Preparation and characterization of affinity sorbents based on isoalloxazine-like ligands for separation of flavoenzymes

Affinity ligands for flavoenzymes were synthesized based on the natural structure of flavo-coenzymes. Two typical flavoenzymes, cholesterol oxidase from Brevibacterium sp. and xanthine oxidase from bovine milk, were employed as standard enzymes. Fluorescent probes were synthesized from eight isoalloxazine-like chemicals and 5-aminofluorescein. Probe-enzyme interactions were analyzed via fluorescence spectra. Chemicals with high binding abilities to flavoenzymes were coupled with Sepharose through spacers composed of epichlorohydrin, ethylenediamine, 1,3-diaminopropane, 2-hydroxy-1,3-diaminopropane, and 1,4-diaminobutane, and subjected to adsorption analysis with flavoenzymes. The results indicated that ligands synthesized from 2,4-dioxohexahydropyrimidine-5-carboxylic acid, cytosine, 7-chloroalloxazine, and 8-chloroalloxazine had high binding abilities to the flavoenzymes. The affinity sorbent based on these ligands revealed a high theoretical maximum adsorption (Q(max)). Protein and bioactivity recoveries were tested after one step of affinity binding via chromatographic analysis on small columns. Results showed that ligands linked with sorbents through long hydrophilic spacers had higher activity recoveries.