ALKALINE PHOSPHATASE from Calf intestine

Orthophosphoric-monoester phosphohydrolase (alkaline optimum) (EC 3.1.3.1)
Orthophosphoric monoester+H₂O→Alcohol+Orthophosphate

PREPARATION and SPECIFICATION
Appearance:	50% glycerol solution
Activity:	GradeⅡ 30,000U/ml or more
Contaminants:	Adenosine deaminase	≤1.0×10⁻⁴ %
	Phosphodiesterase	≤3.0×10⁻³ %
	DNase	No degradation of the fragments is observed by agarose gel electrophoresis, after incubation of 1µ g of λ−DNA with 2 units of alkaline phosphatase for 16 hr at 37℃ in a 50µ l reaction volume.
	RNase	No degradation of the fragments is observed by polyacrylamide gel electrophoresis, after incubation of 2µ g of t-RNA with 2 units of alkaline phosphatase for 16 hr 37℃ in a 50µ l reaction volume.

PROPERTIES ¹· ² ⁾
Stability:	Store at −20ºC (Fig.1)
Molecular weight:	approx. 100,000
Isoelectric point:	5.7
Michaelis constant:	1.7×10‾ ³M (p-Nitrophenyl phosphate)
Inhibitors:	Cu⁺⁺, Ag⁺, Hg⁺⁺, EDTA
Optimum pH :	10.0−10.3(Fig.3)
Optimum temperature:	40℃(Fig.4)
pH Stability:	pH 8.5−10.3 (25℃, 20hr) (Fig.5)
Thermal stability:	below 40℃ (pH 9.5, 30min) (Fig.6)
Effect of various chemicals:	(Table 1)

APPLICATIONS ³· ⁴ ⁾

This enzyme is useful for molecular biology.

LPP-209

ASSAY

Principle:

alkaline phosphatase

p-Nitrophenylphosphate(pNPP) +H₂O ►p-Nitrophenol + Pi

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

Unit definition:

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

Method:

Reagents
A.Diethanolamine buffer, pH10.25:	1M[Dilute 9.66ml of diethanolamine (MW=105.14) in 60ml of H₂O, add 0.25ml of 0.1M MgCl₂ and, after adjusting the pH to 10.25 with 2N HCl, fill up to 100ml with H₂O](Prepare freshly)
B. pNPP solution:	0.1M [Dissolve 371mg of p-nitrophenylphosphate disodium salt (MW=371.16) in 10ml buffer solution A] (Prepare freshly)
C. Enzyme diluent:	0.1M Diethanolamine buffer pH 9.5 contg. 0.25mM MgCl₂

Procedure

Concentration in assay mixture
Diethanolamine buffer	0.97 M
p-Nitrophenylphoshate	10 mM
MgCl₂	0.25mM

1. Prepare the following reaction mixture in a cuvette (d=1.0cm)
and equilibrate at 37℃ for about 5 minutes.

2.6ml Buffer solution (A)
0.3ml Substrate solution (B)

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

3. Record the increase in optical density at 405nm against water for 3 to 5 minutes in a spectrophotometer
thermostated at 37℃, and calculate ΔOD per minute from the intial linear portion of the curve (ΔOD
test).
At the same time, measure the blank rate (ΔOD blank) using the same method as the test except that the
enzyme diluent is added instead of the enzyme solution.

* Dilute 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 :

ΔOD/min (ΔOD test−ΔOD blank) × Vt × df

Volume activity (U/ml) = =ΔOD/min × 1.62 × df

18.5 × 1.0 × Vs

Vt: : Total volume (3.0ml)
Vs: : Sample volume (0.1ml)
18.5: : Millimolar extinction coefficient of p-nitrophenol under the assay condition(㎠/micromole)
1.0: : Light path length (cm)
df: : Dilution factor

REFERENCES

H.N.Ferunley; The Enzymes, Vol.4, (3rd ed.) 417 (1971).
R.K.Morton; Biochem.J, 61, 232 (1955).
F.Dray, E.Dith and C.Rougeot; Methods of Enzymatic Analysis, Vol.9, 348 (1986).
P.Rathman and B.B.Saxena; Methods of Enzymatic Analysis, Vol.9, 396 (1986).

**Table 1. Effect of Various Chemicals on Alkaline phosphatase**
[The enzyme dissolved in 40mM CAPS buffer, pH9.5 (20U/ml) was incubated with each chemical at 25℃ for 1hr.]
Chemical	Concn.(mM)	Residual activity	Chemical	Concn.(mM)	Residual activity
None	−	100%	PCMB	2.0	110%
Metal salt	2.0		MIA	2.0	102
MgCl₂		108	NaF	2.0	104
CaCl₂		99	NaN₃	20	97
FeCl₂		96	EDTA	5.0	19
MnCl₂		76	o-Phenanthroline	2.0	80
CoCl₂		86	Triton X-100	0.1%	101
ZnCl₂		73	Brij 35	0.1%	111
NiCl₂		91	Tween 20	0.1%	95
CuSO₄		56	Span 20	0.1%	82
Pb(OAc)₂		96	Na-cholate	0.1%	94
AgNO₃		0.1	SDS	0.1%	107
HgCl₂		17

Ac, CH₃CO;PCMB, p-Chloromercuribenzoate;MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate.

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