CHOLESTEROL ESTERASE [CENⅡ] (T-250)

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(Diagnostic Reagent Grade)Nagase Diagnostics ENZYMES T-250REACH適合品

CHOLESTEROL ESTERASE [CENII]

from Pseudomonas sp.
(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
: 30kDa (SDS–PAGE)
Isoelectric point
: pH 5.28(estimated from amino acid sequence)
Optimum pH
: 6.5Figure 1
pH stability
: 6.0–11.0(37°C, 60 min, in 0.1% BSA)Figure 2
Optimum temperature
: 35-40°C(Phosphate Buffer)Figure 3
Thermal stability
: Stable at 45°C and below(pH 8.0, 30min)Figure 4
Storage stability
: At least one year at –20°CFigure 6
Effect of coexisting ions
: See Table 2
Effect of detergents
: See Table 3
Activator
: Adekatol TN-100, Adekatol PC-8, Adelatol SO-120,Adekatol SO-135, 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–101).

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

FFA : Free fatty acid

Table 1. Substrate specificity

SubstrateRelative activity (%)
 CENIICEN (T-18)
Cholesterol Acetate C 2:022.621.1
Cholesterol Propionate C 3:027.728.1
Cholesterol Butyrate C 4:078.378.9
Cholesterol Palmitate C 16:037.942.2
Cholesterol Stearate C 18:013.213.0
Cholesterol Oleate C 18:1100.0100.0
Cholesterol Linolate C 18:280.978.1

Table 2. Effect of detergents on CENII activity

AdditivesConcentrationRelative activity (%)
None-100
NiCl1mM67
MnCl21mM101
(NH4)2SO41mM107
MgCl21mM106
ZnCl1mM87
ZnSO41mM81
Ba(CH3COO)21mM106
CaCl21mM114
MoSO41mM69
CuSO40.5mM3
CuCl20.5mM0
FeCl31mM102
CoCl21mM86
Li2CO31mM100
CH3COOT1mM104
EDTA1mM101
KCl0.1M97
NaCl0.1M98
NaN30.05%102
NaF20mM98

Table 3. Effect of detergents on CENII activity

Detergent (0.3%)Relative activity (%)
None100
TritonX-100147
Adekatol TN-100146
Adekatol SO-120142
Newcol 707146
Newcol 710139
Emulgen 120144
Emulgen 705159
Sodium cholate103
Deoxycholic acid128
Sodium dodecyl sulfate1.1
Sucrose fatty acid ester144

Table 4. Effect of detergents on CENⅡ activity when switching from Triton X-100 to other detergents

Detergent (0.3%)Relative activity (%)
TritonX-100100
Adekatol SO-12061
Adekatol SO-13569
Adekatol TN-10082
Adekatol PC-881
Tween 201
Tween 401
Tween 801
Sodium Cholate0
Brij 352
CHAPSO0
Benzyltriethylammonium Chloride1
Sodium dodecyl sulfate0
Emulgen LS-11030
Newcol 71013
None0

Fig. 1 pH Optimum pH


〇: Citrate buffer
●: Phosphate buffer
□: Tris-HCl buffer
■: Glycine-NaOH buffer

Fig. 2 pH Stability


37°C, 60min. in 0.1% BSA
〇: Citrate buffer
●: Phosphate buffer
□: Tris-HCl buffer
■: Glycine-NaOH buffer

Fig. 3 Optimum temperature


pH6.8
40mM Phosphate buffer

Fig. 4 Thermal stability


Tris-HCl buffer pH8.0 30min
CEN II
〇: None
●: + 0.1% Newcol 710
□: + 1mM CaCl2
■: + 0.1% Newcol 710 + 1mM CaCl2
CEN(T-18)
* ： None

Fig. 5 Thermal stability


Tris-HCl buffer pH8.0
+0.1% Newcol 710
+1mM CaCl2 30min
■: CEN II
△: CE from Pseudomonas sp.

Fig. 6 Storage (lyophilized powder)


－20°C

Assay

Principle

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

CENII
Cholesterol ester+H2O→Cholesterol+Fatty acid
CO
Cholesterol+O2→△4–Cholesten–3–one+H2O2
POD
2H2O2 +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°C under the conditions specified in the assay procedure.

Reagents
  1. Reaction mixture
    0.2M KH2PO4–NaOH buffer pH 6.80.60 ml
    0.35%(W/V)4–AA solution0.30 ml
    0.2%(W/V)Phenol solution0.30 ml
    100 U/ml POD solution1)0.30 ml
    3%(W/V)Triton X–100 solution0.30 ml
    0.2 U/ml CONⅡ solution2)0.60 ml

    Substrate solution3)
    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°C.
  2. After 10 min, add 50 μl of enzyme solution and mix to start the reaction at 37°C.
    ※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.
  4. Absorbance sample :As/min
    blank :Ab/min
    △A/min =(As/min−Ab/min)≦ 0.050 Abs/min
Calculation
  1. Activity(U/mg of powder)= {(△A/min)/(12.0×1/2)} × 3.05/0.05 × 1/x
    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°C in the presence of a desiccant is recommended. Enzyme activity will be retained for at least one year under this condition(Figure 5)

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.

CENII 活性測定法（Japanese）

試薬液
  1. 反応試薬混合液
    0.2M KH2PO4–NaOH 緩衝液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–Na2HPO4 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
    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°C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°C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 :H2O22 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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