Analysis of the binding of deoxyribonuclease I to G-actin by capillary electrophoresis.
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 microM 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.
Carter
L K
LK
Institute for Biomedical Research, and Department of Anatomy and Histology, The University of Sydney, New South Wales, Australia. laura@anatomy.su.oz.au
Christopherson
R I
RI
dos Remedios
C G
CG
D000199:Actins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D007834:Lasers; D012822:Silicon Dioxide / Q000737:chemistry
D000199:Actins; D012822:Silicon Dioxide; D003850:Deoxyribonuclease I