Protein charge ladders: a new technique for studying electrostatic interactions in ultrafiltration systems

Although, previous studies have demonstrated the importance of electrostati c interactions in membrane systems, there are few techniques available for the accurate analysis of these effects. This manuscript describes a novel a pproach to study electrostatic interactions during ultrafiltration using pr otein charge ladders. which consist of a series of chemical derivatives of a given protein that differ by single charge units. Charge ladders were for med by reacting myoglobin and bovine carbonic anhydrase with acetic anhydri de to block free lysine groups. The concentration and net electrical charge of the individual "rungs" were determined by capillary electrophoresis. Si eving experiments were performed using a simple stirred ultrafiltration cel l. The results clearly show an increase in electrostatic exclusion of the m ore negatively-charged species in low ionic strength solutions. The membran e surface charge density was estimated from myoglobin sieving data by compa ring the results to model calculations. This surface charge density was in good agreement with independent measurements of the membrane zeta-potential . These results demonstrate that protein charge ladders can be used to stud y electrostatic interactions in much the same way as polydisperse dextrans have been used to study steric interactions in membrane systems. (C) 2001 E lsevier Science B.V. All rights reserved.