Protein polymeric gels

Figure 6.3 Reproducibility of SDS-protein separations utilizing a replaceable polymeric gel. The peaks correspond to mellitic acid (1), a-lactalbumin (2), carbonic anhydrase (3), ovalalbumin (4), bovine serum albumin (5), phosphorylase b (6), /3-galactosidase (7), and myosin (8). (From Ref. 53.)...

By immobilsation, the pH and temperature profiles of the enzymes may be shifted and the stability of the enzymes altered and in most cases the objective is towards enhancement of these properties. Methods used for the immobilization of enzymes fall into the following categories physical adsorption onto an inert carrier, inclusion in the lattices of a polymerized gel, cross-linking of the protein with a bifunctional reagent, and covalent binding to a reactive insoluble support (Figure 6.52). The selection of the carrier depends on the nature of the enzyme itself, as well as the particle size, surface area, molar ratio of hydrophilic to hydrophobic groups and chemical composition. 

Cole C-A, Schreiner SM, Priest JH, Monji N, Hoffman AS. Al-isopropylacrylamide and Al-acryloxysuccinimide copolymer a thermally reversible, water-soluble, activated polymer for protein conjugation. In Russo P, ed. Reversible Polymeric Gels and Related Systems. Washington, DC American Chemical Society, 1987 245-254. 

The above results suggest that the pores that contain the protein behave differently than the free pores. In other words, the presence of the protein dopant somehow alters the behavior of the silicate porous structure. Not only is the protein conformation altered by the silica gel matrix but the structure of the silica network is also affected by the presence of the biological dopant. The biomolecule, according to the spectroscopic data resides in pore that conforms to the size of the protein. The gel network shows a wide distribution of pore sizes, but it appears that the protein is only selectively entrapped by the pores which meet its dimensional requirements. Since the protein is added to the sol and is encapsulated in the growing gel polymeric network, it can be concluded that the pore forms around the protein. The silicate fragments formed as a part of the initial hydrolysis reactions are charged as well H-bonding and the complementary properties of the protein may allow it to interact with the silica polymer. If such interactions are sufficiently attractive the protein is likely to act as a structural template around which the gel network can form. 

Most of the amperometric biosensors described until now are constructed either by crosslinking a suitable redox enzyme within a polymeric gel on the electrode surfiu e or by assembling a preformed enzyme-containing membrane on top of the electrode. Although this approach has led to amperometric biosensors for substrates of most oxidases or enzyme sequences involving oxidases, besides the inherent thermal instability of proteins two major drawbacks have to be fi cused on. 

Sieving techniques are required for separation of species which have no differences in mass-to-charge ratio such as nucleic acids and sodium dodecylsulfate (SDS)-protein complexes. Sieving systems include cross-linked or linear polymeric gels cast in the capillary or replaceable polymer solutions. 

Confining enzymes within the lattices of polymerized gels is another method for immobifization. Such occlusion does not lead to any bond formation between the enzyme and the polymer matrix thus, there is no disruption of the protein molecules. Such biocatalysts are robust and are easy to recover however, appHcation of sol-gel encapsulation has been more related to chemical analysis than bioprocesses. 

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