TOYOBO Ideas & Chemistry Biotechnology Operating
Department

ALKALINE PHOSPHATASE

PREPARATION and SPECIFICATION

Appearance Transparent liquid
Activity GradeⅡ　30,000 U/mL or more
Contaminants Adenosine deaminase≤ 1.0×10-4 %
Phosphodiesterase ≤ 3.0×10-3 %

PROPERTIES

Stability Stable at 4 ℃
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)

APPLICATIONS

This enzyme is useful in molecular biology.

ASSAY

Principle

Principle

The formation of p-nitrophenol is measured at 405 nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of p-nitrophenol per minute under the conditions detailed below.

Method

Reagents

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).
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

Procedure

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
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.

Calculation

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

    Fig.1 pH-Activity

    (in 1M Diethanolamine buffer, pH 8-10.5)

  • Fig.2 Temperature activity

    Fig.2 Temperature activity

    (in 1M Diethanolamine buffer, pH 10.25 )

  • Fig.3 pH-Stability

    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

    Fig.4 Thermal stability

    15 min-treatment with 50 mM K-phosphate buffer, pH 7.0. Enzyme concentration: 10 U/mL

活性測定法（Japanese）

1. 原理

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

  • 1Mジエタノールアミン緩衝液、pH9.8 [9.66 mLのジエタノールアミン (MW=105.14)を蒸留水60 mLで希釈後、0.1M MgCl2 5 mLを添加する。さらに2.0N HClで37℃におけるpHを9.8に調整し、最終液量を100 mLとする] (用時調製)
  • 0.674M pNPP溶液 [2.5gのp-ニトロフェニルリン酸二ナトリウム塩 (MW=371.16)を10 mLの緩衝液Aに溶解する] (用時調製)
  • 酵素溶液: 30mM トリエタノールアミン、1mM MgCl2、0.1mM ZnCl2、0.5 % Sodium cholate, pH7.6で反応直前に0.1～0.3U/mLに希釈する。

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)