Фосфатаза, щелочная из телячьей кишки (CIP) grade II

Химические свойства

solid

Использование

Alkaline Phosphatase is commonly used to remove the 5′-terminal phosphate from nucleic acids.

Общее описание

Alkaline phosphatase from bovine intestinal mucosa is both a phosphomonoesterase and a pyrophosphatase.

Методы очистки

The E.coli supernatant in sucrose (20%, 33mM) in Tris-HCl pH 8.0 is purified through a DEAE-cellulose column and recrystallised. To the column eluates in 0.125M NaCl is added MgCl2 (to 0.01M) and brought to 50% saturation in (NH4)2SO4 by adding the solid (0.20g/mL). The mixture is centrifuged to remove bubbles and is adjusted to pH 8.0 (with 2N NaOH). Saturated (NH4)2SO4 at pH 8.0 is added dropwise until the solution becomes faintly turbid (~61% saturation). It is set aside at ~25o for 1hour (turbidity will increase). The mixture is placed in an ice bath for several minutes when turbidity disappears and a clear solution is obtained. It is then placed in a large ice bath at 0o (~5L) and allowed to warm slowly to room temperature in a dark room whereby crystals are formed appearing as a silky sheen. The crystals are collected by centrifugation at 25o if necessary. The crystalline solutions are stable at ~25o for many months. They can be stored at 0o, but are unstable when frozen. Cysteine at 10-3M and thioglycolic acid at 10-4M are inhibitory. This is reversed on addition of Zn2+ ions. Many organic phosphates are good substrates for this phosphatase. [Molamy & Horecker Methods Enzymol 9 639 1966, Torriani et al. Methods Enzymol 12b 212 1968, Engstrom Biochim Biophys Acta 92 71 1964.] Alkaline phosphatase from rat osteosarcoma has been purified by Me2CO precipitation and chromatography on DEAE-cellulose, Sephacryl S-200, and hydroxylapatite. [Nair et al. Arch Biochem Biophys 254 18 1987.] Phosphoproteins (various). These are purified by adsorbing onto an iminodiacetic acid substituted agarose column to which are bound ferric ions. This chelate complex acts as a selective immobilised metal affinity adsorbent for phosphoproteins. [Muszyfiska et al. Biochemistry 25 6850 1986.]