LECTIN-GLYCOENZYME COLUMN CHROMATOGRAPHY MONITORED BY ENZYME FLOW MICROCALORIMETRY

A method based on the flow microcalorimetric determination of catalyti c activity of immobilized enzyme in a so-called enzyme thermistor was used to monitor the process of lectin affinity chromatography of inver tase on Concanavalin A-bead cellulose. The strong biospecific interact ion between Concanavalin A and invertase was employed to determine the bound enzyme and this principle was used for the investigation of an alternative direct method for monitoring the lectin affinity chromatog raphy of glycoenzymes. The results obtained by flow microcalorimetry s howed that the catalytic activity of invertase immobilized on Concanav alin A-bead cellulose can be compared directly with the thermometric v alue Delta T-max. The validity of the method was also confirmed by the enzyme thermistor post-column method, which is based on the determina tion of the product from the immobilized invertase enzymatic reaction. The adsorption and desorption in the chromatography column were exami ned by flow microcalorimetry in small samples withdrawn from the colum n. Attention has been given to the operating parameters and the storag e stability of the affinity sorbent. The binding ability of the affini ty matrix decreased with the number of consecutive chromatographic run s, although its storage stability was satisfactory.