Affinity capillary electrophoresis for identification and investigation of human Gc-globulin (vitamin D-binding protein) and its isoforms interacting with G-actin.
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 pI of the isoforms; a high pI 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.
Pedersen
Jeppe Trudslev
JT
Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark.
Ostergaard
Jesper
J
Houen
Gunnar
G
Heegaard
Niels H H
NH
D000199:Actins / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry; D012044:Regression Analysis; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014809:Vitamin D-Binding Protein / Q000032:analysis* / Q000737:chemistry
D000199:Actins; D005456:Fluorescent Dyes; D020033:Protein Isoforms; D014809:Vitamin D-Binding Protein