(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-101REACH compliant product

CHOLESTEROL OXIDASE [CONⅡ–FD]

Lyophilized type

from Rhodococcus sp.
(Cholesterol: oxygen oxidoreductase, EC 1.1.3.6)

Cholesterol + O₂ → △⁴–Cholesten–3–one + H₂O₂

Preparation and Specification

Appearance: : Yellowish amorphous powder, lyophilized

Specific activity: : More than 15 U/mg solid

Properties

Substrate specificity: : See Table 1

Molecular weight: : 61.8 KDa (SDS–PAGE)

Isoelectric point: : pH 4.5

Michaelis constants: : Cholesterol 6.0 × 10^-5M

Optimum pH: : 7.0–7.5Figure 1

pH stability: : 5.7–7.8 (65℃, 10 min) Figure 2

Optimum temperature: : 50℃ (Tris–HCl buffer) Figure 3

Thermal stability: : Stable at 65℃ and below (pH 7.0, 10 min) Figure 4 and Figure 5

Effect of detergents: : See Table 2

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of total cholesterol, HDL-C, and LDL-C when coupled with cholesterol esterase (T–18 and T–98) .

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

FFA: Free fatty acid

Table 1. Substrate specificity

Substrate (1mM)	Relative activity (%)
Cholesterol	100
β–Cholesterol	93
Pregnenolone	98
Dehydro–iso–androsterone	10
β–Sitosterol	94
Stigmasterol	66
Androsterol	2
Teststerone	1
Cholic acid	3

Table 2. Effect of detergents on CON Ⅱ -FD activity

Detergents (0.1%)	Relative activity (%)
Triton X–100	100
Emulgen 810	101
Emulgen 911	113
Emulgen 709	107
Emulgen 109P	118
Adekatol SO–120	100
RHEODOL 460	63
SM 1080	122

Fig.1 pH Optimum

〇: MES buffer
▲: PIPES buffer
□: Tris-HCI buffer
●: Glycine-NaOH buffer

Fig.2 pH Stability

65℃, 10 min.
〇: MES buffer
▲: PIPES buffer
□: Tris-HCI buffer
●: Glycine-NaOH buffer

Fig.3 Optimum Temperature

pH 7.0
100 mM Tris-HCI buffer

Fig.4 Thermal Stability

pH 7.0, 10 min.
100 mM Tris-HCI buffer

Fig.5 Thermal Stability

100mM, 10min.
〇: MES buffer(pH5.95)
□: Tris-HCI buffer(pH7.63)
●: Glycine-NaOH buffer(pH8.95)

Assay

Principle

The assay is based on the increase in absorbance at 240 nm as △⁴–cholesten–3–one is produced in the following reaction:

	CON Ⅱ–FD
Cholesterol+O₂	→	△⁴–Cholesten–3–one+H₂O₂

Unit definition

One unit is defined as the amount of enzyme which liberates 1 μmole of △⁴–cholesten–3–one per minute at 37℃ under the conditions specified in the assay procedure.

Reagents

Substrate solution (6 mM cholesterol solution)
Dissolve 232 mg of cholesterol with isopropanol to make a total of 100 ml.

Enzyme dilution buffer
0.1 M KH₂PO₄–Na₂HPO₄ buffer pH 7.0 containing
0.05% (W/V) Triton X–100

※ Prepare the enzyme dilution buffer two days before use and keep it in the refrigerator until use.
Reagents
Cholesterol : NACALAI TESQUE, INC. Special grade

#08721
Triton X–100 : The Dow Chemical Company

Enzyme solution

Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. After 1–1.5 hour incubation at room temperature, dilute it with enzyme dilution buffer to adjust the concentration to within 0.1–0.2 U/ml.

Procedure

Pipette accurately 3.0ml of enzyme dilution buffer and 50 μl of enzyme solution and preincubate at 37℃.

※ In the case of a test blank, add 50 μl of enzyme dilution buffer in place of enzyme sdution.
After 5 min, add 50 μl of substrate solution and mix to start the reaction at 37℃.
After starting the reaction, measure the rate of increase per minute in absorbance at 240nm. The rate must be measured within the linear portion of the absorbance curve.

Absorbance sample : As/min

blank : Ab/min

0.010 Abs/min ≦ △ A/min = (As/min-Ab/min) ≦ 0.060 Abs/min

Calculation

Activity (U/mg) = {(△ A/min)/12.2} × 3.10/0.05 × 1/X

12.2 :	millimolar extinction coefficient of △⁴–Cholesten–3–one at 240 nm ( cm² /μmole)
3.10 :	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. Enzyme activity will be retained for at least one year under this condition.

References

Richmond, W. (1973) Clin. Chem., 19, 1350.
Flegg, H. M. (1973) Ann. Clin. Biochem., 10, 79.
Alain, C. C. et. al. (1973) Clin. Chem., 20, 470.
Tarbutton, P. N. and Gunter, C. R. (1974) Clin. Chem., 20, 724.
Nomoto, S. (1976) Rinsho Kensa, 20, 688.
Kameno, K., Nakano, N. and Baba, S. (1976) Jap. J. Clin. Path., 24, 650.

CON Ⅱ-FD 活性測定法 (Japanese)

試薬液

基質溶液 (6mM コレステロール溶液)
コレステロール232mg をイソプロパノールに溶解して全容100ml とする。
酵素溶解希釈用液
0.05% (W/V) トリトンX–100 を含む
0.1M KH2PO4–Na2HPO4 緩衝液 pH7.0

※ 酵素溶解希釈用液は使用する2 日前に調製し、使用まで冷蔵保存する。
試薬
コレステロール：ナカライテスク製　特級
#08721
トリトンX–100：Dow Chemical 製

酵素試料液

検品約20mg を精密に量り、酵素溶解希釈用液で溶解して全容20ml とする。室温にて1〜1.5 時間放置し、その液を酵素溶解希釈用液で約0.1–0.2U/ml 濃度となるように適宜希釈する。

測定操作法

小試験管に酵素溶解希釈用液3.0ml と酵素試料液50 μl を正確に加え37℃で予備加温する。

※ 盲検は酵素試料液の代りに酵素溶解希釈用液50 μlを加える。

5 分経過後、基質溶液50 μl を正確に加えて混和し、37℃で反応を開始する。
反応開始後、240nm 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。
0.010 Abs/min ≦ △ A/min = (As/min-Ab/min)
≦ 0.060 Abs/min

計算

活性 (U/mg) = {(△ A/min)/12.2} × 3.10/0.05 × 1/X

12.2 :	Δ⁴– コレステン–3– オンの240nm におけるミリモル分子吸光係数 (cm² / μmole)
3.10 :	反応総液量 (ml)
0.05 :	反応に供した酵素試料液量 (ml)
X :	酵素試料液の検品濃度 (mg/ml)

Absorbance sample :	As/min
blank :	Ab/min

CHOLESTEROL OXIDASE [CONⅡ–FD] (T-101)