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Electrophoresis and Electroosmosis in Aerolysin and Hemolysin Nanopores
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Edité par CCSD ; Biophysical Society -
Voltage-dependent interaction of macromolecules with biological nanopores can arise from electroosmotic (EOF) or electrophoretic (EPF) forces. Both poly(ethylene-glycol) (PEG), and cyclodextrins (β- and α-CD, respectively) block the ionic current through α-hemolysin (αHL) and Aerolysin (AeL) pore-forming proteins with well-documented voltage-dependence. Here, we attempt to relate differential effects of the electrolytes KCl and LiCl on the voltage-dependence of frequency and dwell-time of blocks to the relative contributions of EOF and EPF.For AeL, irrespective of electrolyte, blocks by α-CD only occurred from the pore's cis-side and with trans-positive voltages. Blocks in LiCl were much longer than those in KCl (11ms vs. 760μs at 90mV) but dwell times showed a positive, linear voltage dependence in both cases (LiCl:6fold/100mV; KCl:3fold/100mV). In KCl, cis-side PEG blocked AeL with trans-negative voltages only.1 Surprisingly, in LiCl, blocks occurred only at trans-positive voltages and at more than 1000 fold PEG-concentration but we still are in a very diluted regime. Blocks were shorter than in KCl by a factor of 5 (60 vs. 320μs at 100mV), but frequency (3fold/100mV) and dwell time (2fold/100mV) depended linearly on voltage, reminiscent of the behavior of α-CD in both electrolytes.Linear voltage dependence of block frequency might indicate dominance of EOF for pore entry of PEG in LiCl and for α-CD generally, while an exponential voltage-dependence would be expected for an electrophoretic force. Indeed, for PEG in KCl, which may be positively charged due to K+-chelation (see Ref.1 for discussion), we found an exponential increase in frequency for blocks of AeL and αHL with increasing trans-negative and trans-positive voltages, respectively. We cannot, however, exclude voltage and ion-dependent effects on the binding reaction, which, rather than partitioning, may be rate-limiting for the electrophysiologically detectable blockages.
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