酸緩衝液, pH7.0で溶解し,同緩衝液で0.02～0.05U/mlに希釈する。

4.手順

1.試験管に次の反応混液を調製し37℃で約5分間予備加温する。

1.0ml COD-POD溶液 (A)
1.0ml DMA-MBTH溶液 (B)
0.1ml 酵素溶液

2.基質溶液(C)1.0mlを加え反応を開始する。

3.正確に37℃で10分間反応させた後，1.0N HCl (D)1.0mlを加えて反応を停止させる。この液につき590nmにおける吸光度を測定する(OD test)。

4.盲検は,反応混液1(但し酵素溶液の代わりに1.0mlの基質溶液(C)を加えて調整)を37℃で10分間放置後,1.0N HCl (D) 1.0mlを加え，次いで酵素溶液0.1mlを加えて調整し,同様に吸光度を測定する(OD blank)。

5.計算式

  • U/ml =

  • ΔOD/min (OD test−OD blank)×4.1(ml)×希釈倍率

    39.0×10(分)×0.1(ml)

＝ ΔOD×0.105×希釈倍率
U/mg ＝U/ml×1/C
39.0 : Indamine色素の上記測定条件下でのミリモル分子吸光係数 (㎠/micromole)
C : 溶解時の酵素濃度(c mg/ml)