Protein adsorption equilibrium

Modeling of Protein Adsorption Equilibrium at Hydrophobic Solid-Water Interfaces... 

The useful result of a dimensional analysis of protein adsorption equilibrium would be a starting point for the determination of a functional relationship among the variables thought to be pertinent to the process. The relationship can he expressed in the following compact form ... 

In general, the pattern of protein adsorption equilibrium isotherms at solid-liquid interfaces assumes either a Langmuir-type or Freundlich-type shape. The Langmuir-type model is described by... 

The affinity of a protein in single-component solution for a given surface can be characterized by the affinity (or equilibrium) constant, K, given by Eq. (30). This equilibrium constant connotes the total driving force for the process of adsorption. Not only does it constitute the driving force for arrival to the surface but also for surface unfolding, which differentiates protein adsorption equilibrium from that of small molecules. 

As a technique for selective surface illumination at liquid/solid interfaces, TIRF was first introduced by Hirschfeld(1) in 1965. Other important early applications were pioneered by Harrick and Loeb(2) in 1973 for detecting fluorescence from a surface coated with dansyl-labeled bovine serum allbumin, by Kronick and Little(3) in 1975 for measuring the equilibrium constant between soluble fluorescent-labeled antibodies and surface-immobilized antigens, and by Watkins and Robertson(4) in 1977 for measuring kinetics of protein adsorption following a concentration jump. Previous rcvicws(5 7) contain additional references to some important early work. Section 7.5 presents a literature review of recent work. 

By preparing planar lipid monolayers or bilayers on hydrophobically derivatized or native hydrophilic glass, respectively, the adsorption equilibrium constants of a blood coagulation cascade protein, prothrombin, have been examined by TIRF on a surface that more closely models actual cell surfaces and is amenable to alterations of surface charge. It was found that membranes of phosphatidylcholine (PC) that contain some phosphatidyl-serine (PS) bind prothrombin more strongly than pure PC membranes/83... 

Since protein adsorption to an anion exchange resin is reversible and does not constitute a classical immobilization, the ability of the resins to retain activity under various conditions must be determined. Macrosorb KAX DEAE bound -D-glucosidase was tested with solutions of primary interest for their final application. Several batch washes of a 1% w/v slurry were required to ensure complete equilibrium elution for a given concentration, as determined from the absence of pNPG units in subsequent washes. Several salt solutions of typical fermentation media components were tested. These included 3 mM to 50 mM solutions of MgSO, KHgPO, NaQ, and sodium acetate. Also, incubations with cellulase solutions were tested to determine if the proteins present in a cellulose hydrolysis would displace the -D-glucosidase. Both of these displacement studies were carried out at 22°C and 40 C. 

Peppas et al. [16] presented a new method for calculating protein adsorption on polymeric surfaces. In their model, protein adsorption is regarded as an equilibrium reaction, which takes place on the polymer surface in competition with the adsorption of water. 

There has been considerable effort on the prediction of secondary and tertiary structures of protein from the amino acid sequence using computeraided minimal potential energy calculations8). The question as to how a primary amino acid sequence begins to produce secondary and super-secondary structures and fold into its equilibrium tertiary structure and functional domains is a very active field of structural biochemistry. A related problem is the mechanism by which a protein unfolds or denatures 20) which is of fundamental interest in the protein adsorption process. 

In TIRF protein adsorption experiments, it is desirable to correlate the intensity of excited fluorescence with excess protein concentration at the interface. Such an adsorbed layer is often in equilibrium with bulk-nonadsorbed protein molecules which are also situated inside the evanescent volume and thus contributing to the overall fluorescence. Various calibration schemes were proposed, using external nonadsorbing standards40,154 , internal standard in a form of protein solution together with a type of evanescent energy distribution calculation 154), and independent calibration of protein surface excess 155). Once the collected fluorescence intensity is correlated with the amount of adsorbed protein, TIRF can be applied in the study of various interactions between surface and protein. 

Comparison of equilibrium adsorption (Figures 4 and 5) and minute protein adsorption/flocculation as a function of protein concentration, Cp, demonstrates strong but variable effects of pH and salinity.4 The equilibrium adsorption of proteins is as large as 1 mg/m2 (or 300 mg/g) at pH 3.5 (i.e. between pH(IEP) of silica and proteins) for bovine serum albumin (BSA) with 0.9 wt.% NaCl and gelatin without NaCl, or at pH(IEP) of protein for ovalbumin without NaCl. The lowest equilibrium adsorption (0.1-0.2 mg/m2) is typically observed at pH = 2, which is close to pH(IEPS o2) 2.2, and without NaCl (Figure 4). It should be... 

A useful literature relating to polypeptide and protein adsorption kinetics and equilibrium behavior in finite bath systems for both affinity and ion-ex-change HPLC sorbents is now available160,169,171-174,228,234 319 323 402"405 and various mathematical models have been developed, incorporating data on the adsorption behavior of proteins in a finite bath.8,160 167-169 171-174 400 403-405 406 One such model, the so-called combined-batch adsorption model (BAMcomb), initially developed for nonporous particles, takes into account the dynamic adsorption behavior of polypeptides and proteins in a finite bath. Due to the absence of pore diffusion, analytical solutions for nonporous HPLC sorbents can be readily developed using this model and its two simplified cases, and the effects of both surface interaction and film mass transfer can be independently addressed. Based on this knowledge, extension of the BAMcomb approach to porous sorbents in bath systems, and subsequently to packed-, expanded-, and fluidized-bed systems, can then be achieved. 

The average dimensionless solid phase concentration Y can be given409 the numerical value of 0.5, and if the effect of the film mass transfer is negligible, i.e., if K f - 00, then adsorption with the nonporous HPLC sorbent in a well-packed bed is controlled by second-order kinetics.169,399 When the external film resistance Kf controls the adsorption, equilibrium is assumed to exist between the polypeptide or protein and the polypeptide- or protein-ligate complex at each point on the particle surface. 

The effect of salt on the equilibrium and kinetics of protein adsorption on (hydrophobic) butylated surfaces has also been investigated. It was found that increasing the concentration from O.IM to l.OM results in a drop in the rate constant of an order of magnitude and a drop in the degree of adsorption. This again may be explained in terms of a reduction in the hydrophobic attraction between the surface and hydrophobic sites on the proteins. 

The question of whether protein adsorption can be considered as an equilibrium phenomenon has been controversial. The reasons that have led to a widespread belief in the irreversibility of protein adsorption may be summarized as follows. 

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