Relative protein adsorption strength

Figure 7. Relative protein adsorption strength of various types of biochromatography for protein seperation based on dissociation constants.

Simple aqueous buffers of appropriate pH and sufficient Ionic strength are usually capable of preventing protein adsorption by Ionic interactions with the packing. However, certain proteins may still adsorb to packings by hydrophobic Interactions. This problem Is frequently observed with hydro-phobic membrane proteins, which also tend to be relatively insoluble in aqueous buffers thereby resulting In protein aggregation and anomolous behavior in SEC. 

Buffers of relatively high ionic strength (containing 0.1 to 1.0 M NaCl) are used to reduce nonspecific ionic adsorption. A preelution step with a low concentration of imidazole or EDTA can be used for the elution of various contaminating proteins that contain histidine residues [59]. 

The Sepharose-based DEAE CL-6B (Pharmacia) is also a very useful resin for ion exchange of high-mol wt proteins it has a very low nonspecific adsorption of macromolecules, good flow properties, and is relatively stable to changes in ionic strength and pH. Sephadex-based lERs tend to alter volume drastically as a result of changes in ionic strength... 

A segment perpendicular to a surface results in an effective steric barrier, while the number of contact points with the interface influences the strength of adsorption. For example, flexible caseins have numerous proline residues, so they have little ordered secondary structure and no intramolecular crosslink. As a result, caseins are able to adopt a number of different conformational states when being adsorbed at the oil-water interface. They are usually adsorbed at the interface in such a way that considerable portions of their structures protruding into the aqueous phase are available (Dickinson, 1992). On the other hand, serum milk proteins, such as p-lg and a-lactalbumin (a-la), bind relatively close to the interface and do not protrude... 

The adsorption of soy protein at an interface is relatively slow compared to casein, and the rate is affected by ionic strength, being higher at 0.2 M than at zero NaCl where the subunits may be dissociated. Conceivably the reduction of the zeta potential and electrostatic repulsion (from 0 to 0.2 M salt) facilitates penetration and subsequent surface packing (28). The rate of penetration of additional molecules into the film indicated that the soy proteins initially adsorbed and spread easily at the surface ( ). However, this seems inconsistent with the highly stable disulfide linked tertiary structure of soy glycinin (30) and it is perhaps the conglycinin component that forms the initial interfacial film (31). 

Because of the relatively rigid structure of adsorbed globular protein molecules, the adsorption isotherms display well-defined plateau values. The adsorption pattern, the effects of pH (i.e., charge of the protein), and ionic strength are in agreement with those of a polyampholyte the adsorbed mass generally is at a maximum around the isoelectric point of the protein/surface complex, that is, at conditions where the charges on the protein and the surface just compensate each other. 

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