Effect of surface wettability on the adhesion of proteins

Besides significantly broadening the scope of available data on adhesion of proteins on solid substrates, we demonstrate for the first time that all seven proteins (tested here) behave similarly with respect to adhesion exhibiting a step increase in adhesion as wettability of the solid substrate decreases. Also, quantitative measures of like-protein-protein and like-self-assembled-monolayer (SAM)-SAM adhesive energies are provided. New correlations, not previously reported, suggest that the helix and random content (as measures of secondary structure) normalized by the molecular weight of a protein are significant for predicting protein adhesion and are likely related to protein stability at interfaces. Atomic force microscopy (AFM) was used to directly measure the normalized adhesion or pull-off forces between a set of seven globular proteins and a series of eight well-defined model surfaces (SAMs), between like-SAM-immobilized surfaces and between like-protein-immobilized surfaces in phosphate buffer solution (pH 7.4). Normalized force-distance curves between SAMs (alkanethiolates deposited on gold terminated with functional uncharged groups -CH3, -OPh, -CF3, -CN, -OCH3, -OH, -CONH2, and -EG3OH) covalently attached to an AFM cantilever tip modified with a sphere and covalently immobilized proteins (ribonuclease A, lysozyme, bovine serum albumin, immunoglobulin, gamma-globulins, pyruvate kinase, and fibrinogen) clearly illustrate the differences in adhesion between these surfaces and proteins. The adhesion of proteins with uncharged SAMs showed a general "step" dependence on the wettability of the surface as determined by the water contact angle under cyclooctane (thetaco). Thus, for SAMs with thetaco < approximately 66 degrees, (-OH, -CONH2, and -EG3OH), weak adhesion was observed (>-4 +/- 1 mN/m), while for approximately 66 < thetaco < approximately 104 degrees, (-CH3, -OPh, -CF3, -CN, -OCH3), strong adhesion was observed (< or =8 +/- 3 mN/m) that increases (more negative) with the molecular weight of the protein. Large proteins (170-340 kDa), in contrast to small proteins (14 kDa), exhibit characteristic stepwise decompression curves extending to large separation distances (hundreds of nanometers). With respect to like-SAM surfaces, there exists a very strong adhesive (attractive) interaction between the apolar SAM surfaces and weak interactive energy between the polar SAM surfaces. Because the polar surfaces can form hydrogen bonds with water molecules and the apolar surfaces cannot, these measurements provide a quantitative measure of the so-called mean hydrophobic interaction (approximately -206 +/- 8 mN/m) in phosphate-buffered saline at 296 +/- 1 K. Regarding protein-protein interactions, small globular proteins (lysozyme and ribonuclease A) have the least self-adhesion force, indicating robust conformation of the proteins on the surface. Intermediate to large proteins (BSA and pyruvate kinase-tetramer) show measurable adhesion and suggest unfolding (mechanical denaturation) during retraction of the protein-covered substrate from the protein-covered AFM tip. Fibrinogen shows the greatest adhesion of 20.4 +/- 2 mN/m. Unexpectedly, immunoglobulin G (IgG) and gamma-globulins exhibited very little adhesion for intermediate size proteins. However, using a new composite index, n (the product of the percent helix plus random content times relative molecular weight as a fraction of the largest protein in the set, Fib), to correlate the normalized adhesion force, IgG and gamma-globulins do not behave abnormally as a result of their relatively low helix and random (or high sheet) content.