Alkaline Phosphatase (AP)

Description

• General description: 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)
• 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.[2][3] It has also been used to phosphorylate samples to distinguish between phosphate moieties present as phosphorylated glycans and phosphorylated amino acid residues.[4]
• 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.