title: Determination of dissociation constants between polyelectrolytes and proteins by affinity capillary electrophoresis.
abstract: 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. By use of this affinity capillary electrophoresis two K(d) values were observed for the interaction between RNase A and heparin 17kDa, yielding a high affinity binding with K(d1) 0.0075microM, and a lower affinity binding with K(d2) 8.7microM. For dextran sulfate 8kDa these K(d) values were 0.027 and 10.4microM, respectively. Heparin 3kDa only showed a single K(d) value of 0.52microM. The results show that the magnitude of the binding affinity depends on the type of polyelectrolyte and its molecular weight.
authors.affiliation: Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Anderot
authors.forename: Mikael
authors.initials: M
authors.lastname: Nilsson
authors.forename: Akos
authors.initials: A
authors.lastname: Végvári
authors.forename: Eva Horn
authors.initials: EH
authors.lastname: Moeller
authors.forename: Marco
authors.initials: M
authors.lastname: van de Weert
authors.forename: Roland
authors.initials: R
authors.lastname: Isaksson
mesh_terms: D016264:Dextran Sulfate / Q000737:chemistry*; D004573:Electrolytes / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006493:Heparin / Q000737:chemistry*; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry*
chemical_list: D004573:Electrolytes; D012709:Serum Albumin; D006493:Heparin; D016264:Dextran Sulfate; D012259:Ribonuclease, Pancreatic