Protein Adsorption Kinetics Drastically Altered by Repositioning a Single Charge

Abstract

A sterically conservative, neutralizing mutation (glutamic acid to glutamine) in either of two different positions (15 or 48) of the soluble core tryptic fragment of cytochrome b5 results in two proteins with vastly different adsorption propenies. The kinetics of adsorption were measured under well-defined hydrodynamic conditions on a variety of different surfaces, of controlled electrostatic potential. prepared by modifying planar optical waveguides. Repeated measurement of the guided mode spectrum in the presence of protein solution allowed the temporal evolution of the number of adsorbed molecules to be determined. A highly positively charged surface acted as a perfect sink. Le.. adsorption was only limited by transport, adsorption to a highly negatively charged surface was fully reversible, and adsorption to a neuual phospholipid bilayer was very slow and practically irreversible. The macroscopic adsorption behavior can in large part be interpreted in terms of molecular-scale interactions between the protein and the adsorbent surface.

Adsorption Behavior of Cytochrome c, Myoglobin and Hemoglobin in a Quartz Surface Probed Using Slab Optical Waveguide (SOWG) Spectroscopy

“…Through using polarized light in conventionally called TM (transverse magnetic) and TE (transverse electric) modes, and carefully measuring the incident angle, some researchers were able to quantify the adsorbed amount of protein on the waveguide. 30,38 This approach is particularly applicable to systems using a laser line source in the visible region.…”

“…2,3 The reported nomenclature in the literature include slab optical waveguide (SOWG) spectroscopy, 4-17 the integrated optical waveguideattenuated total reflection technique (IOW-ATR) [18][19][20][21][22][23][24][25][26][27] and optical waveguide lightmode spectroscopy (OWLS). 1,[28][29][30][31][32][33][34][35][36][37][38] These techniques have been successfully used in real-time studies on the adsorption of dye molecules 11,12,[14][15][16] and proteins 4,8,[18][19][20][21][22][23][24][25][28][29][30][31][32][33][34][35][36][37]…”

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

“…Ensemble adsorption isotherms, however, suggest the likelihood that protein and nucleic acid separations in ion-exchange columns may involve random ligand clustering (8)(9)(10). Additional support for such an assertion lies in the implementation of stationary phases of very high charge density by polymerization of charged monomers or layer-by-layer deposition (11)(12)(13), and in the demonstration that patches of high charge density on proteins often play a disproportionate role in their adsorption (4,6,(14)(15)(16)(17). In this work, we provide direct evidence of the importance of charge clustering in ion-exchange systems by direct observation of individual adsorption sites.…”

Molecular Engineering of Surfaces for Sensing and Detection

“…However, only electrostatic interaction was considered in their work; this corresponds to the situation when electrostatic interactions dominate in this work. Ramsden et al [31] studied the adsorption of two mutants of cytochrome b5. They found that E15Q adsorbs with its major axis perpendicular to the surface, while E48Q has a more flexible adsorption mode due to the larger dipole moment of E15Q.…”