Covalent attachment methods

Protems can be physisorbed or covalently attached to mica. Another method is to innnobilise and orient them by specific binding to receptor-fiinctionalized planar lipid bilayers supported on the mica sheets [15]. These surfaces are then brought into contact in an aqueous electrolyte solution, while the pH and the ionic strength are varied. Corresponding variations in the force-versus-distance curve allow conclusions about protein confomiation and interaction to be drawn [99]. The local electrostatic potential of protein-covered surfaces can hence be detemiined with an accuracy of 5 mV. 

Currently available proteins are all deficient to greater or lesser extent in one or more of the essential amino acids. The recently advanced plastein reaction (229) has made it possible to use protein itself as substrate and to attach amino acid esters to the protein with high efficiency. By this method, soy bean protein (which is deficient in methionine) has been improved to the extent of having covalently attached L-methionine at 11%. 

Solid-phase peptide synthesis (Section 27.18) Method for peptide synthesis in which the C-terminal amino acid is covalently attached to an inert solid support and successive amino acids are attached via peptide bond formation. At the completion of the synthesis the polypeptide is removed from the support. 

The most reliable methods of the preparation of stable adsorbents involve, however, a covalent attachment of the polymeric stationary phases to the solid supporting material. In addition, the more diffuse interfaces formed in this case (see Sect. 2.2) are often favourable for the separation of proteins. 

The most important physical methods are physical and ionic adsorption on a water-insoluble matrix, inclusion and gel entrapment, and microencapsulation with a liquid or a solid membrane. The most important chemical methods include covalent attachment to a water-insoluble matrix, cross-hnking with the use of a multifunctional, low-molecular weight reagent, and co-cross-linking with other neutral substances, for example proteins. 

A very promising method, immobilized artificial membrane (IAM) chromatography, was developed by Pidgeon and co-workers [299-304,307], where silica resin was modified by covalent attachment of phospholipid-like groups to the surface. The retention parameters mimic the partitioning of drugs into phospholipid bilayers. The topic has been widely reviewed [47,298,307,309-311]. 

The novel concept of synthesizing a molecule while attached to a swollen cross-linked resin bead was introduced and demonstrated by R. B. Merrifield with the solid-phase peptide synthesis method about 20 years ago (1,2). The procedure involves the covalent attachment of an amino-acid residue to the polymer bead followed by the addition of subsequent amino-acid units in a stepwise manner under conditions that do not disrupt the attachment to the support. At the completion of the assembly of the peptide, the product is cleaved from the resin and recovered. The macro-scopically insoluble support provides convenient containment of the desired product so that isolation and purification from soluble co-products in the synthesis can be achieved by simple... 

In addition to the covalent binding, some methods derived from bioaffinity chromatography can be used for non covalent attachment of antibodies to a surface by the inactive Fc portion. The advantage is that antigen binding sites stay undamaged and accessible for the analytes due to the orientation of antibody with the active Fab portions towards the tested medium. 

One of the simplest methods of attaching biomolecules to hydrophobic polymeric particles is the use of passive adsorption. Some of the earliest examples related to the use of particles in immunoassays include the use of non-covalently adsorbed antibody or antigen onto latex microspheres. Protein adsorption onto hydrophobic particles takes place through strong interactions... 

Covalent attachment of proteins to the surface of liposomal bilayers is done through reactive sites created on the head groups of phospholipids with the intermediary use of a crosslinker or other activating agent. The lipid functional groups described in Section 1 of this chapter are modified according to the methods discussed in Section 2 to be reactive toward specific target... 

Martin, F.J., Hubbell, W., and Papahyadjopoulos, D. (1981) Immunospecific targeting of liposomes to cells a novel and efficient method for covalent attachment of Fab fragments via disulfide bonds. Biochemistry 20, 4229-4238. 

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