Protein immobilization methods covalent bonding

Creation of covalent bonds between surface amino acids of the enzyme and an insoluble matrix is perhaps the most frequendy exploited method of immobilization. Polar amino acids, which are likely to be present on the protein surface, have struaures that lend themselves to the chemical manipulation necessary for immobilization. e-Amino groups of lysine residues are the most frequendy employed points of linkages, though cysteine (via -SH), tyrosine, histidine, aspartic and glutamic acids, tryptophan, and arginine can also be used. 

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). 

Chemical immobilization methods may alter the local and net charges of enzymes, through covalent modification of charged residues such as lysine (NH4), aspartate, and glutamate (COO-). Conformational changes in secondary and tertiary protein structure may occur as a result of this covalent modification, or as a result of electrostatic, hydrogen-bonding or hydrophobic interactions with the support material. Finally, activity losses may occur as a result of the chemical transformation of catalytically essential amino acid residues. 

For immobilization studies to date, two distinct modes of immobilization have been used. The first utilizes nonspecific covalent bonding to CNBr-activated Sepharose via primary amino groups on the antibody molecule. Since there are many of these available on the antibody, this is expected to result in random orientation of antibody molecules on the support. The other method involves linkage through immobilized protein A, a protein which binds immunoglobulins in the structural F portion of the molecule. 

---