Proteins chloramine

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. 

Hawkins CL, Pattison DI, Davies MJ (2002) Reaction of Protein Chloramines with DNA and Nucleosides Evidence for the Formation of Radicals, Protein-DNA Cross-Links and DNA Fragmentation. Biochem J 365 605... 

In the presence of reducing agents, protein chloramines are reversed to native proteins, while in the presence of Cl, chloramines disproportionate to yield elementary chlorine (Cl°), which readily substitutes for phenol moieties of tyrosines to yield stable 3 -chloro and 3 5 - dichloro derivatives. The chlorination of tyrosines retentively marks the protein molecule (R15). 

Compounds that can be converted to chloramines [1-4] e.g. amino acids, peptides, proteins and derivatives [5-13]... 

The action of chlorine gas produces, for example, chloramine derivatives from herbicides, amino acids, peptides and proteins the active chlorine of these derivatives then... 

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.

The following protocol is representative of those found in the literature for iodination of protein molecules using chloramine-T. 

Directing the iodination reaction toward histidine residues in proteins, as opposed to principally tyrosine modification, is possible simply by increasing the pH of the lodobeads reaction from the manufacturer s recommended pH 7.0-8.2 (Tsomides et ai, 1991). No reducing agent is required to stop the iodination reaction as is the case with chloramine-T and other methods. 

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] ... 

Whilst chloramines are less reactive than HOC1, they are longer-lived and so can diffuse away from their site of production. Those formed from lipophilic amines are especially toxic because they can permeate membranes. Chloramines are toxic for a number of reasons they can oxidise sulphydryl or sulphur-ether groups, they are unstable and can be hydrolysed to release chlorine in the form of HOC1 or NH2C1, they can react with iodide to form iodine and they can covalently bind proteins. 

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-GEN (l,3,4,6-tetrachloro-3-6-diphenylglycouril) is a better reagent for the lodination of proteins than chloramine-T, is less damaging, and has fewer side reactions than the latter. The insolubility of IODO-GEN in water means that tubes can be precoated with the reagent dissolved in methylene chloride or chloroform. Then the tubes are stored in the dark until required. The reaction is started by adding the protein and radioiodide, and terminated by removing the sample from the reaction vessel. 

Figure 265 IODO-BEADS contains immobilized Chloramine-T functional groups that can react with radioactive iodide in aqueous solution to form a highly reactive intermediate. The active species may be an iodosulfonamide derivative, which then can iodinate tyrosine or histidine residues in proteins.

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