Covalent bonding of protein

Protein is immobilized by combining with the surface of the electrode through a covalent bond, which is called covalent bonding of protein. The process requires low temperature (0°C), low ion intensity, and physiological pH conditions. Although covalent bonding onto the surface of an electrode is more difficult than adsorption, it can provide a more stable immobilized protein. 

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

A number of different molecular mechanisms can underpin the loss of biological activity of any protein. These include both covalent and non-covalent modification of the protein molecule, as summarized in Table 6.5. Protein denaturation, for example, entails a partial or complete alteration of the protein s three-dimensional shape. This is underlined by the disruption of the intramolecular forces that stabilize a protein s native conformation, namely hydrogen bonding, ionic attractions and hydrophobic interactions (Chapter 2). Covalent modifications of protein structure that can adversely affect its biological activity are summarized below. 

The RAHB effect may be illustrated by the ubiquitous C=0- -H—N hydrogen bond of protein chemistry. As shown in Section 5.2.2, the simplest non-RAHB prototype for such bonding, the formaldehyde-ammonia complex (5.31c), has only a feeble H-bond (1.41 kcalmol-1). However, when the carbonyl and amine moieties are combined in the resonating amide group of, e.g., formamide, with strong contributions of covalent (I) and ionic (II) resonance structures,... 

It was hypothesized that the imbibed fibronectin may have been washed out by the medium, and this could have affected adhesion in sections of the disc. To eliminate this possibility, samples of the composite were made with gelatin dissolved in the aqueous phase. They were then emulsified with the hydrophilic prepolymer and applied to the reticulated foam. The result was covalent bonding of some of the protein into the polymer backbone, thus making it immune to washout. Figure 6.6 reports counts of endothelial cells cultured on this formulation and effects of sterilization method compared to controls without gelatin. 

Another type of covalent bond in proteins is the disulfide bond, formed by oxidative coupling of the -SH groups in cysteine. Oxytocin is an example of a cyclic peptide with a S-S bond. 

The epoxide has a strong tendency to covalently bond to protein, DNA, and RNA, and it rearranges to chloroacetaldehyde, a known mutagen. Therefore, vinyl chloride produces two potentially carcinogenic metabolites. Both of these products can undergo conjugation with glutathione to yield products that are eliminated from the body. 

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