Metal-catalyzed oxidative modification

Gomez-Baena, G., Diez, J., Garcia-Pemandez, J. M., ElAlaoui, S., and Humanes, L. (2001). Regulation of glutamine synthetase by metal-catalyzed oxidative modification in the marine oxyphoto-bacterium Prochlorococcus. BBA Gen. Subjects. 1568, 237—244. 

Dissolve the sulfhydryl-containing protein or macromolecule to be modified at a concentration of l-10mg/ml in 50mM Tris, 0.15M NaCl, 5mM EDTA, pH 8.5. EDTA is present to prevent metal-catalyzed oxidation of sulfhydryl groups. The presence of Tris, an amine-containing buffer, should not affect the efficiency of sulfhydryl modification. Not only do amines generally react slower than sulfhydryls, the amine in Tris buffer is of particularly low reactivity. If Tris does pose a problem, however, use 0.1M sodium phosphate, 0.15M NaCl, 5mM EDTA, pH 8.0. 

Transition metals such as iron can catalyze oxidation reactions in aqueous solution, which are known to cause modification of amino acid side chains and damage to polypeptide backbones (see Chapter 1, Section 1.1 Halliwell and Gutteridge, 1984 Kim et al., 1985 Tabor and Richardson, 1987). These reactions can oxidize thiols, create aldehydes and other carbonyls on certain amino acids, and even cleave peptide bonds. The purposeful use of metal-catalyzed oxidation in the study of protein interactions has been done to map interaction surfaces or identify which regions of biomolecules are in contact during specific affinity binding events. 

In earlier studies the in vitro transition metal-catalyzed oxidation of proteins and the interaction of proteins with free radicals have been studied. In 1983, Levine [1] showed that the oxidative inactivation of enzymes and the oxidative modification of proteins resulted in the formation of protein carbonyl derivatives. These derivatives easily react with dinitrophenyl-hydrazine (DNPH) to form protein hydrazones, which were used for the detection of protein carbonyl content. Using this method and spin-trapping with PBN, it has been demonstrated [2,3] that protein oxidation and inactivation of glutamine synthetase (a key enzyme in the regulation of amino acid metabolism and the brain L-glutamate and y-aminobutyric acid levels) were sharply enhanced during ischemia- and reperfusion-induced injury in gerbil brain. 

Sodium metaperiodate has been applied as the stoichiometric oxidant in numerous transition metal catalyzed oxidations. Of particular use is a one-pot oxidative cleavage of olefins to aldehydes by the Os04-NaI04 catalytic system, as exemplified in Scheme 3.374 [1362]. This oxidative cleavage, with some modifications. 

It should be noted that protein carbonylations occur via many different pathways. The hydrazide-coated glass beads are capable of enriching peptides harboring carbonyl modihcations other than those by HNE (e.g., 4-hydroxy-2-hexenal, 4-oxo-2-nonenal, metal-catalyzed oxidation, and others). These modifications can be included in the database search algorithms for in-depth mining of posttranslational protein carbonylations. 

The above data unequivocally established the fact that protein carbonyls stemming from metal catalyzed oxidation are present in the aging human lens, and that these modifications are increased in presence of diabetes. The mechanistic studies with rabbit lenses exposed to hyperbaric oxygen confirm the critical role of oxygen in the formation of allysine. 

The following protocol is a suggested method for labeling a protein with AMCA-HPDP. It is assumed that the presence of a sulfhydryl on the protein has been documented or created. The reaction conditions can be carried out in a variety of buffers between pH 6 and 9. Avoid the presence of extraneous sulfhydryl-containing compounds (such as disulfide reductants) that will compete in the reaction. The inclusion of EDTA in the modification buffer prevents metal-catalyzed sulfhydryl oxidation. Optimization for a particular labeling experiment should be done to obtain the best level of fluorophore incorporation. 

A number of aglycone flavonoids are potent inhibitors of in vitro oxidative modification of LDL [44]. Phenolic compounds isolated from red wine inhibit the copper-catalyzed oxidation of LDL in vitro (10 mmol/L), significantly more than a-tocopherol [45], possibly by regenerating a-tocopherol [44], Alternatively, chelation of divalent metal ions by flavonoids may reduce formation of free radicals induced by Fenton reactions [42]. Hydroxylation of the flavone nucleus appears to be advantageous because flavone itself is a poor inhibitor of LDL oxidation, whereas polyhydroxylated flavonoids such as quercetin, morin, hypoleatin, fisetin, gossypetin, and galangin are potent inhibitors of LDL oxidation [44],... 

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