Mechanism of protein binding to spherical polyelectrolyte brushes studied in situ using two-photon excitation fluorescence fluctuation spectroscopy.
We used two-photon excitation fluorescence fluctuation spectroscopy with photon counting histogram (PCH) analysis as a new tool to study the binding of globular proteins to colloidal particles in situ. Whereas fluorescence fluctuations are traditionally evaluated by calculating the autocorrelation function (fluorescence correlation spectroscopy), a complementary PCH analysis has been performed in this study which is advantageous when particle concentrations of a multicomponent system are of interest and the particles can be distinguished through particle brightness differences. The binding of two proteins, staphylococcal nuclease (SNase) and bovine serum albumin (BSA), to spherical polyelectrolyte brushes (SPB) was measured as a function of protein concentration and ionic strength of the solution at pH-values where SNase and BSA are positively and negatively charged, respectively. It has been found that SNase and BSA strongly bind to the SPB regardless of the protein charge. When the ionic strength of the solution is raised to 100 mM, the SPB become resistant to both proteins. These findings provide further evidence for a binding mechanism where the proteins are mainly driven to the SPB by the "counterion evaporation" force, while Coulomb interactions play a minor role. The results of this study characterize the potential of SPB as a new class of carrier particles for proteins whose use in biotechnological applications appears to be rewarding.
Physikalische Chemie I, Universität Dortmund, D-44221 Dortmund, Germany.
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C
Czeslik
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Jansen
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M
Ballauff
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Wittemann
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Royer
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Gratton
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Hazlett
D000180:Acrylic Resins / Q000737:chemistry*; D000327:Adsorption; D000465:Algorithms; D001665:Binding Sites; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D004573:Electrolytes / Q000737:chemistry*; D046911:Macromolecular Substances; D008836:Micrococcal Nuclease / Q000737:chemistry*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D043942:Nanotubes / Q000737:chemistry*; D011137:Polystyrenes / Q000737:chemistry*; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000737:chemistry*
D000180:Acrylic Resins; D020099:Coated Materials, Biocompatible; D004573:Electrolytes; D046911:Macromolecular Substances; D011137:Polystyrenes; D012710:Serum Albumin, Bovine; C006903:carbopol 940; D008836:Micrococcal Nuclease