ALKALINE PHOSPHATASE from Microorganism
Appearance | Transparent liquid | |
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Activity | GradeⅡ 30,000 U/mL or more | |
Contaminants | Adenosine deaminase | ≤ 1.0×10-4 % |
Phosphodiesterase | ≤ 3.0×10-3 % |
Stability | Stable at 4 ℃ |
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Molecular weight | approx. 104,000 |
Optimum pH | 9.5(Fig. 1) |
Optimum temperature | ≧ 60 ℃ (Fig. 2) |
pH Stability | pH 5.5-10.4 (25 ℃, 16 hr)(Fig. 3) |
Thermal stability | below 65 ℃ (pH 7.0, 60 min) (Fig. 4) |
This enzyme is useful in molecular biology.
The formation of p-nitrophenol is measured at 405 nm by spectrophotometry.
One unit causes the formation of one micromole of p-nitrophenol per minute under the conditions detailed below.
A. Diethanolamine buffer | 1 M: Dilute 9.66 mL of diethanolamine (MW = 105.14) in 60 mL of H2O, add 5 mL of 0.1 M MgCl2, and, after adjusting the pH to 9.8 with 2 N HCl, make up to 100 mL with H2O (should be freshly prepared). |
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B. pNPP solution | 0.674 M: 2.5g of p-nitrophenylphosphate disodium salt (MW = 371.16) in 10 mL of diethanolamine buffer (A). Should be freshly prepared. |
C. Enzyme diluent | 30 mM Triethanolamine, 1 mM MgCl2, 0.1 mM ZnCl2, 0.5 % sodium cholate, pH 7.6 |
1.Prepare the following working solution (30.5 mL) in a brownish bottle and store on ice (should be freshly prepared).
30 mL | Diethanolamine buffer | (A) |
0.5 mL | pNPP solution | (B) |
Concentration in assay mixture | |
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Diethanolamine buffer | 0.97 M |
p-Nitrophenylphosphate | 11 mM |
MgCl2 | 4.8 mM |
2.Pipette 3.0 mL of working solution into a cuvette (d = 1.0 cm) and equilibrate at 37 ℃ for approximately 5 minutes.
3.Add 0.1 mL of the enzyme solution* and mix by gentle inversion.
4.Record the increase in optical density at 405 nm against water for 3 to 5 minutes with a spectrophotometer thermostated at 37 ℃, and calculate the ∆OD per minute from the initial linear portion of the curve (∆OD test).
At the same time, measure the blank rate (∆OD blank) using the same method as the test except that the enzyme diluent (C) is added instead of the enzyme solution.
*Dilute the enzyme preparation to 0.1-0.3 U/mL with ice-cold enzyme diluent (C), immediately before the assay.
Activity can be calculated by using the following formula:
Volume activity (U/mL) =
∆OD/min(∆OD test-∆OD blank)×Vt×df
18.5×1.0×Vs
= ∆OD/min×1.676×df
Weight activity (U/mg) = (U/mL)×1/C
Vt | : Total volume (3.1 mL) |
Vs | : Sample volume (0.1 mL) |
18.5 | : Millimolar extinction coefficient of p-Nitrophenol under the assay condition (cm2/micromole) |
1.0 | : Light path length (cm) |
df | : Dilution factor |
Fig.1 pH-Activity
(in 1M Diethanolamine buffer, pH 8-10.5)
Fig.2 Temperature activity
(in 1M Diethanolamine buffer, pH 10.25 )
Fig.3 pH-Stability
25 ℃, 16 hr-treatment with 0.1 M buffer solution: pH 4-6, dimethylglutaric acid-NaOH; pH 6-8, K-phosphate; pH 8-9, Tris-HCl; pH 9-10, glycine-NaOH. Enzyme concentration: 10 U/mL
Fig.4 Thermal stability
15 min-treatment with 50 mM K-phosphate buffer, pH 7.0. Enzyme concentration: 10 U/mL
1. 原理
p-Nitrophenolの生成量を405nmにおける吸光度の変化で測定する。
2.定義
下記条件下で1分間に1マイクロモルのp-Nitrophenolを生成する酵素量を1単位(U)とする。
3.試薬
4.手順
1.下記反応混液(30.5mL)を調製する。
30 mL | ジエタノールアミン緩衝液 | (A) |
0.5 mL | pNPP溶液 | (B) |
2.3.0 mLの反応混液をキュベット(d=1.0cm)に移し、37℃で約5分間予備加温する。
3.酵素溶液0.1 mLを添加し、ゆるやかに混和する。
4.水を対照に37℃に制御された分光光度計で405nmの吸光度変化を3~5分間記録し、その初期直線部分から1分間当たりの吸光度変化を求める(∆OD test)。盲検は酵素溶液に代えて酵素希釈液を加え、上記同様に操作を行って1分間当たりの吸光度変化を求める(∆OD blank)。
5.計算式
U/mL =
ΔOD/min (ΔOD test - ΔOD blank) × 3.00(mL) × 希釈倍率
18.5 × 1.0 × 0.1 (mL)
= ∆OD/min × 1.676 × 希釈倍率 | |
Vt | : 総液量 (3.1 mL) |
Vs | : 試料総量 (0.1 mL) |
18.5 | : p-Nitrophenolのミリモル分子吸光係数 |
1.0 | : 光路長(cm) |
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