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 (17 kDa), heparin (3 kDa) and dextran sulfate (8 kDa) were 33 and 307 mu M, 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 17 kDa, yielding a high affinity binding with K-d1 0.0075 mu M, and a lower affinity binding with K-d2 8.7 mu M. For dextran sulfate 8 kDa these K-d values were 0.027 and 10.4 mu M, respectively. Heparin 3 kDa only showed a single K-d value of 0.52 mu M. The results show that the magnitude of the binding affinity depends on the type of polyelectrolyte and its molecular weight. (C) 2009 Elsevier B.V. All rights reserved.

Ämnesord

NATURVETENSKAP  -- Kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

TEKNIK OCH TEKNOLOGIER  -- Medicinteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Medical Engineering (hsv//eng)

Nyckelord

Dissociation constant

Polyelectrolytes

Heparin

Dextran sulfate

Human serum albumin

Capillary electrophoresis

Ribonuclease A

RNase

Partial filling

Affinity

Binding

Chemistry

Kemi

dissociation constant

heparin

dextran sulfate

human serum albumin

capillary electrophoresis

ribonuclease A

RNase

affinity binding

partial filling

polyelectrolytes

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