TOYOBO BIO

진단시약원료 효소

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ALKALINE PHOSPHATASE

PREPARATION and SPECIFICATION

class="darkGray">Appearance Transparent liquid Activity GradeⅡ 30,000U/ml or moreContaminantsAdenosine deaminase≤ 1.0×10^-4%Phosphodiesterase≤ 3.0×10^-3%

PROPERTIES

< table class="darkGray">Stability Stable at 4℃Molecular weightapprox. 104,000 Optimum pH 9.5(그림 1)Optimum temperature ≧ 60℃ (그림 2)pH Stability pH 5.5-10.4 (25℃, 16hr)(Fig. 3)Thermal stabilitybelow 65℃ (pH 7.0, 60min)( 그림 4)

APPLICATIONS

This enzyme is useful for molecular biology.

ASSAY

Principle

The appearance of p-Nitrophenol is measured at 405nm by spectrophotometry.

< h2 class="ttl_box">Unit definition

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

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Method

Reagents

A. Diethanolamine buffer	1M[Dilute 9.66ml of diethanolamine (MW=105.14) in 60ml of H₂O, add 5ml of 0.1M MgCl₂ and, after adjusting the pH to 9.8 with 2N HCl, fill up to 100ml with H₂O](Prepare fleshly)
B. pNPP solution	0.674M [2.5g of p-Nitrophenylphosphate disodium salt (MW=371.16) in 10ml Diethanolamine buffer (A)](Prepare fleshly)
C. Enzyme diluent	30mM Triethanolamine , 1mM MgCl₂, 0.1mM ZnCl₂, 0.5% Sodium cholate, pH7.6

Procedure

1. Prepare the following working solution (30.5ml) in a brownish bottle and store on ice (Prepare freshly).

< td>pNPP solution

30ml	Diethanolamine buffer	(A)
0.5ml	(B)

Concentration in assay mixture
Diethanolamine buffer	0.97 M
p-Nitrophenylphosphate	11 mM
MgCl₂	4.8 mM

2.피펫 3.0ml of working solution into a cuvette (d=1.0cm) and equilibrate at 37℃ for about 5 minutes.

3.Add 0.1ml of the enzyme solution^* and mix by gentle inversion.

4.Record the increase in optical density at 405nm against water for 3 to 5 minutes in 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) by 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 0.1-0.3U/ml with ice-cold enzyme diluent (C), immediately before 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 (3.1ml)
Vs	: Sample volume (0.1ml)
18.5	: Millimolar extinction coefficient of p-Nitrophenol under the assay condition (cm^2/micromole)
1.0	: Light path length (cm)
df< /td>	: Dilution factor

Fig.1 pH-Activity

그림. 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℃, 16hr-treatment with 0.1M 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: 10U/ml

Fig.4 Thermal stability

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

활성 측정법(Japanese)

1. 원리

2. 정의

하기 조건 하에서 1분에 1 마이크로몰의 p-Nitrophenol을 생성하는 효소량을 1단위( U)로 한다.

3. 시약

1M 디에탄올아민 완충액, pH 9.8 [9.66ml의 디에탄올아민 (MW=105.14) 증류수 60ml로 희석 후, 0.1M MgCl₂ 5ml를 첨가한다. 또한 2.0N HCl로 37℃에서의 pH를 9.8로 조정하고, 최종 액량을 100ml로 한다] (용시 조제)
0.674M pNPP 용액 [2.5g의 p-니트로페닐린 산 이나트륨 염 (MW = 371.16)을 10ml의 완충액 A에 용해시킨다. 0.1mM ZnCl₂, 0.5% Sodium cholate, pH 7.6에서 반응 직전에 0.1~0.3U/ml로 희석한다.

4. 절차

1.하기 반응 혼합물(30.5ml)을 제조한다. 한다.

30ml	디에탄올아민 완충액	(A)
0.5ml	pNPP 용액	(B)

2.3.0ml의 반응 혼합물을 큐벳(d=1.0cm)으로 옮기고 37℃에서 약 5분 동안 예비 가온한다.

3. 효소 용액 0.1ml를 첨가하고 완만하게 혼합한다.

4.물을 대조에 37℃로 제어된 분광광도계로 405nm의 흡광도 변화를 3~5분간 기록하고, 초기 직선 부분으로부터 분당 흡광도 변화를 구한다 (∆OD test). 맹검은 효소 용액 대신에 효소 희석액을 첨가하고, 상기와 같이 조작을 행하여 1분당 흡광도 변화를 구한다(∆OD blank).

5. 수식

U/ml=
ΔOD/min (ΔOD test − ΔOD blank) × 3.00(ml) × 희석 배율
18.5 × 1.0 × 0.1 (ml)

	= ∆OD/min × 1.676 × 희석 배율
Vt	: 총 액량(3.1ml)
Vs	: 샘플 총량 (0.1ml)
18.5	: p- Nitrophenol의 밀리몰 분자 흡광 계수
1.0	: 광로 길이(cm)