You are performing an in vitro experiment in which you will expose a material yo

Question

You are performing an in vitro experiment in which you will expose a material you are considering for a medical device to synovial fluid, which contains the proteins albumin, transferrin, and IgM at concentrations of 5,0.5, and 0.05 mg/ml, respectively. Each of these components has a particular affinity for your material, with IgM being the highest and albumin being the lowest. Describe the kinetics of protein adsorption to your material and how the surface concentration of each protein will change with time.

Explanation / Answer

A widely-held belief within the biomaterials community is that adsorbed protein catalyzes, mediates, or moderates the biological response to artificial materials [1–10]. In light of this seemingly incontrovertible fact, it is self evident that responsible proposition of evidence-based biochemical mechanisms for the biological response to materials requires quantitative knowledge of the exact composition of protein that becomes adsorbed to biomaterials in contact with complex biological mixtures such as blood. This composition must be known both in terms of protein identity and concentration. It also seems self evident that prospective materials engineering for various medical applications requires detailed knowledge that relates biomaterial properties to protein adsorption. Otherwise, structure-property relationships cannot be formulated, leaving biomaterials engineering dependent on design-directed or trial-and-error approaches [11]. Thus, the entirety of biomaterials surface science seems critically dependent on a thorough understanding of protein adsorption. Our work in protein adsorption is aimed at experimental resolution of some fundamental aspects of protein adsorption, motivated by the expectation that clarification of the physical chemistry of adsorption will ultimately lead to a predictive basis for cardiovascular biomaterials design. These fundamentals include the reversibility/irreversibility of protein adsorption, mechanism of the so-called Vroman effect [1, 17, 20, 24–43], capacity of proteins to adsorb in multilayers [6, 38, 44–60], energetics of protein adsorption [6, 14, 22, 43, 45, 49–51, 61–63], and the applicability of thermodynamic/computational models [47]. In particular, we have focused on obtaining energy [6, 14, 22, 43, 50, 51, 61–63] and mass [44, 45, 47] balance for the adsorption of a wide variety of purified blood proteins to a broad span of materials with different surface chemistry/energy. Results of this survey have been illuminating (see brief review in the introduction of ref. [45]) and strongly implicate a controlling role for water in the adsorption process. In very brief summary, experimental results have led us to adopt the classical Guggenheim interphase model of the surface region [64, 65] and interpret protein adsorption as a partitioning between bulk solution and a three-dimensional (3D) interphase region that separates bulk solution from the physical adsorbent surface. The 3D interphase paradigm is hardly new to surface science but has not been widely applied in the study of protein adsorption, or to the general problem of adsorption for that matter. Significant advantages of the interphase paradigm are retention of the concept of chemical activities (concentrations) essential for a complete understanding of adsorption energetics and consistency with standard surface thermodynamics [48]. Importantly, the interphase model easily accommodates multilayer-protein adsorption that has been shown to occur by a number of investigators using a variety of experimental methods over the last twenty years or so [6, 38, 49–60]. This so-called ‘volumetric interpretation’ of adsorption has it that proteins are expelled from aqueous solution to the interphase by what amounts to be the hydrophobic effect. Expelled protein can partition into the interphase if-and-only-if it is energetically favorable to displace a volume of interphase water equal to that of the adsorbing hydrated protein [51]. In this way, water-wettability substantially controls adsorption because it requires more energy to displace water from the interphase of relatively hydrophilic surfaces than from the interphase of more hydrophobic counterparts [45]. Efforts to understand competition between two proteins adsorbing from mixed solution [43, 46] based on this overarching mechanism have been less satisfactory because, for reasons detailed herein, we have failed to fully appreciate the role that adsorption kinetics plays in protein-adsorption selectivity. It has been widely (but not universally) assumed that protein adsorption occurs over a relatively long timeframe that is directly related to tens-of-minutes-to-hours change in interfacial energetics observed when a protein solution is brought into contact with a surface [6, 14, 22, 43, 50, 51, 61]. Furthermore, it is popularly held that selective adsorption of certain proteins from a mixture is due, at least in part, to a time-dependent adsorption-displacement phenomenon involving proteins engaged in adsorption competition (a.k.a Vroman Effect). Recent work shows that the former perspective is substantially incorrect for concentrated (mg/mL) protein solutions [48, 66]. This finding, in turn, suggests that the interpretation of the Vroman effect is also problematic. A more correct view seems to be that protein molecules rapidly diffuse into an inflating interphase volume that is initially formed upon contact of an adsorbent with concentrated protein solution [48]. Through this process, the interphase captures a fixed mass of protein in proportion to solution concentration. Mass transport from concentrated solution is effectively complete within seconds, if not milliseconds, but certainly well before the tens-of-minutes-to-hours associated with interfacial energetic changes. Indeed, protein-adsorption kinetics are difficult to follow unless solution concentrations are quite low in the µg/mL range. This initially-formed interphase slowly shrinks in volume by efflux of interphase water, causing interphase protein concentrations to increase and interfacial tensions to concomitantly decrease over the aforementioned tens-of-minutes-to-hours time scale. Unfolding of proteins (changing occupied volume, denaturation) at a surface [67] can also lead to concentration of protein within the interphase region. Curious to understand ramifications of the above-described adsorption kinetics on protein-adsorption competition from mixed-protein solutions, we adapted the standard solution-depletion method to measure adsorption competition [46] between two proteins as a function of time, ranging from 5 minutes to 90 minutes and, in one test case, 24 hours. Herein we show that adsorption competition is very rapid, leading to unanticipated selectivity that persists long after the initial burst of protein arriving at the adsorbent surface. This outcome has significant ramifications in understanding both protein adsorption and how protein adsorption controls the biological response to materials. Changes in time: Conformational changes of two types of proteins, water-soluble proteins (BSA and myoglobin) and membrane-associated protein (cytochrome c), induced by a negatively charged supported planar phospholipid monolayer were studied. The water-soluble proteins lost most of their ordered secondary structure and formed a random conformation in the initial stage of adsorption, and but in the later stage they retained more of the a-helical structure which was their main native secondary structure in solution. The membrane-associated protein, cytochrome c, showed a different conformational change in the adsorption process. In the initial stage, it showed an increase in ß-structures but not random coils. In the later stage, it contained more a-helixes than that in solution.

Related Questions

Navigate

• Browse (All)
• Browse Y
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.