CHOLESTEROL ESTERASE [CEN] (T-18)

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(Diagnostic Reagent Grade) Nagase Diagnostics ENZYMES T-18REACH compliant product

CHOLESTEROL ESTERASE [CEN]

from Pseudomonas sp.
(Steryl-ester acylhydrolase, EC 3.1.1.13)
(Sterol esterase)

Cholesterol ester + H2O → Cholesterol + Fatty acid

Preparation and Specification

Appearance
: White to pale brownish amorphous powder, lyophilized
Specific activity
: More than 100 U/mg solid

Properties

Substrate specificity
: See Table 1
Molecular weight
: 29.5 kDa (SDS–PAGE)
31.0 kDa (Sephadex G–100)
Isoelectric point
: pH 4.25
Michaelis constants
: Cholesterol linolate 1.28 × 10-3M
Cholesterol ester of calf serum 7.5 × 10-4M
Optimum pH
: 6.5Figure 1
pH stability
: 6.5–10.0 (37℃, 60 min) Figure 2
Thermal stability
: Stable at 55℃ and below (pH 8.0, 10 min) Figure 3
Storage stability
: At least one year at –20℃Figure 4
Activator
: Triton X–100

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of total cholesterol,HDL-C, and LDL-C coupled with cholesterol oxidase T–84 and T–101.

 POD
2 H2O2 + 4－AA + Phenol→Quinoneimine dye + 4 H2O
 CONⅡ
Cholesterol + O2→Cholestenone + H2O2
 CEN
Cholesterol ester + H2O→Cholesterol + FFA

FFA: Free fatty acid

 

Table 1. Substrate specificity

SubstrateRelative activity
(%)
Cholesterol acetateC 2:03.2
propionate3:012.3
butylate4:026.7
palmitate16:025.7
stearate18:014.9
oleate18:1100.0
linoleate18:2534.8

Fig.1 pH Optimum


〇: Phosphate buffer
●: 3,3-Dimethylglutarate-NaOH buffer
□: Tris-HCl buffer

Fig.2 pH Stability


37℃, 60 min
〇: 3,3-Dimethylglutarate-NaOH buffer
●: Phosphate buffer
□: Tris-HCl buffer
△: Glycine-NaOH buffer

Fig.3 Thermal Stability


pH 8.0, 10 min.
Tris-HCl buffer

Fig.4 Storage (lyophilized powder)


〇: －20℃
□: 5℃

Assay

Principle

The assay is based on the increase in absorbance at 493 nm as the formation of quinoneimine dye proceeds in the following reactions:

 CEN
Cholesterol ester+H2O→Cholesterol+Fatty acid
 CO
Cholesterol+O2→△4 –Cholesten–3–one+H2O2
 POD
2 H2O2+4 –AA+Phenol→Quinoneimine dye+4 H2O

CO : Cholesterol oxidase
Unit definition

  1. One unit is defined as the amount of enzyme which liberates 1 μmole of cholesterol per minute at 37℃ under the conditions specified in the assay procedure.

Reagents
  1. Reaction mixture
    0.2M KH2PO4–NaOH buffer　pH 6.8
    		0.60 ml
    0.35% (W/V) 4–AA solution0.30 ml
    0.2% (W/V) Phenol solution0.30 ml
    100 U/ml POD solution 1) 0.30 ml
    3% (W/V) Triton X–100 solution0.30 ml
    0.2 U/ml CONⅡ solution 2) 0.60 ml
  1. Substrate solution 3)
    		0.30 ml
    Distilled water0.30 ml
    1) :
    		100 U/ml POD solution
    Dissolve 1000 U (PPU) of POD with 10 ml of distilled water.
    2) :0.2 U/ml CONⅡ solution
    Dissolve 2 U of CONⅡ with CONⅡ dilution buffer※)
    ※) :CONⅡ dilution buffer
    0.1 M KH2PO4–Na2HPO4 buffer pH 7.0
    containing 0.05% (W/V) Triton X–100.
    3) :Substrate solution
    Calf serum
  2. Enzyme dilution buffer
    10 mM KH2PO4–NaOH buffer pH 7.5 containing 0.1%
    (W/V) bovine serum albumin (BSA)
  3. Reagents
    Triton X–100 : The Dow Chemical Company
    CONⅡ : Nagase Diagnostics Co., Ltd.　#T–84
    Calf serum: GIBCO Co. (USA)
    BSA: Millipore Fraction V pH 5.2　#81–053
    4–AA: NACALAI TESQUE, INC. Special grade #01907–52
    POD: Sigma Chemical Co. Type Ⅱ #P–8250
Enzyme solution

  1. Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml.

    Dilute it with enzyme dilution buffer to adjust the concentration to within 0.3–0.5 U/ml.

Procedure
  1. Pipette accurately 3.0 ml of reaction mixture into a small test tube and preincubate it at 37℃.
  2. After 10 min, add 50 μl of enzyme solution and mix to start the reaction at 37℃.
    ※In the case of a test blank, add 50 μl of enzyme dilution buffer in place of enzyme solution.
  3. After starting the reaction, measure the rate of increase per minute in absorbance at 493 nm. The rate must be measured within the linear portion of the absorbance curve.
    Absorbance sample :As/min
    blank :Ab/min
    △A/min = (As/min－Ab/min) ≦ 0.050 Abs/min
Calculation
Activity (U/mg of powder) = {(△A/min) /(12.0×1/2)}× 3.05/0.05 × 1/x
Calculation
  1. 12.0 :millimolar extinction coefficient of quinoneimine dye at 493 nm
    ( cm2 /μmole)
    1/2 :a multiplier derived from the fact that 2 mole of H2O2 produce 1 mole of quinoneimine dye
    3.05 :final volume (ml)
    0.05 :volume of enzyme solution (ml)
    X :concentration of the sample in enzyme solution
    ( mg/ml)
Storage

  1. Storage at –20℃ in the presence of a desiccant is recommended. Enzyme activity will be retained for at least one year under this condition (Figure 4)

References
  1. Bradford, M. B., (1976) Anal. Biochem., 72, 248–254.
  2. Allain, C. C., Poon, L. S., Chan, C. S. G., Richmond, W. and Fu, P.C. (1974) Clin. Chem., 20, 470–475.
  3. Kameno, Y., Nakano, N. and Baba, S. (1976) Japanese Journal of Clinical Pathology, 24, 650.

CEN 活性測定法 (Japanese)

試薬液
  1. 反応試薬混合液
    0.2M KH2PO4–NaOH 緩衝液　pH6.8
    		0.60 ml
    0.35% (W/V) 4–AA 溶液
    		0.30 ml
    0.2% (W/V) フェノール溶液
    		0.30 ml
    100U/ml POD 溶液1)
    		0.30 ml
    3% (W/V) トリトンX–100 溶液
    		0.30 ml
    0.2U/ml CONⅡ溶液2)
    		0.60 ml
    基質溶液3)
    		0.30 ml
    精製水0.30 ml
    1) :100U/ml POD 溶液
    POD 1,000 単位 (PPU) を精製水10ml で溶解する。
    2) :0.2U/ml CONⅡ溶液
    CONⅡ 2 単位 (U) をCONⅡ溶解用液※) 10ml で溶解する。
    ※) :CONⅡ溶解用液
    0.05% (W/V) トリトンX–100 を含む0.1M
    KH2PO4–Na2HPO4A 緩衝液pH7.0
    3) :基質溶液
    仔牛血清液
  2. 酵素溶解希釈用液
    0.1% (W/V) BSA を含む10mM KH2PO4–NaOH
    緩衝液pH7.5
  3. 試薬
    トリトンX–100：Dow Chemical 製
    CONⅡ (コレステロール酸化酵素) ：
    ナガセダイアグノスティックス製 #T–84
    仔牛血清液 (Calf serum) ：GIBCO (USA) 製
    BSA: Millipore 製　Fraction V pH5.2 #81–053
  1. 4–AA：ナカライテスク製　特級　#01907-52
    POD：シグマ製　Type Ⅱ　#P–8250
酵素試料液
  1. 検品約20mg を精密に量り、酵素溶解希釈用液に溶解して全容20ml とする。
    その液を酵素溶解希釈用液で0.3～0.5U/ml 濃度となるように適宜希釈する。
測定操作法
  1. 小試験管に反応試薬混合液を3.0ml 正確に分注して37℃で予備加温する。
  2. 10 分経過後、酵素試料液50 μl を正確に加えて混和し、37℃で反応を開始する。
    ※盲検は酵素試料液の代わりに酵素溶解希釈用液50 μl を加える。
  3. 反応開始後、493nm における吸光度を測定して直線的に反応している1 分間当たりの吸光度変化を求める。
    求められた吸光度変化を試料液はAs/min、盲検液はAb/min とする。
    ΔA/min = (As/min－Ab/min) ≦ 0.050 Abs/min
計算
活性 (U/mg) = {(△ A/min)/ (12.0 × 1/2)}× 3.05/0.05 × 1/x
12.0 :キノンイミン色素の493nm におけるミリモル分子吸光係数
(cm2 / μmole)
1/2 :H2O2 2 モルからキノンイミン色素1 モルが生成することによる係数
3.05 :反応総液量 (ml)
0.05 :反応に供した酵素試料液量 (ml)
X :酵素試料液中の検品濃度 (mg/ml)
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