Protein oxidation chloramine

Figure 12.3 The strong oxidant chloramine-T can react with iodide anion in aqueous solution to form a highly reactive mixed halogen species. 125IC1 then can modify tyrosine and histidine groups in proteins to form radiolabeled products.

HOCl-mediated protein oxidation accelerates under pathophysiological conditions. Thus, proteins from extracellular matrix obtained from advanced human atherosclerotic lesions contained the enhanced levels of oxidized amino acids (DOPA and dityrosine) compared to healthy arterial tissue [44], It was also found that superoxide enhanced the prooxidant effect of hypochlorite in protein oxidation supposedly by the decomposition of chloramines and chlor-amides forming nitrogen-centered free radicals and increasing protein fragmentation [45], In addition to chlorination, hypochlorite is able to oxidize proteins. The most readily oxidized amino acid residue of protein is methionine. Methionine is reversibly oxidized by many oxidants including hypochlorite to methionine sulfide and irreversibly to methionine sulfone [46] ... 

Pattison DI, Hawkins CL, Davies MJ (2007) Hypochlorous Acid-Mediated Protein Oxidation How Important Are Chloramine Transfer Reactions and Protein Tertiary Structure Biochemistry 46 9853... 

Despite that the MPO-catalyzed oxidation of halides seems to employ the same mechanisms active in primary production of the respective hypohalous acids, the final products of peptide and protein oxidation vary, depending on the halide ion employed iodides and bromides when used as substrates yield stable bromo-and iodotyrosine derivatives, whereas direct chlorination of the available free amino moieties and semistable chloramines formation predominate when Cl is oxidized as the substrate (S54, Z3). The chlorination is a unique function of polymorphonuclear neutrophilic leukocytes (W6, Z2). 

During the iodination with the oxidizer chloramine T, all reactants are present in solution (one-phase system). Pierce offers oxidizers that were applied to a solid phase (two-phase system iodobeads, iodogen). lodobeads are N-chlorobenzene sulfonamides attached to polystyrene beads. Iodogen is a hydrophobic chloramine T derivative applied to the wall of the reaction vessel. After the reaction with iodobeads and iodogen, the solid phase with the oxidizer can easily be separated from the reaction mixture. Hence, the addition of reducing agent (bisulfite) is unnecessary, which spares the sensitive disulfide bridges of some proteins. In addition, N-chlorobenzene sulfonamide is a milder oxidizer than chloramine T. 

Furthermore, it was found that stimulated human neutrophils are able to produce 5-chloro-2 -deoxycytidine and that the myeloperoxidase system generates just the same levels of 5-chlorocytosine in DNA and RNA in vitro (Reaction (4), Figure 28.3). It is possible that myeloperoxidase-generated chlorinated products may modify nuclear acids of pathogens and nuclear acids in host cells during inflammation. Hawkins et al. [48] suggested that DNA oxidation may be initiated by protein chloramines formed in the reaction of HOCl with histones in the nucleosome. 

Iodine becomes incorporated into proteins either oxidatively, or enzymatically, or electrochemically. Oxidative incorporation uses organochemical oxidants, such as, for example, chloramine T or lodo-gen (l,3,4,6-tetrachloro-3a,6a-diphenyl glycouril). Enzymatic incorporation is done by means of lactoperoxidase. By these methods iodine is introduced into phenyl (tyrosyl) residues of the protein. 

IODO-BEADS reaction from the manufacturer s recommended pH 7.0 to 8.2 (Tsomides et al., 1991). No reducing agent is required to stop the iodination reaction, as is the case with chloramine-T and other methods. Simple removal of the bead(s) from the reaction is enough to eliminate the iodination process. The mild nature of the IODO-BEADS iodination reaction can result in better recovery of active protein than using soluble oxidants (Lee and Griffiths, 1984). 

At neutral and slightly alkaline pH, N-chlorosuccinimide (NCS) and N-chloro-p-toluenesulfonamide (chloramine-T) oxidize methionine residues in peptides and proteins to methionine sulfoxides (31). Chlor-amine-T is more selective than NCS it does not react with tryptophan whereas NCS does. However both of these reagents react with cysteine. Treating the Ca2+-dependent protein modulator with NCS in the presence of Ca2+ resulted in selective oxidation of methionines 71, 72, 76, and possibly 109 in the modulator sequence with concomitant loss in interaction with cyclic nucleotide phosphodiesterase (32). Methionine residues have been implicated in the activation of cyclic nucleotide phosphodiesterase by the Ca2+-dependent protein modulator. 

Recently it was found that the reaction employs intermediary nitrogen-centered free radical formation via thermal homolysis of the N—Cl bond (H11). The stable chloramine T was effectively used for relatively specific oxidation of cysteinyl residues exposed to the surface of the protein molecule (S25). 

The other amino acid residue present in proteins that is susceptible to oxidation is the indole moiety of tryptophan (Fig. 11). The reducing potential of tryptophan is considerably less than that of cysteine and methionine, so oxidation of tryptophanyl residues usually does not occur until all exposed thiol residues are oxidized. Also, the spontaneous oxidation of tryptophanyl residues in proteins is much less probable than that of cysteinyl and methionyl residues. Tryptophan residues are the only chromophoric moieties in proteins which can be photooxi-dized to tryptophanyl radicals by solar UV radiation, even by wavelengths as long as 305 nm (B12). Tryptophanyl residues readily react with all reactive oxygen species, hypochlorite, peroxynitrite, and chloramines. Oxidative modifications of other amino acid residues require use of strong oxidants, which eventually are produced in the cells. Detailed mechanisms of action of these oxidants is described in subsequent sections of this chapter. 

Chloramines have a particular affinity to the protein surface-oriented thiol and thioether residues. Oxidation of the particular methionine residues in human hemoglobin with chloramine T have shown that chloramine almost exclusively attacks the exposed methionyl residues (at 1 5 mol/mol ratio), yielding methionine sulfoxide. The first oxidized were three methionyl residues located at positions 032P13, 076EF5, and 551)(,. The buried cysteinyl residues, located at positions 01104G11 and pi 12O14, remained unaffected. Also, methionine 032613 is fully protected, and methionine a76EF5 appears to be oxidized only partially (A 10). 

When the taurine chloramine uptake rate exceeds the rate of NADPH-dependent regeneration of GSH, there is a net loss of cellular GSH level, causing protein-thiol oxidation, ATP loss, and disruption of cellular metabolism. Heme moieties are the other target of chloramine attack on cellular constituents. Oxidation of hemoglobin to methemoglobin (and other hemoproteins to their oxidized derivatives) occurs at 10-fold excess of chloramine taurine molar concentration compared... 

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