HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF PROTEINS ON DEFORMED NONPOROUS AGAROSE BEADS - FAST BORONATE AFFINITY-CHROMATOGRAPHY OF HEMOGLOBIN AT NEUTRAL PH

Aminophenylboronic acid was attached to epoxy-activated non-porous agarose beads with diameters of 12-15 μm and this boronate gel column was used for the fractionation of glycosylated from non-glycosylated haemoglobin. By varying the experimental conditions it was shown that the ratio between the second peak (glycosylated haemoglobin) and the first peak (non-glycosylated haemoglobin) was virtually independent of pH in the range 7-8, ionic strength, flow-rate and sample load (up to at least 160 μl ofhaemolysate on a 0.7-ml column). It is therefore not necessary to control thoroughly these parameters in order to obtain reproducible results, which is a great advantage in fast routine analyses of glycosylated haemoglobin. At a flow-rate of 4.0 ml/min an analysis was finished within 2 min on a 2.5 cm X 0.6 cm I.D. column. The total time of an analysis was also short because a sample from a droplet of blood could be applied directly onto the column after haemolysis for 1 min without removal of cell debris by time-consuming centrifugation. The experiments were performed at a pH close to the isoelectric point of haemoglobin, because at this pH haemoglobin has a negligible net charge and will therefore not interact with the charged groups of the ligands, the matrix and proteins adsorbed in previous runs. © 1990.