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Affinity capillary electrophoresis for identification and investigation of human Gc‐globulin (vitamin D‐binding protein) and its isoforms interacting with G‐actin
Author(s) -
Pedersen Jeppe Trudslev,
Østergaard Jesper,
Houen Gunnar,
Heegaard Niels H. H.
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700618
Subject(s) - vitamin d binding protein , gene isoform , capillary electrophoresis , chemistry , biochemistry , chromatography , affinity electrophoresis , globulin , identification (biology) , actin , vitamin , affinity chromatography , biology , enzyme , immunology , gene , botany
A CE procedure was established for the nondenaturing separation and identification of the isoforms of the actin‐binding human plasma protein Gc‐globulin. To characterize interactions with globular actin (G‐actin), a novel method was developed for the simultaneous qualitative assessment of the binding interaction between the three major isoforms of Gc‐globulin and G‐actin using pre‐equilibrium affinity CE and UV detection. Evidence was found that some difference in binding affinity existed among the isoforms, although the quantification of this difference was not feasible by UV detection because of the high affinity nature of the binding. The difference in affinity appeared to be related to the p I of the isoforms; a high p I corresponding to a high affinity. For quantitative binding studies Gc‐globulin was fluorescently labeled with 5‐(and‐6)‐carboxyfluorescein, succinimidyl ester (CFSE). Data suggested that extensive labeling interfered with actin binding but with moderately labeled Gc‐globulin it was possible to determine a dissociation constant of K d = 21 ± 1 nM for the binding between labeled Gc‐globulin and G‐actin using pre‐equilibrium affinity CE and LIF detection.