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

Anderot, Maria; Nilsson, Mikael; Végvári, Ákos LU ; Moeller, Eva Horn; van de Weert, Marco and Isaksson, Roland (2009) In Journal of Chromatography. B 877(10). p.892-896
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, Kd, 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 μM, 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 Kd values were... (More)
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, Kd, 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 μM, 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 Kd values were observed for the interaction between RNase A and heparin 17 kDa, yielding a high affinity binding with Kd1 0.0075 μM, and a lower affinity binding with Kd2 8.7 μM. For dextran sulfate 8 kDa these Kd values were 0.027 and 10.4 μM, respectively. Heparin 3 kDa only showed a single Kd value of 0.52 μM. The results show that the magnitude of the binding affinity depends on the type of polyelectrolyte and its molecular weight. (Less)
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author
organization
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type
Contribution to journal
publication status
in press
subject
keywords
dissociation constant, heparin, dextran sulfate, human serum albumin, capillary electrophoresis, ribonuclease A, RNase, affinity binding, partial filling, polyelectrolytes
in
Journal of Chromatography. B
volume
877
issue
10
pages
892 - 896
publisher
Elsevier
external identifiers
  • scopus:62249218883
ISSN
1873-376X
DOI
10.1016/j.jchromb.2009.02.021
language
English
LU publication?
yes
id
95aac6f6-2496-4b42-ae68-c7ae49c9e62a (old id 1314221)
date added to LUP
2009-03-26 09:31:57
date last changed
2017-07-09 03:46:22
@article{95aac6f6-2496-4b42-ae68-c7ae49c9e62a,
  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, Kd, 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 μM, 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 Kd values were observed for the interaction between RNase A and heparin 17 kDa, yielding a high affinity binding with Kd1 0.0075 μM, and a lower affinity binding with Kd2 8.7 μM. For dextran sulfate 8 kDa these Kd values were 0.027 and 10.4 μM, respectively. Heparin 3 kDa only showed a single Kd value of 0.52 μM. The results show that the magnitude of the binding affinity depends on the type of polyelectrolyte and its molecular weight.},
  author       = {Anderot, Maria and Nilsson, Mikael and Végvári, Ákos and Moeller, Eva Horn and van de Weert, Marco and Isaksson, Roland},
  issn         = {1873-376X},
  keyword      = {dissociation constant,heparin,dextran sulfate,human serum albumin,capillary electrophoresis,ribonuclease A,RNase,affinity binding,partial filling,polyelectrolytes},
  language     = {eng},
  number       = {10},
  pages        = {892--896},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography. B},
  title        = {Determination of dissociation constants between polyelectrolytes and proteins by affinity capillary electrophoresis},
  url          = {http://dx.doi.org/10.1016/j.jchromb.2009.02.021},
  volume       = {877},
  year         = {2009},
}