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API-RO

Roche

Alkaline Phosphatase (AP)

grade I, from calf intestine

Synonym(s):
AP, Alkaline Phosphatase
Enzyme Commission number:

biological source

bovine (calf) intestine

Quality Level

form

suspension

specific activity

~2000 units/mg protein (At 37 °C with 4-nitrophenyl phosphate as the substrate, and diethanolamine as the buffer (400 U/mg at 25 °C with 4-nitrophenyl phosphate as the substrate, and glycine as the buffer))

packaging

pkg of 1,500 U (10108138001)
pkg of 7,500 U (10108146001)

manufacturer/tradename

Roche

optimum pH

8.0-10.5

absorption

7.6 at 278 nm (10 mg AP/ml)

storage temp.

2-8°C

General description

Orthophosphoric-monoester phosphohydrolase (alkaline optimum)
Alkaline phosphatase catalyzes the removal of phosphate group from various compounds that are phosphorylated. It hydrolyzes 5′-monophosphate groups from both DNA and RNA. It can also hydrolyze 5′-diphosphate and 5′-triphosphate groups from RNA.

Specificity

Heat inactivation: AP activity is destroyed irreversibly by incubating the enzyme at 65 °C.
Denaturation is dependent on protein concentration - using less than 10 U/ml (0.1 mol/l glycine buffer, pH 10.5, 1 mmol/l MgCl2 and 0.1 mmol/l ZnCl2), AP is inactivated by a 10 minutes incubation at 65 °C.

Application

Selective cleavage of terminal phosphate groups from oligonucleotides and in protein nucleotide exchange and deliverance. It has also been used to phosphorylate samples to distinguish between phosphate moieties present as phosphorylated glycans and phosphorylated amino acid residues.

Quality

Contaminants: <0.0004% PDE, <0.004% ADA and A-5-MP deaminase, each

Sequence

AP is a dimer of identical or nearly identical subunits; each of which contains 2 molecules of Zn2+ , one tightly bound and necessary for structural stability, the other loosely bound and required for caytalytic activity. The active site contains a reactive serine.
In some mammals (e.g., humans), there are at least 3 distinguishable isoenzymes: the intestinal, the placental and the form found in bone/liver/kidney.

Unit Definition

Unit Conversion: 1 Roche unit (tested at +25 °C) corresponds to ~2 to 2.5 Sigma units (tested at +37 °C).
AP activity is not expressed in Armstrong units; a conversion factor for international units is not known.
Approx. 3.8 U (grade I AP) or 4.0 U (grade II AP) [+37 °C, 4-NPP as substrate, diethanolamine as buffer, pH 9.8] = 1 U [+25 °C, 4-NPP as substrate, glycine as buffer, pH 10.5].

Biochemica Information. 87 (grade I AP) :
1.0 U [+37 °C, QC assay conditions] = 0.79 U [+30 °C, QC assay conditions] = 0.57 U [+25 °C, QC assay conditions]

Physical form

Suspension in 3.2 M ammonium sulfate solution, 1 mM MgCl2, 0.1 mM ZnCl2, pH approximately 7

Preparation Note

  • Activator: Divalent metal ions (Mg2+, Co2+, Mn2+)
  • Amino alcohols (2-amino-2-methyl-1-propanol, diethanolamine)

Note that high concentrations of amino alcohols can as well act as competitive inhibitors (e.g., monoethanolamine in diethanolamine).

Storage conditions (working solution): The suspension is offered at a c= approx. 5 mg/ml.
If this suspension is diluted only 5 to 10-fold with further 3.2 M ammonium sulfate solution, 1 mM MgCl2, 0.1 mM ZnCl2, pH approx. 7, we would expect from general experience that a stability for some weeks might be given provided it is stored at 4 °C (no guarantee!).

Other Notes

For life science research only. Not for use in diagnostic procedures.

Storage Class Code

12 - Non Combustible Liquids

WGK

WGK 1

Flash Point(F)

does not flash

Flash Point(C)

does not flash

Certificate of Analysis

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Certificate of Origin

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