PMC

Core-shell model of a hydrated spherical protein with hydrated radius R that is 1.3X larger than the packed amino-acid core with radius r_v , creating an excluded diameter 2R. The packed radius scales as MW^1/3 so that protein volume scales directly with MW. The hydration shell surrounding a polyelectrolyte protein is an osmolaric barrier to physical contact between two proteins.

Adsorbent capacity (D_i)^max of different silanized glass particles for HSA decreases monotonically with adsorbent hydrophilicity to detection limits (LOD) near the τ° = 30 mJ/m² (θ = 65°) pivot point (see Section 4.2 and , ). Lines through the data are guides to the eye.

Diffusion coefficient ratio as a function of molecular weight ratio follows the relationship predicted by the Stokes-Einstein-Sutherland equation (line through data) applied to a spherical model of proteins with good fidelity for the proteins listed in the table annotation. Proteins from different sources with different shape exhibit a spherical excluded volume in solution.

Adsorption isotherms for human serum albumin (HSA, 66kDa) plotting the solution depletion D_i against initial solution concentration (mg/mL) for five different silanized glass-particle adsorbents exhibiting 113°-60° advancing buffer contact angles θ_a obtained using the solution-depletion method (see ref. [] for details). The adsorbent capacity (D_i)^max and initial solution concentration at which the adsorbent was saturated by HSA (see star annotations) decreased monotonically with adsorbent hydrophilicity at fixed total surface area (see ). Lines through the data are guides to the eye.

Graphical illustration of the kinetics of single-protein adsorption (upper half figure) and binary-adsorption competition between i, j proteins for the same hydrophobic adsorbent (lower half figure). Essential steps depicted in both cases are: (A) instantaneous creation of a thin interface between adsorbent (Physical Surface) and protein solution (Bulk Solution); (B) rapid diffusion of proteins from solution into an inflating interphase region with concomitant displacement of interphase water; (C) reorganization and concentration of protein within an interphase that is shrinking by expulsion of either interphase water (upper half figure) or both interphase water and initially-adsorbed protein (lower half figure); and (D) attainment of steady-state interphase protein concentration by entrapment of initially-adsorbed protein in a minimal volume interphase.

Rendering of a hypothetical 50×50×50 nm cube of blood plasma showing distribution and relative sizes of spheres representing the 5 most abundant blood proteins. A much larger cube would be required to capture all of the 30 classical blood proteins. Numbers of molecules of each protein type that would fall within the cube are listed on the bottom right. Human serum albumin (HSA) greatly out numbers all other blood protein molecules; immunoglobulins type G (IgG), transferrin (Tr), Fibrinogen (Fib), and immunoglobulins type A (IgA).

Sequence of events leading to the adsorption of human serum albumin (HSA, gray spheres) to a liquid-air surface that interprets neutron reflectometry (NR) data (Section 4.8.2). Panel A is derived from by removing all other proteins from the hypothetical 50X50X50 nm cube of blood plasma. Panel B represents the instant of creating an air interface on top of Panel A but before any movement of HSA occurs. Panel C releases the stop-motion constraint allowing HSA molecules to diffuse into an inflating interphase, ultimately achieving the interphase concentration detected by NR. Calculations suggest that a packed hexagonal array of molecules is required to achieve NR concentration, as depicted in Panel C.

Free energy of dehydrating a surface ΔG_E as a function of surface site wettability measured by cosine of the advancing contact angle, cosθ_a. Wetting energy increases as surface sites become more hydrophilic (left-to-right on the abscissa), crossing a boundary near a θ_a = 65° nominal contact angle (cosθ_a = 0.42, dotted vertical line within the gray band) that differentiates hydrophobic from hydrophilic and where biological responses to materials pivot from high-to-low or low-to-high (see Section 4.2). The wetting energy corresponding to this pivot point (dotted horizontal line) defines hydrophilicity on an energetic basis: hydrophilic wetting expends > 1.3 kJ/mole-of-surface-sites whereas hydrophobic wetting expends < 1.3 kJ/mole-of-surface-sites (see vertical arrow annotations, left-hand axis). The dashed line annotation running diagonally along the ΔG_E trend emphasizes the linear-like increase in ΔG_E through the hydrophobic range of water wettability which increases sharply increases through the hydrophilic range.

Partitioning of a spherical protein into the interphase separating bulk solution from the physical surface of a biomaterial adsorbent. The thin unit area of surface a in contact with bulk solution is expanded to reveal a three-dimensional interphase containing two hypothetical protein layers occupying an interphase volume V_I at weight concentration W_I (mg/mL) adsorbed from bulk solution at concentration W_B. Curved arrows indicate that a protein partitioning into the interphase from bulk solution must displace a volume of interphase water equivalent to the volume of the hydrated protein because two objects cannot occupy the same space at the same time. The volume of displaced interphase water depends on the size of the protein (MW) and may involve hundreds-to-thousands of water molecules per adsorbed protein molecule. Steady-state is controlled by the partition coefficient .

Time-and-concentration interfacial tensions γ_lv (Panel A,B) and advancing contact angles θ_a (Panel C,D) for human serum albumin (fatty-acid free HSA) and prothrombin (blood factor FII), respectively. Panels A,B plot γ_lv as a function of logarithmic (natural) solution concentration C_B scaled as picomoles/L []. Panels C,D plot θ_a of FII solutions on a methyl-terminated SAM surface []. Panels A,C display results in 3D coordinates plotting γ_lv or θ_a as a function of concentration and analysis time (drop age). Panels B,D show only selected time slices taken from Panels A,C (Panel B: filled circle = 0.25 sec, open circle = 900 sec, filled triangles = 1800 sec, open triangles = 3594 sec. Panel D: filled circle = 0.25 sec, open circle = 900 sec, open triangles = 1800 sec, and open squares = 3594 sec). Notice that kinetics dominates early protein adsorption, requiring 30 min to 1 hr to reach steady state and that surface hydration (Panel D) slowly increases SAM surface wettability (vertical arrow annotation, Panel D).

A provocative representation of protein-adsorption theories positing that all such theories fall into one of two categories. The leftmost category collects all theories premised on the idea that adsorption is controlled by strong protein/surface interactions whereas the rightmost category collects all theories premised on the idea that the hydrophobic effect is the significant driving force underlying protein adsorption. Subscribers to the former category greatly outnumber the current subscription to the latter. Importantly, the left category has it that protein adsorption is complex at the molecular scale, requiring computations at the statistical-mechanics level to understand the essential biophysics of protein adsorption. By strong contrast, the right category has it that water (solvent) controls protein adsorption and, as a consequence, adsorption of blood proteins can be understood on a much more generic basis. Distinguishing between the two categories is important because most practical biomaterials problems are intractable at the molecular level. Group 1 type analytical methods (Section 3.1) reinforce adherence to the left category through a circular self-fulfilling prediction that protein adsorption is mediated by strong protein/surface interactions (see Section 4.5).

Apparent Gibbs’ surface excess scaled as a function of adsorbent surface water wettability (surface energy) as measured by the advancing contact angle θ_a of phosphate buffered saline (PBS) solution, expressed as water (buffer) adhesion tension τ° = γ_lv cosθ_a for incrementally sampling the full range of observable water wettability (where buffer interfacial tension γ_lv = 71.97 mJ/m²; SiO_x = oxidized silicon semi-conductor wafer, APTES = aminopropyltriethoxysilane silanized SiO_x, PS = polystyrene spun-coated onto SiO_x, SAM = 1-hexadecanethiol self-assembled monolayer on gold-coated SiO_x). Symbols and error bars represent mean and standard deviation of ten different proteins spanning three orders of MW (ubiquitin, 10.7 kDa; thrombin (FIIa), 35.6 kDa; FV HSA, 66.3 kDa; Hageman factor (FXII), 78 kDa; fibrinogen, 340 kDa; IgG, 160 kDa; C1q, 400 kDa; IgM, 1000 kDa). See ref. [] for details. Panel A shows that Gibbs’ surface-excess parameter [Γ_sl − Γ_sv] decreases monotonically with increasing adsorbent-surface hydrophilicity, projecting [Γ_sl − Γ_sv] = 0 near the τ° = 30 mJ/m² pivot point (θ = 65°, see Section 4.2 and ). Likewise, the ratio decreases from +1 to −1 (Panel B) as [Γ_sl − Γ_sv] decreases from a maximum [Γ_sl − Γ_sv] = −Γ_lv at the liquid-vapor (lv) interface and hydrophobic SAM surface (τ° = −15 mJ/m²) to a minimum [Γ_sl − Γ_sv] = −Γ_lv at the water-wetted (τ° → 73 mJ/m² surfaces. Smoothed curves drawn through the data are guides to the eye.