Superhydrophobic effect on the adsorption of human serum albumin

Analytical protocol greatly influences the measurement of human serum albumin (HSA) adsorption to commercial expanded polytetrafluororethylene (ePTFE) exhibiting superhydrophobic wetting properties. Degassing of buffer solutions and evacuation of ePTFE adsorbent to remove trapped air immediately prior to contact with protein solutions are shown to be essential. Results obtained with ePTFE as a prototypical superhydrophobic test material suggest that vacuum degassing should be applied in the measurement of protein adsorption to any surface exhibiting superhydrophobicity. Solution depletion quantified using radiometry ((125)I-labeled HSA) or electrophoresis yield different measures of adsorption, with nearly 4-fold higher surface concentrations of unlabeled HSA measured by the electrophoresis method. This outcome is attributed to the influence of the radiolabel on HSA hydrophilicity which decreases radiolabeled-HSA affinity for a hydrophobic adsorbent in comparison to unlabeled HSA. These results indicate that radiometry underestimates the actual amount of protein adsorbed to a particular material. Removal of radiolabeled HSA adsorbed to ePTFE by 3x serial buffer rinses also shows that the remaining "bound fraction" was about 35% lower than the amount measured by radiometric depletion. This observation implies that measurement of protein bound after surface rinsing significantly underestimates the actual amount of protein concentrated by adsorption into the surface region of a protein-contacting material.