Preparation of high purity alkaline phosphatase from calf intestine using dye-ligand chromatography.
For the effective application of alkaline phosphatase from calf intestine in Molecular Biology research highly purified enzyme and free from contaminating DNases, DNA nicking, ribonuclease and phosphodiesterase activities is required. We now report the use of a two-step procedure which involves chromatography on a Mimetic Blue AP-Agarose, a commercially available adsorbent and Heparin Sepharose to purify calf intestinal alkaline phosphatase from a crude commercial preparation to homogeneity. Purified enzyme preparations were free from contaminating DNases, DNA nicking, ribonuclease and phosphodiesterase activities and exhibited a specific activity (3.800 units/mg) which is one of the highest reported among the existing high purity commercial preparations. It is therefore concluded that the reported purification protocol can be used routinely to prepare high purity alkaline phosphatase suitable for use in Molecular Biology research.
Pozidis
C
C
Institute of Molecular Biology and Biotechnology, Enzyme Technology Division, Crete, Greece.
Bouriotis
V
V
D000345:Affinity Labels; D000469:Alkaline Phosphatase / Q000302:isolation & purification*; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D002847:Chromatography, Agarose / Q000379:methods; D004396:Coloring Agents; D007422:Intestines / Q000201:enzymology*; D008024:Ligands; D012685:Sepharose / Q000031:analogs & derivatives
D000345:Affinity Labels; D004396:Coloring Agents; D008024:Ligands; C037405:heparin-sepharose; D012685:Sepharose; D000469:Alkaline Phosphatase