Fenton chemistry. Amino acid oxidation.

E.R Stadtman,B.S Berlett

doi:10.1016/s0021-9258(19)47359-6

Abstract

The oxidation of amino acids by Fenton reagent (H2O2 + Fe(II] leads mainly to the formation of NH+4, alpha-ketoacids, CO2, oximes, and aldehydes or carboxylic acids containing one less carbon atom. Oxidation is almost completely dependent on the presence of bicarbonate ion and is stimulated by iron chelators at levels which are substoichiometric with respect to the iron concentration but is inhibited at higher concentrations. The stimulatory effect of chelators is not due merely to solubilization of catalytically inactive polymeric forms of Fe(OH)3 nor to the conversion of Fe(II) to complexes incapable of scavenging hydroxyl radicals. The results suggest that an iron chelate and another as yet unidentified form of iron are both required for maximal rates of amino acid oxidation. The metal ion-catalyzed oxidation of amino acids is likely a "caged" process, since the oxidation is not inhibited by hydroxyl radical scavengers, and the relative rates of oxidation of various amino acids by the Fenton system as well as the distribution of products formed (especially products of aromatic amino acids) are significantly different from those reported for amino acid oxidation by ionizing radiation. Several iron-binding proteins, peptides, and hemoglobin degradation products can replace Fe(II) or Fe(III) in the bicarbonate-dependent oxidation of amino acids. In view of their ability to sequester metal ions and their susceptibility to oxidation by H2O2 in the presence of physiological concentrations of bicarbonate, amino acids may serve an important role in antioxidant defense against tissue damage.

Highlights

(H,O, Fe(I1)) leads mainly to the formation ofNH:, involve the conversion of histidine residues toasparagine residues (5), of proline residues to glutamic semialdehyde and a-ketoacids, CO, oximes, and aldehydes or carboxylic to pyroglutamic or glutamicacidresidues (6), of arginine acids containing one less carbon atom.Oxidation is residues to glutamicsemialdehyderesidues (6), and of all almost completely dependenton the presenceof bicar- theseandotheramino acid(especially lysine) residues to bonate ion and is stimulatedby iron chelators at levels carbonyl derivatives (6, 7 ) .Such oxidations are marking steps which are substoichiometric with respect to the iron concentration but is inhibitaetdhigher concentrations
A more extensive analysis of the products various combinations of products as shown in column 2 of formed in the oxidation of amino acids other than phenylalof products (Table11, column 4)is approximately equalto the We could obtain no evidence for the formation of phenolic actual amount of H20, which was consumed (26.5 Fmol/ml) derivatives as judged by spectrophotometric and HPLC analas determined by direct measurement
H202)is almost completely dependent upon the presence of bicarbonate ion. In this regard it is noteworthy that seinries of articles beginning with the first issue of The Journal of Biological Chemistry, published in 1905, Dakin summarized the results of studies on the oxidationof amino acids by the Fenton reagent(37-39)

Summary

AMINO ACID OXIDATION*

From the Labf1rator.yo/ Biochemistry, National Heart, Lung, and Blood Institute, National Institutesof Health. Dinitrophenylhydrazone derivative of carbonyl compoundsformed in Unless otherwise stated, the complete reaction mixtures (2.0-ml vol- the oxidation of leucine were separated by HPLC on a 25-cm IBM umes) contained 23.5 mM NaHCO: mM amino acid, 30 mM H,O,, C18 column and were identified by comparison of the retention times and either 1.5 or 100 pM FeS04 and3.75 or 250 p~ ADP, respectively; with thoseof authentic standards. 2. Manometric measurement of gas produced during the oxidation of leucine in the presence and absence of oxsorvented arm of the Warburg vessels as described under “Ex- bant. The FeS04 and H20, were mixed, in that order, with either by decarboxylation of the a-ketoisocaproic acid inter- thecontents of themaincompartments,andthechangesin gas mediate (Reaction 2 ) or by oxidation of the isovaleraldehyde pressure were monitored at various time intervals.

Pathway C

Of particular significance is the demonstration here that

Initial rate

Themultiphasic response of amino acid oxidationto

Findings

We reported earlier that bicarbonaitoen is required for the