SELECTIVE ADSORPTION OF PROTEINS

Figure 1.12 Schematics illustrating interfacial interactions between bone tissne or cell and implanted biomaterials viewed from the host tissne perspective (a) protein adsorption from blood and tissue fluids, (b) protein desorption, (c) substrate surface changes and material release, (d) inflammatory and connective tissue cells approach the implant, (e) possible targeted release of matrix proteins and selected adsorption of proteins, (f) formation of lamina limitans and adhesion of osteogenic cells, (g) bone deposition on both the exposed bone and implant surfaces, and (h) remodeling of newly formed bone.

Morin C, Hitchcock AP, Cornelius RM, Brash JL, Urquhart SG, A. Scholl A, Doran A. (2004) Selective adsorption of protein on polymer surfaces studied hy soft X-ray photoemission electron microscopy. ]El Spec RelPhenom 137—140 785—794. 

Recent reports describe the use of various porous carbon materials for protein adsorption. For example, Hyeon and coworkers summarized the recent development of porous carbon materials in their review [163], where the successful use of mesoporous carbons as adsorbents for bulky pollutants, as electrodes for supercapacitors and fuel cells, and as hosts for protein immobilization are described. Gogotsi and coworkers synthesized novel mesoporous carbon materials using ternary MAX-phase carbides that can be optimized for efficient adsorption of large inflammatory proteins [164]. The synthesized carbons possess tunable pore size with a large volume of slit-shaped mesopores. They demonstrated that not only micropores (0.4—2 nm) but also mesopores (2-50 nm) can be tuned in a controlled way by extraction of metals from carbides, providing a mechanism for the optimization of adsorption systems for selective adsorption of a large variety of biomolecules. Furthermore, Vinu and coworkers have successfully developed the synthesis of... 

Hydroxylapatite column chromatography relies on the selective adsorption of the protein onto the surface of calcium phosphate. The final stage of purification, on a high-performance DEAE column, is carried out just prior to crystallization of the protein. 

Other modifications of capillary electrophoresis techniques, described below, have been adopted for the improvement of separation of several substances, including proteins, peptides and aminoacids. Furthermore, new procedures are also being developed to avoid the adsorption of proteins and peptides onto the walls of the capillary and consequently improving their selectivities. 

Separations as Influenced by Temperature. Variations in temperature were also investigated as a means of probing the selection of more refined reversed-phase conditions for the chromatography of rhGH. It is generally recognized that both the adsorption of proteins onto the nonpolar solid... 

The simple Alexander—de Gennes theory, which assumed a steplike monomer density in the brush, captured the dependence of the interaction on the physical parameters (length of the polymer, density of grafting, quality of the solvent) and provided a satisfactory approximation for the calculation of the steric repulsion. However, new applications of grafted polymers on surfaces, such as the control of the catalytic selectivities of some chemical reactions by varying the thickness of a brush,4 the prevention of the adsorption of proteins on surfaces (a condition required for biocompatibility),5 or the control of... 

Brena, B. M., and Batista-Viera, F. (1992). Selective adsorption of immunoglobulins and glucosylated proteins on phenylboronate-agarose.. Chromatogr. 604, 109-115. 

Immunologically based sensors show great potential, but there are a number of problems that may limit their performance. For example, the nonspecific adsorption of proteins and other large molecules can adversely affect the a rent sensitivity and selectivity. Strategies for minimizing this effect include the use of a reference crystal coated with a protein that does not specifically interact with the antigen or compound of interest [27], and deactivation of nonspecific adsorption sites. 

Solid phase extraction. With the availability of pre-prepared cartridges of silica-based adsorbents, the use of solid phase extraction has increased in the last few years although the technique has been in use for many years for the isolation of many biochemicals, e.g. amino acids, catecholamines. In essence it is a version of chromatography conditions for the selective adsorption of the analytes (column, solvent, pH, etc.) are chosen, the sample is applied to a column, washed and the analytes selectively eluted with appropriate solvents. Since the columns are disposable there is no need to worry about protein contamination or infection. The adsorbents available cover an even wider range than HPLC materials since they are not required to withstand high back pressures. It is possible... 

RNA, which are anionic biorelated polymers with phosphoric acid groups, were adsorbed very well by all the adsorbents. By contrast, the adsorption of protein was more dependent on the Mn of the adsorbent than its anion-exchange capacity (AEC). The adsorption of BSA (Mw 6.9 X 10" ), an acidic protein, increased from 5% to 68% with an increase in the from 2 x 10 to 1 x lO". The adsorption of y-globulin (M 1.6 x 10 ), a hydrophobic protein, increased from 2% to 22% with an increase in the Miim from 1 x 10 to >2 x 10. Polymyxin-Sepharose with large pore size (Mn >2x 10 ) also adsorbed BSA (78%) and y-globulin (26%), as shown in Table 1. Very little of the other neutral or basic proteins adsorbed onto the adsorbents under similar conditions. As a result, only when the PL cellulose (10 ), with a of 2 x 10 and AEC of 0.6 meq/g, was used as the adsorbent at pH 7.0 and ionic strength of fi=0.05 were LPS and DNA selectively well adsorbed. 

Physical immobilization methods do not involve covalent bond formation with the enzyme, so that the native composition of the enzyme remains unaltered. Physical immobilization methods are subclassified as adsorption, entrapment, and encapsulation methods. Adsorption of proteins to the surface of a carrier is, in principle, reversible, but careful selection of the carrier material and the immobilization conditions can render desorption negligible. Entrapment of enzymes in a cross-linked polymer is accomplished by carrying out the polymerization reaction in the presence of enzyme the enzyme becomes trapped in interstitial spaces in the polymer matrix. Encapsulation of enzymes results in regions of high enzyme concentration being separated from the bulk solvent system by a semipermeable membrane, through which substrate, but not enzyme, may diffuse. Physical immobilization methods are represented in Figure 4.1 (c-e). 

The vial selected should be of type I glass, as stated in the USP. It is highly recommended that all experimental work begin with this type of glass, and that early in the process the interactions of the proteins with the glass surfaces should be determined. Glass is not an inert material. Glass surfaces must be taken into consideration to study the adsorptive properties of the respective protein. Adsorption of proteins will be treated later in this chapter. 

Adsorption chromatography, in which separation occurs due to the selective adsorption of the components of a mixture onto active sites on the surface of an adsorbent, has not been widely applied to the separation of proteins by HPLC. This may be because an organic mobile phase must be used and the presence of any water deactivates the column hence, aqueous solutions of proteins cannot be analyzed. 

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