Protein covalent

Certain proteins are found to be covalently linked to lipid molecules. For many of these proteins, covalent attachment of lipid is required for association with a membrane. The lipid moieties can insert into the membrane bilayer, effectively anchoring their linked proteins to the membrane. Some proteins with covalently linked lipid normally behave as soluble proteins others are integral... 

Fusion protein Covalent linkage of two distinct protein entities, e.g. j3-galactosidase and somatostatin. A fusion protein need not retain the different biological properties of its two components. 

Liu, J. Li, Q. Yang, X. van Breemen, R. B. Bolton, J. L. Thatcher, G. R. Analysis of protein covalent modification by xenobiotics using a covert oxidatively activated tag raloxifene proof-of-principle study. Chem. Res. Toxicol. 2005, 18, 1485-1496. 

Rhodopsin is a seven ot-helix trans-membrane protein and visual pigment of the vertebrate rod photoreceptor cells that mediate dim light vision. In this photoreceptor, retinal is the chromophore bound by opsin protein, covalently linked to Lys296 by a Schiff base linkage. Kpega et al.64 have studied NMR spectra of Schiff bases being derivatives of all-frans retinal and amino-p-cyclodextrins as a model of rhodopsin, where p-cyclodextrin plays a role of a binding pocket. On the basis of analysis of the chemical shift differences for the model compound in the presence and in the absence of adamantane carboxylate, it has been shown that the derivative of 3-amino-p-cyclodextrin forms dimer in water and retinoid is inserted into p-cyclodextrin cavity [31]. 

The presence of chemically reactive structural features in potential drug candidates, especially when caused by metabolism, has been linked to idiosyncratic toxicity [56,57] although in most cases this is hard to prove unambiguously, and there is no evidence that idiosyncratic toxicity is correlated with specific physical properties per se. The best strategy for the medicinal chemist is avoidance of the liabilities associated with inherently chemically reactive or metabolically activated functional groups [58]. For reactive metabolites, protein covalent-binding screens [59] and genetic toxicity tests (Ames) of putative metabolites, for example, embedded anilines, can be employed in risky chemical series. 

Weetall, H.H. (1993) Preparation of immobilized proteins covalently coupled through silane coupling agents to inorganic supports. Applied Biochemistry and Biotechnology, 41, 157-188. 

Day, S.H., Mao, A., White, R., Schulz-Utermoehl, T., Miller, R. and Beconi, M.G. (2005) A semi-automated method for measuring the potential for protein covalent binding in drug discovery. Journal of Pharmacological and Toxicological Methods, 52 (2), 278-285. 

Leone, A.M. et al. (2007) Evaluation of felbamate and other antiepileptic drug toxicity potential based on hepatic protein covalent binding and gene expression. Chemical Research in Toxicology, 20 (4), 600-608. 

For estimation of proteins covalently bound to chromatographic matrices hydrolyze the sample for 6 h at 37 ° C in solution D. After centrifugation, use an aliquot for protein determination. 

Particularly suitable for insoluble proteins, protein in foods and protein covalently immobilized on chromatographic supports. 

Oxidation of catechols in the presence of a protein may lead to extensive catechol-protein covalent coupling (Figure 9.4) as demonstrated in the case of the chlorogenic acid-BSA couple. Autoxidation of EGCG at pH 4.9 in the presence of Zn(II) cations was shown to generate semiquinone radicals (stabilized by Zn(II) binding) mainly on the B ring moiety. 

More generally, one-electron oxidation of protein-bound phenols to form reactive ary-loxyl radicals is a possible pro-oxidant mechanism since these radicals can propagate H-atom or electron transfers within the protein. In addition to phenol protein covalent coupling, these phenol-mediated oxidative damages to proteins could be detrimental to their function as enzymes, receptors, and membrane transporters. For instance, investigations by capillary electrophoresis have shown that quercetin in concentrations lower than 25 pM potentiates HSA degradation by AAPH-derived peroxyl radicals. 

Cross-linking of proteins. Covalent cross-linking of caseins is evident (by gel electrophoresis) after even 2 min at 140°C and it is not possible to resolve the heat-coagulated caseins by urea- or SDS-PAGE. 

Iressa (gefitinib, 2) is a reversible inhibitor, thus mechanistically it is possible to excert less toxicity in comparison to similar irreversible inhibitors, which bond to the protein covalently. Gefitinib (2) is also quite selective. It is at least 100-fold selective against other tyrosine kinases such as erbB-2, KDR, c-flt or serine/threonine kinases such as PKC, MEK-1, and ERK-2. 

The radiolabel was bound to liver protein covalently and to a much greater extent than in other tissues such as the muscle (Table 7.4). 

Hence, concerning the interaction with plasma proteins, covalently immobilized heparin performs identically to heparin in solution, and this results in the enrichment of the HCP surface with the most thrombogenic plasma components fibrinogen and thrombin. 

In addition to finding small organic molecules that bind to a protein, covalent capture methods can identify peptides that interact with proteins. Kohda and colleagues used this approach to study the mitochondrial protein Tom20, an import receptor that recognizes an epitope on proteins targeted for the mitochondria.1301 Previous work had characterized this epitope as a five-residue peptide that assumes an amphiphilic helical conformation, and coarse sequence preferences had been worked out. However, Tom20 has both low affinity... 

Cross-linking High stability Strong binding force Used for stabilization of proteins covalently bound to a solid support Possible protection from microbiological attack Reaction difficult to control High amounts of biomolecule are required Possible loss of protein activity... 

Park, S. Wanna, L. Johnson, M. E. Venton, D. L. 2000. A mass spectrometry screening method for antiaggregatory activity of proteins covalently modified by combinatorial library members application to sickle hemoglobin. /. Comb. Chem., 2,314-317. 

Evans DC, Watt AP, Nicoll-Griffith DA, et al. Dmg-protein covalent adducts an industry perspective on minimizing the potential for drag bioactivation in drag discovery and development. Chem Res Toxicol 2004 17 3-16. 

Although the transition state analog approach is suitable for enzymes that bind their transition state noncovalendy, many natural enzymes achieve rate accelerations through covalent catalysis. For example, in the mechanism of most esterases and amidases, a functional group (e.g., a serine hydroxyl) of the protein covalently interacts with the substrate to form a protein bound intermediate. Furthermore, nature s most fundamental carbon-carbon bond-forming enzymes, class I aldolases, use... 

Huang LS, Ortiz de Montellano PR (2006) Heme-protein covalent bonds in peroxidases and resistance to heme modification during halide oxidation. Arch Biochem Biophys 446 77-83... 

Huang LS, Wojciechowski G, Ortiz de Montellano PR (2006) Role of heme-protein covalent bonds in mammalian peroxidases - Protection of the heme by a single engineered heme-protein link in horseradish peroxidase. J Biol Chem 281 18983-18988... 

Colas C, Ortiz de Montellano PR (2004) Horseradish peroxidase mutants that autocataly-tically modify their prosthetic heme group. Insights into mammalian peroxidase heme-protein covalent bonds. J Biol Chem 279 24131-24140... 

Wojciechowski G, Huang L, Ortiz de Montellano PR (2005) Autocatalytic modification of the prosthetic heme of horseradish but not lactoperoxidase by thiocyanate oxidation products. A role for heme-protein covalent crosslinking. J Am Chem Soc 127 15871-15879... 

Carbohydrates and lipids are often covalently associated with proteins. These modifica-tions/additions to the protein occur following translation they are termed post-translational modifications. Proteins covalently associated with lipids or carbohydrates are termed... 

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