Electrostatic Interactions Influence Protein Adsorption (but Not

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Electrostatic Interactions Influence Protein Adsorption – but not Desorption – at the Silica-Aqueous Interface Aaron C. McUmber, Theodore W. Randolph, and Daniel K. Schwartz J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.5b00933 • Publication Date (Web): 16 Jun 2015 Downloaded from http://pubs.acs.org on June 17, 2015

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The Journal of Physical Chemistry Letters

Electrostatic Interactions Influence Protein Adsorption – but not Desorption – at the Silica-Aqueous Interface

Aaron C. McUmber, Theodore W. Randolph, and Daniel K. Schwartz* Department of Chemical and Biological Engineering University of Colorado Boulder, Boulder, CO 80309

*To whom correspondence should be addressed: [email protected]

1 ACS Paragon Plus Environment


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Abstract High-throughput single-molecule total internal reflection fluorescent microscopy was used to investigate the effects of pH and ionic strength on bovine serum albumin (BSA) adsorption, desorption, and interfacial diffusion at the aqueous – fused silica interface. At high pH and low ionic strength, negatively-charged BSA adsorbed slowly to the negatively-charged fused silica surface. At low pH and low ionic strength, where BSA was positively charged, or in solutions at higher ionic strength, adsorption was approximately 1,000 times faster. Interestingly, neither surface residence times nor the interfacial diffusion coefficients of BSA were influenced by pH or ionic strength. These findings suggested that adsorption kinetics were dominated by energy barriers associated with electrostatic interactions, but once adsorbed, protein-surface interactions were dominated by short-range non-electrostatic interactions. These results highlight the ability of single-molecule techniques to isolate elementary processes (e.g. adsorption and desorption) under steady state conditions, which would be impossible to measure using ensemble-averaging methods.

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Keywords: Electrostatic, hydrophobic, van der Waals interactions, DLVO, TIRF, single molecule



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Introduction The dynamic behavior of proteins at solid-liquid interfaces is critically important to various areas of biotechnology and biomaterials,1 including biopharmaceuticals,2 vaccines,3 bioseparations,4 and biosensing.5 Silica (i.e., glass) surfaces are particularly relevant to pharmaceutical, chromatographic and sensing technologies. Due to their complex molecular structure, proteins interact with surfaces via a combination of long-range (e.g. electrostatic) and short-range noncovalent (van der Waals, hydrophobic, hydrogen bonding) interactions.6 As the only interaction that can be repulsive, electrostatic effects have received particular attention, since they can be controlled (using pH and ionic strength) and exploited to stabilize protein solutions against aggregation7,8 or to reduce the rate of interfacial adsorption.6,9 While many models have been developed to describe the combined effects of surface-protein interactions,1,10,11 the typical experiments (surface plasmon resonance, quartz crystal microbalance, etc.) that are used to study dynamic behavior of proteins at interfaces rely on ensemble-averaging, and thus measure the net effect of many elementary processes (adsorption, desorption, surface-mediated protein associations, conformational changes, etc.), making it difficult to isolate the influence of specific environmental conditions on each elementary process. Even the apparently simple process of adsorption is difficult to measure directly, since rates determined using transient ensembleaverage measurements actually reflect a net adsorption rate involving both elementary adsorption and desorption rates (the latter of which depends on surface coverage). While these elementary rates can be extracted by assuming specific theoretical kinetic models,12 it is desirable to measure the elementary rates directly, preferably under steady-state conditions. Single-molecule total internal reflection fluorescence microscopy (SM-TIRFM) provides this capability, permitting direct observations of individual molecular processes such as adsorption, interfacial diffusion,


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and desorption.1,13 In particular, by separating net adsorption into its elementary components – adsorption and desorption – the effects of electrostatic interactions (a long-range interaction) can be disentangled from those of short-range interactions.

Proteins are formed from diverse amino acids. Their topologically complex surfaces are composed of amino acid residues that may be charged (positively or negatively), uncharged but polar, or hydrophobic.14,15 Proteins often display multiple positively and negatively charged surface patches. Since these charged groups are acidic or basic, they may be neutralized depending on the local environment and the pH of the environment. To first order, the overall electrostatic character of a protein can be inferred by measuring the isoelectric point (pI), which is the pH at which the protein’s net charge is equal to zero.15 In solutions with pH that is above the pI, protein molecules exhibit a net negative charge, while the net charge is positive below the pI. However, buffer choice, salt concentration, and protein composition all affect the actual magnitude of the charge at a given pH.15

Using SM-TIRFM, we investigated the interfacial dynamics of bovine serum albumin (BSA) at the aqueous – fused silica (FS) interface as a function of pH and ionic strength. Under these conditions we measured adsorption rates, residence times, and interfacial diffusivity of large numbers of individual molecules for each experimental condition. Under the conditions of these experiments, the protein and surface had opposite charges at low pH and like charges at high pH, so we expected to measure the effects of electrostatic repulsion and attraction on molecular adsorption, which should be sensitive to long-range protein-surface interactions. By comparing the dynamics as a function of ionic strength, the influence of charge-screening could be



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characterized. Moreover, these experiments enabled us to explore the influence of electrostatic interactions on desorption and interfacial diffusion, phenomena that are dominated by the shortrange interactions of proteins and surfaces in intimate contact.

Results and Discussion Steady-state adsorption rate coefficients, kads, for fluorescently-labeled BSA were calculated from single-molecule TIRFM. These adsorption rate constants represented the absolute adsorption rate, as opposed to the net adsorption rate, which is uniquely measured using singlemolecule observations. At pH values below 5, when the protein and surface had opposite net charges, fast adsorption was observed, with kads > 1000 nm/s (Figure 1). Intuitively, an adsorption rate coefficient of 1000 nm/s implies that the number of solute molecules in a 1000 nm thick slab of solution adsorb the surface each second. Similar values of kads were observed previously for a fluorescently labeled fatty acid on a hydrophobic surface at both low and high ionic strength,9 suggesting that this order of magnitude of kads reflects conditions where strong repulsion is absent. At pH values above 4.7, where both the surface and protein were expected to exhibit negative net charges, kads decreased sharply by several orders of magnitude, reaching a value of 2.9±0.3nm/s at pH=7.4 (Figure 1). Under conditions of high ionic strength (100 mM NaCl was added to the solution), as indicated by the open symbols in Figure 1, kads at pH=2.6 remained similar to the values measured under low ionic strength conditions. However, in solutions with pH=7.4, kads increased almost 1000 fold, to 2000±200 nm/s, compared to the kads measured at low ionic strength.


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Figure 1: Adsorption rate coefficients of BSA at FS surface as a function of pH in 10 mM CP (closed circles) and 10 mM CP with 100 mM NaCl (open diamonds) obtained from single-molecule adsorption observations made using TIRF. Error bars in plot represent the standard deviation between three replicate experiments.

The isoelectric point (pI) of unlabeled BSA is at 4.715 and the pI of fluorescently-labeled BSA can be assumed to be very similar, particularly given the resolution of pH values explored in this work. Changes in pI have shown to be minimal when BSA is fluorescently-labeled at the labeling efficiency used within this study, showing a decrease in pI by only ~0.1 pH units.16 In solutions with pH < pI, the net charge of BSA is expected to be positive, whereas the net charge of BSA is expected to be negative for pH>pI.15 The surface charge of FS is expected to be negatively charged above pH≈2,17 i.e. for all the conditions explored here. Under the low ionic strength conditions associated with 10 mM citric acid – sodium phosphate buffer (CP), the effective Debye length is ~2.5 nm, and strong electrostatic interactions are expected between BSA and FS. In particular, for pH>pI, DLVO theory suggests that electrostatic repulsion provides a strong energetic barrier to adsorption, overwhelming the short-range van der Waals attraction that becomes dominant at close contact, resulting in a barrier to adsorption of roughly 10–30



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kT.18 This is a reasonable explanation for the dramatic decrease in the adsorption rate at high pH and have been observed in net adsorption rate measurements.19 However, under the higher ionic strength conditions associated with the addition of 100 mM NaCl, the Debye length for the system decreased to 0.65 nm, and electrostatic interactions between BSA and FS are therefore greatly weakened relative to van der Waals attraction, resulting in a much lower barrier to adsorption on the order of kT. As a result, under high ionic strength conditions, the adsorption rate remained high even for pH>pI, when surface and protein had like charges. These observations were consistent with an adsorption rate that was dominated by electrostatic interactions within the context of electrical double-layer (DLVO) theory.20

Interestingly, for pH

Electrostatic Interactions Influence Protein Adsorption (but Not

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