Evaluation of a newly developed continuous protein separation system based on a difference in the isoelectric points of the proteins

Abstract

Several advanced glycation end products (AGEs) were identified as potential uremic toxins, which were accumulated in patients with end-stage renal disease. Current blood purification technology, however, can not provide a method for separating AGE-modified proteins from normal proteins. The aim of this study was to develop a continuous protein separation system capable of separating AGE-modified proteins from normal proteins. We focused on the change in the isoelectric point of AGE-modified proteins and evaluated a manufactured prototype device based on a free flow electrophoresis. We use myoglobin (isoelectric point: 7.35) solution as a test solution. Protein concentration and pH were measured at varying flow rates (1.3, 3.4, 7.2 mL/min) and voltages (10 and 50 V).When the voltage = 50 V, pH gradient was formed in the electrophoretic chamber after 15 min and myoglobin concentration increase at the center of the electrophoretic chamber. Myoglobin was focusing on sample outlet where the pH of the solution near the isoelectric point. Concentration ratio (maximum / average) was 1.25 -1.30 and had a maximum value at a flow rate of 3.4 mL/min. This newly developed device for continuous protein separation was capable of concentrating myoglobin at the specific position in the electrophoretic chamber and continuously collecting the concentrated myoglobin. However, the concentration ratio of myoglobin was low. The optimization of the operating condition required to improve the selectivity.