Analysis of the binding of deoxyribonuclease I to G-actin by capillary electrophoresis
- 1 January 1997
- journal article
- research article
- Published by Wiley in Electrophoresis
- Vol. 18 (7), 1054-1058
- https://doi.org/10.1002/elps.1150180704
Abstract
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 (Kd 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 Kd value of approximately 0.03 μM was obtained by simulation of binding data. We conclude that CZE combined with a LIF detector has the capacity to extend the determination of Kd values from the micromolar range to the nanomolar range. Subsequent determination of Kd values for other actin-binding proteins should provide information on interactions between the binding sites on actin for these proteins and their spatial relationship.Keywords
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