CHOLESTEROL ESTERASE from Schizophyllum commune
Appearance | Light brown amorphous powder, lyophilized |
---|---|
Activity | GradeⅢ 2.0 U/mg-solid or more (containing approx. 20 % of stabilizers) |
Stabilizer | Na-Cholate |
Stability | Stable at −20 ℃ for at least one year(Fig.1,2) |
---|---|
Molecular weight | approx. 130,000 |
Isoelectric point | 4.1±0.1 |
Michaelis constants | 3.9×10-5 M (Linoleate),9.2×10-5 M (Palmitate), |
6.3×10-5 M (Decylate),8.8×10-5 M (Propionate) | |
Inhibitors | Heavy metal ions (Hg2+, Ag+, Fe3+) |
Optimum pH | 4.8−8.0 (Cholesterol linoleate), 5.0 (serum)(Fig.5) |
Optimum temperature | 55−60 ℃(Fig.6) |
pH Stability | pH 2.5−7.5 (25 ℃, 20 hr)(Fig.7) |
Thermal stability | below 55 ℃ (pH 5.5, 10 min)(Fig.8) |
Substrate specificity | (Table 1) |
This enzyme is useful for enzymatic determination of cholesterol in combination with cholesterol oxidase (COO-311, COO-321, COO-331) in clinical analysis.
The formation of indamine is measured at 590 nm by spectrophotometry.
One unit causes the hydrolysis of one micromole of cholesterol ester per minute under the conditions detailed below.
A. COD-POD solution | 14.4 g Na2HPO4・12H2O, 31g boric acid, 15,000 U COD (GradeⅢ), 25,000 PUPOD, 290 mg sodium cholate/1,000 mL of H2O (adjusting the pH to 6.5; stable for 1 month if stored at 0−5 ℃) |
---|---|
B. DMA-MBTH solution | 400 mg EDTA-Na2, 0.7 mL DMA, 150 mg MBTH/1,000 mL of 0.5 M acetate buffer, pH4.7 (stable for 1 week if stored at 0−5 ℃ in a brownish bottle) |
C. Cholesterol linoleate solution | 0.6 mM[Dissolve 40 mg of cholesterol linoleate in 8.0 mL of absolute ethanol on a hot plate, mix 90 mL of 1.0 % (V/V) of Triton X-100 solution, keep at 70 ℃ in a hot water bath for 15 minutes and, after cooling to room temperature in running water, make up to 100 mL with 1.0 % (V/V) Triton X-100 (Should be prepared fresh) |
D. HCl solution | 1.0 N |
E. Enzyme diluent | 10 mM Phosphate buffer, pH 7.0. |
1.Prepare the following reaction mixture in a test tube and equilibrate at 37℃ for about 5 minutes.
1.0ml | COD-POD solution | (A) |
---|---|---|
1.0 mL | DMA-MBTH solution | (B) |
0.1 mL | Enzyme solution* |
2.Add 1.0 mL of the substrate solution (C) and mix.
3.After exactly 10 minutes at 37℃, add 1.0 mL of HCl solution (D) to stop the reaction and measure the optical density at 590 nm against water (OD test).
At the same time, prepare the blank by mixing the reaction mixture (1.0 mL of substrate solution is used instead of the enzyme solution) with 1.0 mL of HCl solution (D) after 10 min-incubation at 37℃, followed by addition of the enzyme solution (OD blank).
*Dissolve the enzyme preparation in ice-cold enzyme diluent and dilute to 0.02〜0.05U/mL with the same buffer.
Activity can be calculated by using the following formula :
Volume activity (U/mL) =
ΔOD(OD test−OD blank)×Vt×df
39.0×t×Vs
= ΔOD×0.105×df
Weight activity (U/mg) = (U/mL)×1/C
Vt | : Total volume (4.1 mL) |
Vs | : Sample volume (0.1 mL) |
39.0 | : Millimolar extinction coefficient of indamine dye under the assay conditions (cm2/micromole) |
t | : Reaction time (10 minutes) |
df | : Dilution factor |
C | : Enzyme concentration in dissolution (c mg/mL) |
1)W.Richmond; Clin.Chem., 19, 1350 (1973).
2)H.M.Flegg; Ann.Clin.Biochem., 10, 79 (1973).
3)C.C.Allain et al.; Clin.Chem., 20, 470 (1974).
4)P.N.Tarbutton and C.R.Gunter; Clin.Chem., 20, 724 (1974).
5)S.Nomoto; Rinsho Kensa, 20, 688 (1976).
6)Y.Kameno et al.; Jap.J.Clin.Path., 24, 650 (1976).
[The reaction was carried out at 37℃ for 10min in 0.1M acetate buffer, pH 5.0, contg. 0.2mM each cholesterol ester and 0.33 % Triton X-100.]
Cholesterol ester | Relative activity(%) |
---|---|
Linoleate(18 :2) | 100 |
Acetate(2 :0) | 9 |
Propionate(3 :0) | 40 |
Butyrate(4 :0) | 38 |
Crotonate(4 :1) | 0 |
Valerate(5 :0) | 18 |
Caproate(6 :0) | 63 |
Heptanoate(7 :0) | 32 |
Caprylate(8 :0) | 94 |
Nonanoate(9 :0) | 120 |
Decylate(10 :0) | 143 |
10-Undecenoate(11 :1) | 141 |
Cholesterol ester | Relative activity(%) |
---|---|
Laurate(12 :0) | 108 |
Tridecanoate(13 :0) | 59 |
Myristate(14 :0) | 57 |
Pentadecanote(15 :0) | 76 |
Palmitate(16 :0) | 111 |
Heptadecanoate(17 :0) | 99 |
Stearate(18 :0) | 39 |
Oleate(18 :1) | 59 |
Lelaidate(18 :3) | 43 |
Linolenate(18 :3) | 91 |
Arachidonate(20 :4) | 3 |
Fig.1. Stability (COE-301)(Powder form)
(kept under dry conditions)
Fig.2. Stability (COE-302)(Powder form)
(kept under dry conditions)
Fig.3.Stability (Powder form)
(kept under dry conditions)
Fig.4. Stability (Liquid form at 40℃)
Enzyme concentration: 20mg/mL Buffer composition :0.2M boric acidborax contg. 0.1% sodium cholate and 0.6 % Trinton X-100, pH5.5.
Fig.5.pH-Activity
37℃, 10 min-reaction in 0.1M buffer solution: pH3.0-6.0,acetate;pH6.0-8.0,phosphate; pH8.0-10.0,boric acid-KCI-sodium carbonate
Fig.6. Temperature activity
10min-reaction in 0.1M acetate buffer, pH5.5.
Fig.7. pH-Stability
25℃,20hr-treatment with 50mM buffer solution:pH2.0-4.0,citrate; pH4.0-6.0, acetate;pH6.0-8.0, phosphate;pH8.0-9.0,boric acid-KCI-sodium carbonate
Fig.8. Thermal stability
10min-treatment with 50mM acetate buffer,pH5.5 and 50mM phosphate buffer, pH7.0
1. 原理
MBTHとDMAの酸化縮合生成物であるIndamine色素を590nmで測定し,上記反応で生成したH2O2量(加水分解されたCholesterol esterの量)を定量する。
2.定義
下記条件下で1分間に1マイクロモルのcholesterol esterを加水分解する酵素活性を1単位 (U)とする。
3.試薬
酵素溶液:酵素標品を予め氷冷した10mMリン酸緩衝液, pH7.0で溶解し,同緩衝液で0.02~0.05U/mLに希釈する。
4.手順
1.試験管に次の反応混液を調製し37℃で約5分間予備加温する。
1.0 mL | COD-POD溶液 | (A) |
1.0 mL | DMA-MBTH溶液 | (B) |
0.1 mL | 酵素溶液 |
2.基質溶液(C)1.0 mLを加え反応を開始する。
3.正確に37℃で10分間反応させた後,1.0N HCl (D)1.0mlを加えて反応を停止させる。この液につき590nmにおける吸光度を測定する(OD test)。
4.盲検は,反応混液1(但し酵素溶液の代わりに1.0mlの基質溶液(C)を加えて調整)を37℃で10分間放置後,1.0N HCl (D) 1.0 mLを加え,次いで酵素溶液0.1 mLを加えて調整し,同様に吸光度を測定する(OD blank)。
5.計算式
U/mL =
ΔOD/min (OD test−OD blank)×4.1(mL)×希釈倍率
39.0×10(分)×0.1(mL)
= ΔOD×0.105×希釈倍率 | |
U/mg | =U/mL×1/C |
39.0 | : Indamine色素の上記測定条件下でのミリモル分子吸光係数 (㎠/micromole) |
C | : 溶解時の酵素濃度(c mg/mL) |
For inquiries and cosultations regarding our products, please contact us through this number.