Description

Alkaline phosphatases are ubiquitous enzymes, present in all species from bacteria to humans. In humans, four loci encode the different alkaline phosphatase isoenzymes.

For laboratories that require sensitive and reproducible alkaline phosphatase determination, the key practical consideration is the reaction behavior of the enzyme. In vitro, at neutral pH, alkaline phosphatase behaves as a phosphotransferase rather than a hydrolase. Consequently, the reaction rate — and therefore the sensitivity of alkaline phosphatase determination — is considerably increased by using phosphate-accepting molecules, such as diethanolamine, triethanolamine, Tris, and 2-amino-2-methyl-1-propanol.

This phosphate-acceptor requirement is the basis for the sensitivity of alkaline phosphatase determination, and it should be taken into account whenever setting up a dephosphorylation or conjugation reaction with this enzyme.

Specification

Property	Value
Source	Calf intestine
Grade	Conjugation Grade
Form	Ammonium sulfate suspension
Solubility	Distilled water or diluted buffer
Stability	Store at 4 °C (39 °F); do not freeze
Specific activity	~3,000–6,000 U/mg protein
Catalog No.	BF-1404500
Price	$10.00/mg

Unit definition: One unit (U) is the amount of enzyme that releases 1 μmol of p-nitrophenol per minute from p-nitrophenyl phosphate at 37 °C in diethanolamine buffer, pH 9.8.

Features

Conjugation Grade — suitable for conjugation applications.
High specific activity — approximately 3,000–6,000 U/mg protein.
Phosphotransferase activity at neutral pH — the reaction rate is considerably increased by phosphate-accepting molecules (diethanolamine, triethanolamine, Tris, 2-amino-2-methyl-1-propanol).
Ammonium sulfate suspension — stable storage format.
Soluble in distilled water or diluted buffer.
Stable at 4 °C (39 °F) — do not freeze.

Application

Alkaline Phosphatase, Calf Intestine (Conjugation Grade) is intended for conjugation applications, in which the enzyme is coupled to antibodies and proteins. The enzyme catalyzes the dephosphorylation of phosphate groups and, at neutral pH, behaves as a phosphotransferase.

FAQ

What is the source and grade of this alkaline phosphatase?

This alkaline phosphatase is obtained from calf intestine and is supplied at Conjugation Grade.

What is the specific activity of this alkaline phosphatase?

The specific activity is approximately 3,000–6,000 U/mg protein.

What is the unit definition of alkaline phosphatase?

One unit (U) is the amount of enzyme that releases 1 μmol of p-nitrophenol per minute from p-nitrophenyl phosphate at 37 °C in diethanolamine buffer, pH 9.8.

How is alkaline phosphatase activity measured?

Alkaline phosphatase activity is measured by following the increase in absorbance at 405 nm, which corresponds to the release of p-nitrophenol from p-nitrophenyl phosphate.

What reagents are required for the activity assay?

Reagent	Preparation
Diethanolamine buffer (1 mol/L, pH 9.8; 0.5 mmol/L MgCl₂)	9.7 mL diethanolamine (99%) + distilled water; add 0.05 mL MgCl₂ solution; adjust to pH 9.8 (37 °C) with HCl (≥2 mol/L); make up to 100 mL.
MgCl₂ solution (1 mol/L)	20.3 g MgCl₂·6H₂O dissolved in 100 mL distilled water.
4-Nitrophenyl phosphate solution (0.67 mol/L)	250 mg 4-nitrophenyl phosphate sodium salt + 1.0 mL distilled water.
Dilution buffer (0.1 mol/L, pH 7.6)	1.86 g TEA·HCl in distilled water; add 0.1 mL MgCl₂ solution + 0.1 mL ZnCl₂ (0.1 mol/L, freshly prepared); adjust to pH 7.6 with NaOH (1 mol/L); make up to 100 mL.
Sample solution	Dilute with dilution buffer to 0.05–0.06 U/mL; stand for 15–20 min at room temperature before assay.

What is the assay procedure for alkaline phosphatase?

Set the spectrophotometer to 405 nm and 37 °C.
Into a cuvette, add 2.90 mL diethanolamine buffer and 0.05 mL 4-nitrophenyl phosphate solution.
Incubate at 37 °C for 6–8 minutes (temperature equilibration and blank rate).
Add 0.1 mL diluted enzyme solution, mix, and record the absorbance increase at 405 nm for 5 minutes.
Use the linear portion of the curve to calculate ΔE₄₀₅/min.

How is the activity of alkaline phosphatase calculated?

Activity (U/mg) = (ΔE₄₀₅/min × total volume × enzyme dilution) / (18.5 × enzyme volume × mg enzyme/mL).

Here, 18.5 is the millimolar extinction coefficient of p-nitrophenol at 405 nm.

How should this alkaline phosphatase be stored?

Store at 4 °C (39 °F). Do not freeze.

In what form is this alkaline phosphatase supplied?

The product is supplied as an ammonium sulfate suspension.

What is the solubility of this alkaline phosphatase?

The enzyme is soluble in distilled water or diluted buffer.

Why does alkaline phosphatase behave as a phosphotransferase at neutral pH?

In vitro, at neutral pH, alkaline phosphatase behaves as a phosphotransferase rather than a hydrolase. This behavior means that the reaction rate depends on the availability of a phosphate-accepting molecule.

Which phosphate-accepting molecules increase the reaction rate?

Phosphate-accepting molecules such as diethanolamine, triethanolamine, Tris, and 2-amino-2-methyl-1-propanol considerably increase the reaction rate. This is the basis for the sensitivity of alkaline phosphatase determination.