Abstract
A number of metal-catalyzed oxidation (MCO) systems mediate the oxidative inactivation of enzymes. This oxidation is accompanied by conversion of the side chains of some amino acid residues to carbonyl derivatives (for review, see Stadtman, E. R. (1986) Trends Biochem. Sci. 11, 11-12). To identify the amino acid residues which are sensitive to MCO oxidation, several enzymes/proteins and amino acid homopolymers were exposed to various MCO systems. The carbonyl groups which were formed were converted to their corresponding 3H-labeled hydroxy derivatives. After acid hydrolysis, the labeled free amino acids were separated by ion exchange chromatography. Each protein or polymer gave rise to several different labeled amino acids. The elution profiles of the labeled amino acids obtained from preparations of Escherichia coli glutamine synthetase which had been oxidized by MCO systems comprised of either Fe(II)/O2 or ascorbate/Fe(II)/O2 both in the presence and absence of EDTA were qualitatively the same. From a comparison of the elution profiles of labeled amino acids from various proteins with those obtained from homopolymers, it is evident that the side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation by the MCO systems. This conclusion is supported also by direct amino acid analysis of acid hydrolysates which shows that the oxidation of glutamine synthetase, enolase, and phosphoglycerate kinase is associated with the loss of at least 1 histidine residue per subunit. From the results of studies with homopolymers, it is apparent that glutamic semialdehyde is a major product of both proline and arginine residues. In addition, hydroxyproline and unlabeled glutamic acid were identified among the hydrolysis products of oxidized poly-L-proline, and unlabeled aspartic acid was identified as a product of poly-L-histidine oxidation.
Highlights
A number of metal-catalyzed oxidation (MCO) sys- A number of metal ion-catalyzed oxidation (MCO)’. systems mediate the oxidative inactivation of enzymes. tems catalyze the inactivationof enzymes (1-5)
Each study, we found that when HAVA is heated with 6 N HC1 at of the three basic amino acids and proline must be considered 155 "C for 2 h, it is partly converted to a compound with a as apossible target for oxidation of the proteins by the Fe(I1) retention time of 41 min when assayed under the conditions MCO system
Glutamic and aspartic acids were identified among unlabeled products inacid hydrolysates of poly-L-proline and poly
Summary
Oxidation of Proteins and Polypeptides-Reaction mixtures (2.0 ml) containing 2 mgof protein or polypeptide, 100 mM potassium phosphate buffer (pH 6.8), 10 mM MgC12,90 mMKC1, 4 mM FeSO,, and 8 mM EDTA were incubated in air in 25-ml Erlenmeyer flasks for 2 hat 37 “Cwith shaking (200 rpm). The reaction was stopped by adding trichloroacetic acid to a final concentration of IO%,and the protein or polypeptides were separated by centrifugation. After 30 min a t 37 ”C, trichloroacetic acid was added to a final concentration of 10% (in thecase of polylysine, to 25%),and theprecipitated protein was collected by centrifugation. As will be shown later, under these experimental conditions Mn2+ catalyzes the exchange of 3H into the phenylalanine, tyrosine, and histidine residues of the native forms of some proteins To prevent this oxidation-independent labeling of proteins, EDTA was always present during the treatment with [3H]NaBH4.The precipitate was washed twice with 1.0 ml of 10% trichloroacetic acid and dissolved in 300 p1 of 70% formic acid. Properties of the solid sample of HAVA were: m.p. 228 “C (decomposition) (reported (20) 225 “C (decomposition));TLC (Eastman silica gel sheet, EtOH/NH40H, 15:5)R p 0.34; ‘H NMR in DzO: 6(ppm) 1.58 (m, 2H, H-3), 1.82 (m, 2H, H-4), 3.58 (t, 2H, H-5), 3.67 (t, IH, H-2)
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