Expanding the scope of CE reactor to ssDNA-binding protein-ssDNA complexes as exemplified for a tool for direct measurement of dissociation kinetics of biomolecular complexes.
CE reactor (CER), which was developed as a tool for direct measurement of the dissociation kinetics of metal complexes, was successfully applied to the complexes of Escherichia coli ssDNA-binding protein (SSB) with ssDNA. The basic concept of CER is the application of CE separation process as a dissociation kinetic reactor for the complex, and the observation of the on-capillary dissociation reaction profile of the complex as the decrease of the peak height of the complex with increase of the migration time. The peak height of [SSB-ssDNA] decreases as the migration time increases since the degree of the decrease of [SSB-ssDNA] through the on-capillary dissociation reaction is proportional to the degree of the decrease of the peak height of [SSB-ssDNA]. The dissociation degree-time profiles for the complexes are quantitatively described by analyzing a set of electropherograms with different migration times. Dissociation rate constants of [SSB-ssDNA] consisting of 20-mer, 25-mer and 31-mer ssDNA were directly determined to be 3.99x10(-4), 4.82x10(-4) and 1.50x10(-3)/s, respectively. CER is a concise and effective tool for dissociation kinetic analysis of biomolecular complexes.
Takahashi
Toru
T
Division of Environmentally Benign Systems, Graduate School of Environmental Studies, Tohoku University, Sendai, Japan.
Ohtsuka
Kei-Ichirou
KI
Division of Environmentally Benign Systems, Graduate School of Environmental Studies, Tohoku University, Sendai, Japan.
Tomiya
Yoriyuki
Y
Division of Environmentally Benign Systems, Graduate School of Environmental Studies, Tohoku University, Sendai, Japan.
Iki
Nobuhiko
N
Division of Environmentally Benign Systems, Graduate School of Environmental Studies, Tohoku University, Sendai, Japan.
Hoshino
Hitoshi
H
Division of Environmentally Benign Systems, Graduate School of Environmental Studies, Tohoku University, Sendai, Japan.
D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances
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