(Diagnostic Reagent Grade) ASAHI KASEI 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, and LDL-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 Acetate	C 2:0	0.4
Cholesterol Propionate	C 3:0	9.3
Cholesterol Butyrate	C 4:0	27.8
Cholesterol Palmitate	C16:0	19.8
Cholesterol Stearate	C18:0	2.0
Cholesterol Oleate	C18:1	100.0
Cholesterol Linoleate	C18:2	80.6

Table 2 Effect of coexisting ions on CEBPⅡ activity

Coexisting ion (20 mM)	Relative activity (%)
None	100
NaCl	101
KCl	100
NH₄Cl	100
CaCl₂	101
MgCl₂	101
MnCl₂	103
ZnCl₂	105
EDTA	102
NaN₃	100
NaF	98

All samples contain 0.1% BSA

Table 3 Effect of detergents on CEBP II activity

Detergent (0.1%)	Relative activity (%)
None	100
Triton X-100	115
Adekatol TN-100	117
Adekatol SO-120	142
Newcol-707	115
Newcol-710	113
Emulgen 120	141
Emulgen 705	135
Deoxycholic acid	129
Tauroursodeoxycholic acid	134
Sodium dodecyl sulfate	127

Table 4 Light stability

Form of CEBP II	Light source	Residual activity (%)
Powder	LED	96.5
	Fluorescent	98.7
Solution	LED	94.1
(1mg/ml, water)	Fluorescent	98.4

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

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

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

Reaction mixture

0.2M KH₂PO₄‒NaOH buffer　pH 6.8	0.60 ml
0.35% 4‒AA solution	0.30 ml
0.2% (W/V) Phenol solution	0.30 ml
100U/ml POD solution¹⁾	0.30 ml
3% (W/V) Triton X‒100 solution	0.30 ml
0.2U/ml CONⅡ solution²⁾	0.60 ml
Substrate solution³⁾	0.30 ml
Distilled water	0.30 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

Enzyme dilution buffer
10mM KH₂PO₄‒NaOH buffer pH 7.5 containing 0.1%
(W/V) BSA.
Reagents
Triton X‒100: The Dow Chemical Company
CONⅡ : Asahi Kasei Pharma Corporation　#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

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

Pipette accurately 3.0 ml of reaction mixture into a small test tube and preincubate it at 37℃.
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.
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) ≦0.040 Abs/min

Calculation

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

Storage at －20℃ in the presence of a desiccant is recommended.

References

Bradford, M. B., (1976) Anal. Biochem., 72, 248‒254.
Allain, C. C., Poon, L. S., Chan, C. S. G., Richmond, W. and Fu, O.C. (1974) Clin. Chem., 20, 470‒475.
Kameno, Y., Nakano, N. and Baba, S. (1976) Jap. J. Clin. Path., 24, 650.

CEBPⅡ 活性測定法 (Japanese)

試薬液

反応試薬混合液

0.2M₂ KHPO₄‒NaOH 緩衝液 pH6.8	0.60 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