Alkaline phosphatase is a broad term associated with non-specific phosphomonoesterases with activity optima at alkaline pH.
E. coli alkaline phosphatase is a valuable reagent for removal of terminal monoesterified phosphate from both ribo- and deoxyribo-oligonucleotide.
Characteristics of Alkaline Phosphatase from E. coli :
Molecular weight: 80,000 Taylor and Coleman (1972). 86,000 Lazdunski and Lazdunski (1969). 89,000 Anderson et al. (1975).
Composition: E. coli alkaline phosphatase is a dimeric, zinc and magnesium containing protein (Anderson et al. 1975; Bosron et al. 1975). Though the subunits are believed to be coded by the same gene (Garen and Garen 1963), they develop molecular heterogeneity after translation (Bosron and Vallee 1975; Bridgen and Secher 1973). See also Schlesinger (1974), Halford et al. (1972). For amino acid composition see Reid and Wilson (1971) and Kelley et al. (1973).
Fernley (1973) and Chappelet-Tordo et al. (1974) indicate there to be two active sites, only one is functional at a time. Two Zn2+ are needed for activity. A stable apoenzyme can be reactivated with Zn2+ , Mn, Co, Ni, Cu, Cd and Hg have been substituted for Zn but only Co restores significant activity (Taylor and Coleman 1972). Magnesium does not activate the apoenzyme but enhances activity of the enzyme containing two gram atoms of zinc (Anderson et al. 1975). See also Anderson and Vallee (1975), Lilja et al. (1975), Brown et al. (1974), Daemen and Riordan (1974), Kaden et al. (1972).
Optimum pH: 8.0 (Garen and Levinthal 1960).
Extinction coefficient: E278 = 7.2 (Anderson et al. 1975).
Isoelectric point: pH 4.5 (Garen and Levinthal 1960).
Activity: Bloch and Schlesinger (1974) report on kinetic studies. Bock and Sheard (1975) indicate that the E. coli enzyme binds phosphate tightly over a wide range of pH forming complexes that may be intermediate in the hydrolytic action. See also Bosron and Vallee (1975), Zukin and Hollis (1975), and Chlebowski and Coleman (1974).
Specificity: See review by Reid and Wilson (1971). Also Mushak and Coleman (1972) and Snyder and Wilson (1972).
Inibibitors: The enzyme is inhibited by chelating agents and inorganic phosphates.
Assay
The assay is that of Garen and Levinthal (1960) in which the reaction velocity is determined by measuring an increase in absorbance at 410 nm resulting from the hydrolysis of p-nitrophenylphosphate to p-nitrophenol. One unit releases one micromole of p-nitrophenol per minute at 25°C, pH 8, under the specified conditions.
Reagents
1.5 M Tris⋅HCl buffer, pH 8.0
0.003 M p-nitrophenylphosphate (PNP). Care must be exercised to use an analytical grade and the correct molecular weight.
Enzyme
Dilute in reagent grade water to obtain a rate of 0.02-0.04 ΔA/minute.
Procedure
Adjust the spectrophotometer to 410 nm and 25°C.
Pipette into each cuvette as follows:
0.003 M PNP 1.0 ml
1.5 M Tris⋅HCl, pH 8.0 2.0 ml
Mix well and incubate in the spectrophotometer for 4-5 minutes to achieve temperature equilibration and to establish blank rate, if any. Add 0.1 ml of diluted enzyme and record. Determine A410 for 3-5 minutes from linear portion of the curve.
Calculation
where 3500 is the molar extinction coefficient of salicylic acid at 300 nm.
H2611416
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