Determination of dissociation constants between polyelectrolytes and proteins by affinity capillary electrophoresis.

In this manuscript we report the binding affinity between two model proteins, human serum albumin (HSA) and ribonuclease A (RNase A), and negatively charged polyelectrolytes, two different heparin fractions and dextran sulfate, by means of partial filling and affinity capillary electrophoresis. The apparent dissociation constants, K(d), obtained by use of the partial-filling method, between HSA and heparin (17kDa), heparin (3kDa) and dextran sulfate (8kDa) were 33 and 307microM, respectively. A new method was developed to determine affinities that take in account different migration directions between the protein and the polyelectrolyte, which was required to study RNase A.