Study On The Purification Of Alkaline Phosphatase By Novel 'Bio-mimetic' Dye Affinity Ligand And Its Characteristics

Eight novel "bio-mimetic" azo dye affinity ligands which bear competitive inhibitors of alkaline phosphatase (AKP, EC.3.1.3.1)) were designed and synthesized. Ligand V was found to have good affinity for AKP. Affinity chromatography parameters for the purification of AKP from calf intestine by ligand V were optimized. AKP from Ulva pertusa kjellmwas was firstly purified to single band in SDS-PAGE by ligand V, and its characteristics were studied. EPR,VIS, and XRS were firstly employed to investigate the micro-inhibitory mechanism of activity of AKP, which was exerted by kinds of metal ions. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme described by Tsou was applied to the study on the kinetics of the course of inactivation of the AKP from calf intestine and Ulva pertusa kjellm respectively. The main results are as follows.1. Ligand V has the best affinity for AKP among the eight newly-synthesized dye ligands, and its selectivity to AKP is higher than Cibacron Blue 3FGA.2. The optimal conditions for the coupling reaction between ligand V and Sepharose were that the primary concentration of ligand is 1.0%; optimal reaction temperature and reaction time are 60 癈 and 6hours respectively; the selectivity of media is the highest when the density of ligand on gel is 4.55mg ligand/g moist gel; with 0.1M NaCl and 30mM Na2HPO4 to elute AKP in turn, the resulting enzyme has a specific activity of 990Unit/mg with a recovery of 89%.3. The Ulva pertusa kjellm AKP, whose molecular weight is 76kD,is composed of four same subunits. The optimal temperature and pH is 37癈 and 9.9 respectively. Km and Vmax are 0.95mM and 5.00uM/min. Mg2+ has no effect on the enzyme activity, while Co2"1", Cu 2+ and Ni2+inhibit the enzyme activity obviously. The enzyme activity can be inhibitedcompletely when the concentration of DTT is 2.5mM, which illustrates S-S bond in the molecule is important for the preservation of enzyme activity. The NBS can also inhibit the enzyme activity, which demonstrates that enzyme activity is associated with Trp.4. The data of enzyme activity demonstrates that Na"1", K+ and Li have no effect on the activity of AKP, while Cu2+, Co2*, Ni2+, Hg2*, Ag+, Zn2+ have inhibitory effect on the activity of AKP. Especially, Zn2+, Hg2+, Ag+ can completely inhibit the activity when their concentrations are high enough. The results of EPR, VIS and XRS illustrate there exist a direct interaction between the Cu2+, Co2"1" and the metal ions of the active centers of AKP, as a result, part of the Zn2+ in AKP were replaced by Cu2+, Co2"1" and the corresponding Co-AKP, Cu-AKP derivatives were produced and the catalytic activity of AKP decreased. The result of XRS demonstrate that Zn2"1" replaces the Mg2+in the activity center of AKP, while Hg2"1" replaced both Zn2"1" and Mg2+completely, consequently the activity of AKP was inhibited completely, this result confirmed that Mg2"1" is important for the preservation of AKP activity.5. Mg2+ also inhibit the activity of AKP from calf intestine, this may be because the Mg2+ replaced the Zn2* of activity center of the enzyme, and this suggests that the synergetic effects between Mg2+ and Zn2"1" be essential to the activity preservation of AKP from calf intestine.6. Although the kinetics of inactivation of alkaline phosphatase from calf intestine and Ulva pertusa kjellm by EDTA are both complexing irreversible inhibition, the experiment result illustrates there exists difference between them. This may be because that the metal ions in calf intestine AKP is more close to the surface of enzyme molecule than in Ulva pertusa kjellm , EDTA can exerted inhibitory effect on the AKP from calf intestine more easily and quickly.