CHOLESTEROL ESTERASE [CEBPⅡ] (T-243)

CholesterolT-243 PDF download
(Diagnostic Reagent Grade) Nagase Diagnostics ENZYMES T-243REACH適合品

CHOLESTEROL ESTERASE [CEBPII]

from Microorganism
(Sterol‒ester acylhydrolase, EC 3.1.1.13)
(Sterol esterase)

Cholesterol Ester + H2O → Cholesterol + Fatty Acid

★ Advantage
High liquid stability

Preparation and Specification

Appearance
: White to off white lyophilized powder
Specific activity
: More than 10.0 U/mg solid

Properties

Substrate specificity
: See Table 1
Molecular weight
: 63 kDa(SDS‒PAGE)
Isoelectric point
: 5.1 (estimated from amino acid sequence)
Optimum pH
: 5.6Figure 1
pH stability
: 4.0‒8.0 (37°C, 60 min)Figure 2
Optimum temperature
: 45℃Figure 3
Thermal stability
: Stable at 45°C and below(pH6.0, 30 min) Figure 4
Liquid stability
: See Figure 5
Adsorption on glass
: See Figure 6
Effect of coexisting ions
: See Table 2
Effect of detergents
: See Table 3
Light stability
: See Table 4

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of total cholesterol, HDL-C,andLDL-C coupled with cholestreol oxidase (T‒84 and T‒101) .
This enzyme is suitable for assembling in liquid reagens.

 CEBPII
Cholesterol ester + H2O→Cholestero + FFA
 CON Ⅱ
Cholesterol + O2→△4 -Cholesten-3-one + H2O2
 POD
2 H2O2 + 4-AA + Phenol→Quinoneimine dye + 4 H2O

FFA: Free fatty acid

 

Table 1 Substrate specificity

Substrate (0.95mM)Relative activity (%)
Cholesterol AcetateC 2:00.4
Cholesterol PropionateC 3:09.3
Cholesterol ButyrateC 4:027.8
Cholesterol PalmitateC16:019.8
Cholesterol StearateC18:02.0
Cholesterol OleateC18:1100.0
Cholesterol LinoleateC18:280.6
  

 

Table 2 Effect of coexisting ions on CEBPⅡ activity

Coexisting ion (20 mM)Relative activity (%)
None100
NaCl101
KCl100
NH4Cl100
CaCl2101
MgCl2101
MnCl2103
ZnCl2105
EDTA102
NaN3100
NaF98

All samples contain 0.1% BSA

 

Table 3 Effect of detergents on CEBP II activity

Detergent (0.1%)Relative activity (%)
None100
Triton X-100115
Adekatol TN-100117
Adekatol SO-120142
Newcol-707115
Newcol-710113
Emulgen 120141
Emulgen 705135
Deoxycholic acid129
Tauroursodeoxycholic acid134
Sodium dodecyl sulfate127

 

Table 4 Light stability

Form of CEBP IILight sourceResidual activity (%)
PowderLED96.5
 Fluorescent98.7
SolutionLED94.1
(1mg/ml, water)Fluorescent98.4

After 24 hours of incubation

Fig. 1 Optimum pH


〇: 3.3-Dimethylglutarate-NaOH buffer
●: Phosphate buffer
□: Tris-HCl buffer
■: Glycine-NaOH buffer

Fig. 2 pH stability


37℃, 60 min
〇: 3.3-Dimethylglutarate-NaOH buffer
●: Phosphate buffer
□: Tris-HCl buffer
■: Glycine-NaOH buffer

Fig. 3 Optimum temperature


pH 7.5
40 mM Phosphate buffer

Fig. 4 Temperature stability


pH 6.0, 30 min
〇: Phosphate buffer
●: Phosphate buffer + 0.1% Emulgen 705
□: Phosphate buffer
+ 0.1% Emulgen 705 & 10 mM NH4Cl

Fig. 5 Stability of CEBPⅡ in a liquid reagent


● : 4℃Reagent composition:0.5 U/mL CEBP II
○ : 37℃ 50 mM BES buffer pH 6.5
  0.4% Triton X-100
  0.05% NaN3
  0.6 mM ADOS
  7.5 U/mL POD

Fig. 6 Adsorption on glass


pH 7.0
10 mM Phosphate buffer containing 0.1% BSA
Solution of CEBPⅡ was repeatedly transferred into another test tube, and then the residual activity in the solution of each tube was measured.

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:

 CEBPⅡ
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 6.80.60 ml
    0.35% 4‒AA solution0.30 ml
    0.2% (W/V) Phenol solution0.30 ml
    100U/ml POD solution1) 0.30 ml
    3% (W/V) Triton X‒100 solution0.30 ml
    0.2U/ml CONⅡ solution2) 0.60 ml
    Substrate solution3) 0.30 ml
    Distilled water0.30 ml
    1) :100U/ml POD solution
    Dissolve 1000 U (PPU) of POD with 10 ml of distilled water.
    2) :0.2U/ml CONⅡ solution
    Dissolve 2 U of CONⅡ with CONⅡ dilution buffer※)
    ※) :CONⅡ dilution buffer
    0.1M KH2PO4‒Na2HPO4 buffer pH 7.0 containing
    0.05% (W/V) Triton X‒100.
    3) :Substrate solution
    Calf serum
  2. Enzyme dilution buffer
    10mM KH2PO4‒NaOH buffer pH 7.5 containing 0.1%
    (W/V) 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 pH5.2 #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 0.35 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.
  4. Absorbance sample :As/min
    blank :Ab/min
    △A/min = (As/min－Ab/min) ≦0.040 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℃ in the presence of a desiccant is recommended.

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, O.C. (1974) Clin. Chem., 20, 470‒475.
  3. Kameno, Y., Nakano, N. and Baba, S. (1976) Jap. J. Clin. Path., 24, 650.

CEBPⅡ 活性測定法 (Japanese)

試薬液
  1. 反応試薬混合液
    0.2M2KHPO4‒NaOH 緩衝液 pH6.80.60 ml
    0.35% 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
    POD1,000 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 社製
    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
    POD：シグマ製　Type Ⅱ　#P‒8250
    4‒AA：ナカライテスク製　特級　#01907‒52
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約0.35U/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50μl 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。
    Δ A/min = (As/min－Ab/min) ≦ 0.040Abs/min
計算
活性 (U/mg）= {(ΔA/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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