Protein adsorption reversibility

Owing to the weak hydrophobicity of the PEO stationary phases and reversibility of the protein adsorption, some advantages of these columns could be expected for the isolation of labile and high-molecular weight biopolymers. Miller et al. [61] found that labile mitochondrial matrix enzymes — ornitine trans-carbomoylase and carbomoyl phosphate synthetase (M = 165 kDa) could be efficiently isolated by means of hydrophobic interaction chromatography from the crude extract. 

Norde, W. and C.A. Haynes. 1995. Reversibility and the mechanism of protein adsorption. In Proteins at Interfaces II Fundamentals and Applications. T.A. Hor-bett and J.L. Brash, editors. American Chemical Society, Washington, D.C., 26 40. 

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. 

In contrast, on the surface of the amino-containing polymeric materials, protonated amino groups introduced in a small proportion under physiological conditions, destroy their surrounding hydrogen bonds to produce, here and there, gaps in the network [127, 128]. Thus, the network structures are considered to become more or less unstable. As a consequence, the residence time of protein molecules trapped by these defective networks will be shorter than in the case of polyHEMA or cellulose. On the surface of these amino-containing materials, reversible protein adsorption and desorption, and also replacement (Vroman effect) - or even protein rejection - will become possible. 

Fig. 12. Hypothetical 3-D plot of protein adsorption isotherm (D plotted against [P]B) as a function of surface ligand concentration, [A]s. Note that the system is reversible only up to a critical [A]s and then behaves irreversibly for higher ligand surface concentrations. The right arrows (— ) denote adsorption the left-facing ones ( -), desorption...

Fig. 20. Schematic adsorption isotherms with a constant surface site concentration ([A]s in Fig. 12 is here constant), but with adsorption time as a variable. At very short times, adsorption is diffusion controlled. At short times, the protein has insufficient time to conformationally adjust to the interface, thus adsorption can be reversible and of the Langmuir type. At longer times, conformational adjustments begin leading to the commonly observed semi- orir-reversible behavior of protein adsorption. Other nomenclature same as Fig. 12...

Rejection of protein adsorption to the outermost grafted surface is attributed to a steric hinderance effect due to the tethered chains. A grafted surface in contact with an aqueous medium, a good solvent of the chains, has been identified to have a diffuse structure [57,151,152]. Reversible deformation of the tethered... 

It is important to note that proteins tend to denature during such an adsorption process on noble metals or carbon electrodes. In addition, the stability of the adsorbed sensing layer is highly dependent on the pH value and ionic strength of the solution as well as the temperature, the electrode material, and other additional factors. For instance, as early as 1972 direct ET was observed on mercury electrodes employing cytochrome c as redox protein [108]. Reversible electrochemical behavior of cytochrome c was not observed because the protein denatured on the surface. 

The surface is divided into areas of irreversible binding sites (POPS-enriched) and reversible binding sites (POPC-enriched). Protein adsorption takes place on both areas, however, at different rates. Protein desorption is allowed only from the reversible binding sites. Mass transport of the proteins to the surface is considered by using a mean field ansatz of a stagnation point flow in accordance with the experimental setup. The kinetics of reversible... 

By variation of the rate constants fcon> koff> and hrr, the course of the simulated frequency shifts can be fitted to the QCM results for a given munber and area of domains. The reversible adsorption rate constant (fcon) governs the frequency difference before and after rinsing with protein-free buffer, whereas the desorption rate constant (fcotr) is solely responsible for the time course after rinsing, fcoff can be approximated independently from the QCM data by fitting a monoexponential function to the time course after the wash. The final frequency shift after buffer rinsing is exclusively determined by irreversible protein adsorption on the POPS-enriched domains (kirr)-... 

Early experiments of MacRitchie Alexander (1963) showed that the adsorption behaviour of different proteins can be described by a simple reaction relation given by Eq. (4.80). Important investigation of the reversibility of protein adsorption... 

It is likely that experimentally found values of molecular cross-sectional areas do not correspond to the equilibrium states of the protein layers but reflect only the transition state configuration, as assumed by MacRitchie [16] and depicted in Fig. 1. The problem of adsorption reversibility is basic for understanding the protein behavior at interfaces. The belief in the protein adsorption irreversibility is mainly based on drastic conformational changes in interfacial film and the great difficulty of desorbing a protein from this film [15], However, these criteria are not always a proof of irreversibility. It was shown in many cases [3,24,39-41] that proteins can be desorbed... 

Data on the reversibility of the protein adsorption process and thus the applicability of different models for its description are contradictory. Norde et al. [17] consider protein adsorption as a nonequilibrium irreversible process, whose description should be, generally, based on the laws of irreversible thermodynamics. 

Figure 8 Schematic illustration of four different adsorption/displacement models proposed by Wahlgren and Amebrant [63] for protein and surfactant adsorption to solid surfaces. The three diagrams for each model show protein adsorption, surfactant addition, and state after rinsing. Figure A represents the case where surfactant binds to the protein and the protein-surfactant complex desorbs. Figure B represents protein displacement by the surfactant. Figure C represents reversible adsorption of the surfactant by the protein. Figure D represents reversible adsorption by the surfactant resulting in partial desorption of the protein. The figures relate to a hydrophilic surface at a hydrophobic surface the orientation of the surfactant molecules with respect to the surface will be different. (Reproduced from [63] with permission from Academic Press.)...

By the same token, the chemical composition of the substrate may play a major role in protein adsorption, since highly hydrophilic (hydrogel) surfaces tend to adsorb plasma proteins reversibly with little damage, whereas hydrophobic surfaces cause strong and partially irreversible adsorption leading to extensive damage of adsorbed proteins. 

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