Synthesis of new ‘biomimetic’ dye-ligands and their application in the purification of alkaline phosphatase

A series of new ‘biomimetic’ dye-ligands were synthesized for the enzyme alkaline phosphatase. The biomimetic dye-ligands were immobilized on Sepharose CL-6B and the affinity adsorbents, together with the non-biomimetic adsorbent bearing Cibacron Blue 3F-GA, were studied for their ability to purify alkaline phosphatase from calf intestine. The results showed that all synthesized dye-ligands have certain affinity to alkaline phosphatase, but the affinity is varying with different molecular structures (such as functional groups or their positions) of the dye-ligands. In particular, the dye V has the higher affinity to alkaline phosphatase. Detailed investigation on the elution conditions of alkaline phosphatase on synthesized dye V-immobilized Sepharose CL-6B showed that pulse elution by 0.1 M NaCl and 30 mM Na2HPO4 in turn could lead to 65-fold purification in a single step with high enzyme recovery (84%) of alkaline phosphatase. Kinetic inhibition studies showed that dye V inhibits alkaline phosphatase in a competitive manner with a KI of 1 mM.