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Analysis of the binding of deoxyribonuclease I to G‐actin by capillary electrophoresis
Author(s) -
Carter Laura K.,
Christopherson Richard I.,
dos Remedios Cristobal G.
Publication year - 1997
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.1150180704
Subject(s) - capillary electrophoresis , chemistry , deoxyribonuclease i , deoxyribonuclease , fluorescence , actin , affinity electrophoresis , monomer , dissociation constant , capillary action , polymerization , chromatography , biophysics , analytical chemistry (journal) , polymer , biochemistry , dna , affinity chromatography , materials science , enzyme , biology , organic chemistry , physics , receptor , quantum mechanics , composite material , base sequence
Deoxyribonuclease I (DNase I) is an actin monomer‐sequestering actin binding protein (ABP) that inhibits the rate and extent of actin polymerisation in vitro by forming a high affinity, stoichiometric 1:1 complex. Using capillary zone electrophoresis (CZE), we have studied the interaction between G‐actin and DNase I to evaluate the capability of CZE to determine the dissociation constant ( K d value) for this interaction. We used (i) an uncoated fused‐silica capillary and ultraviolet (UV) detection at 214 nm; (ii) a hydrophilic‐coated capillary with UV detection at 214 nm; and (iii) a hydrophilic‐coated capillary with laser‐induced fluorescence (LIF) detection. Using procedure (ii), a K d value of approximately 0.03 μ M was obtained by simulation of binding data. We conclude that CZE combined with a LIF detector has the capacity to extend the determination of K d values from the micromolar range to the nanomolar range. Subsequent determination of K d values for other actin‐binding proteins should provide information on interactions between the binding sites on actin for these proteins and their spatial relationship.