Measurement of 5-Hydroxy-2-aminovaleric Acid as a Specific Marker of Iron-Mediated Oxidation of Proline and Arginine Side-Chain Residues of Low-Density Lipoprotein Apolipoprotein B-100

doi:10.1006/bbrc.2000.2533

Biochemical and Biophysical Research Communications

Volume 270, Issue 3, 21 April 2000, Pages 852-857

https://doi.org/10.1006/bbrc.2000.2533 Get rights and content

Abstract

An alteration of apolipoprotein (apo) B-100 structure by direct oxidative modification is supposed to be an important mechanism involved in atherogenesis. There is difficulty in quantifying this type of modification owing to a lack of specific assays. We evaluated a methodology based on the oxidation of protein arginine and proline to γ-glutamyl semialdehyde which by reduction forms 5-hydroxy-2-aminovaleric acid (HAVA). We determined HAVA by using derivatization to N(O)-ethoxycarbonyl ethyl esters and gas chromatography–mass spectrometry in different low-density lipoprotein preparations subjected to oxidative damage in the presence of iron. Results suggest that apoB-100 proline and arginine residues are highly reactive toward oxygen radicals ex vivo. Femtomole levels of HAVA can be reproducible measured. HAVA determination compares well with the measurement of carbonyl group formation used as a generally accepted but nonspecific index of protein oxidation. Thus, HAVA could prove to be a sensitive assay for studying specific modification of apoB-100.

References (38)

J.A. Berliner et al.
Free Radical Biol. Med.
(1996)
M.J. Davies et al.
Free Radical Biol. Med.
(1999)
A. Gießauf et al.
FEBS Lett.
(1996)
A. Van der Vliet et al.
Arch. Biochem. Biophys.
(1995)
H. Kaur et al.
Methods Enzymol.
(1994)
A. Ayala et al.
Free Radical Biol. Med.
(1996)
A. Ayala et al.
Free Radical Biol. Med.
(1996)
R.F. Adams
J. Chromatogr.
(1974)
J.F. Thompson et al.
J. Biol. Chem.
(1964)
A. Amici et al.
J. Biol. Chem.
(1989)

J. Pietzsch et al.

Biochim. Biophys. Acta

(1995)

S. Kopprasch et al.

Atherosclerosis

(1998)

G. Cohen et al.

Anal. Biochem.

(1998)

A.J. Matthews et al.

J. Surg. Res.

(1997)

J. Pietzsch et al.

J. Lipid Res.

(1996)

R.L. Levine et al.

Methods Enzymol.

(1990)

Z.-H. Huang et al.

J. Chromatogr.

(1993)

B.S. Berlett et al.

J. Biol. Chem.

(1997)

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An array of products is formed when proteins undergo oxidative damage. Protein carbonylation is the result of damage caused to a wide range of amino acids such as sulfur containing amino acids, aromatic and aliphatic amino acids [28–31]. Protein carbonyls are considered a marker of overall protein oxidation as they are formed early in stress situations and are a result of the action of a wide range of oxidants [32].
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These are major intermediates in radical-mediated protein oxidation (reviewed in: Refs. [40,50]), and can undergo metal-ion catalysed (pseudo-Fenton) reactions to give alkoxyl radicals. The latter can then undergo hydrogen atom abstraction reactions (to give alcohols, which have been detected in a range of biological samples [42–47]), or fragmentation, some of which give rise to additional carbonyl groups. These alkoxyl fragmentation reactions provide a mechanism for the observed loss of the carboxyl groups from aspartic and glutamic acids (Fig. 6; D-30 and E-30 species).
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Proteomic tools provide a promising way to decode disease mechanisms at the protein level and help to understand how carbonylation affects protein structure and function. The challenge for future research is to identify the type and nature of oxidised residues to gain a deeper understanding of the mechanism(s) governing carbonylation in cells and organisms and assess their role in disease.
Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions
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Oxidative damage to proteins results in a multitude of products (for reviews see [2–6]), arising from modification of a wide range of amino acids. These include damage to sulfur-containing, aromatic, and aliphatic amino acids [7–10] (Table 1). Protein carbonyls represent an irreversible form of protein modification and have been demonstrated to be relatively stable (degradation/clearance in hours/days) in contrast to lipid peroxidation products that are removed within minutes [11,12].
Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples.
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Citation Excerpt :
In the paper, we concentrated our work on tetrapeptide Arg-Gly-Asp-Cys (RGDC). In this peptide, the side chain of arginine is far more basic than any other sites in a peptide (the pKa of Arg is about 12.5) [29–35]. The peptide RGDC with a Cys residue (quite easy to oxidize, the rate constant for reaction of Cys with HO− is 3.5 × 1010 M−1 S−1) [36] was subjected to γ-ray oxidation.
The influence of charge distribution on the cleavage of the peptides was investigated by fragmentation efficiency curves and quantum chemical calculations in order to clarify the fragmentation mechanism in this paper. The peptide Arg-Gly-Asp-Cys (RGDC) was oxidized to change the charge distribution, but its main sequence was retained. Under this study, it was illustrated that the fragmentation of the peptide RGDC became easier with each addition of an O atom to the Cys hydrosulfide group and the relative charge ratios between O and N (Q_O/Q_N) in the amide bonds had much to do with the cleavage of the peptide RGDC. For each amide bond, the situations coincided with overall conclusion: the increase of the Q_O/Q_N values results in a higher fragmentation efficiency and vice versa. The methods which combined fragmentation efficiency curves with the charge distribution of peptides provided a way to refine the mobile proton model for peptide fragmentation and to probe the discrepant fragmentation of peptides in peptide/protein identification.
Mapping oxidations of apolipoprotein B-100 in human low-density lipoprotein by liquid chromatography-tandem mass spectrometry
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Citation Excerpt :
As mentioned earlier, tryptic peptides were searched against a full-length cDNA sequence of apo B-100 protein (P04114) listed in the IPI human database [1] using the SEQUEST algorithm. For large proteins such as apo B-100, it is difficult to get high sequence coverage via LC–MS/MS analyses of the peptides derived from tryptic digestion [19,23]. We achieved 48 ± 1% (two samples) sequence coverage from untreated LDL samples.
Human low-density lipoprotein (LDL) is a major cholesterol carrier in blood. Elevated concentration of low-density lipoprotein, especially when oxidized, is a risk factor for atherosclerosis and other cardiac inflammatory diseases. Past research has connected free radical initiated oxidations of LDL with the formation of atherosclerotic lesions and plaque in the arterial wall. The role of LDL protein in the associated diseases is still poorly understood, partially due to a lack of structural information. In this study, LDL was oxidized by hydroxyl radical. The oxidized protein was then delipidated and subjected to trypsin digestion. Peptides derived from trypsin digestion were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Identification of modified peptide sequences was achieved by a database search against apo B-100 protein sequences using the SEQUEST algorithm. At different hydroxyl radical concentrations, oxidation products of tyrosine, tryptophan, phenylalanine, proline, and lysine were identified. Oxidized amino acid residues are likely located on the exterior of the LDL particle in contact with the aqueous environment or directly bound to the free radical permeable lipid layer. These modifications provided insight for understanding the native conformation of apo B-100 in LDL particles. The presence of some natural variants at the protein level was also confirmed in our study.
The oxidative environment and protein damage
2005, Biochimica et Biophysica Acta - Proteins and Proteomics
Proteins are a major target for oxidants as a result of their abundance in biological systems, and their high rate constants for reaction. Kinetic data for a number of radicals and non-radical oxidants (e.g. singlet oxygen and hypochlorous acid) are consistent with proteins consuming the majority of these species generated within cells. Oxidation can occur at both the protein backbone and on the amino acid side-chains, with the ratio of attack dependent on a number of factors. With some oxidants, damage is limited and specific to certain residues, whereas other species, such as the hydroxyl radical, give rise to widespread, relatively non-specific damage. Some of the major oxidation pathways, and products formed, are reviewed. The latter include reactive species, such as peroxides, which can induce further oxidation and chain reactions (within proteins, and via damage transfer to other molecules) and stable products. Particular emphasis is given to the oxidation of methionine residues, as this species is readily oxidised by a wide range of oxidants. Some side-chain oxidation products, including methionine sulfoxide, can be employed as sensitive, specific, markers of oxidative damage. The product profile can, in some cases, provide valuable information on the species involved; selected examples of this approach are discussed. Most protein damage is non-repairable, and has deleterious consequences on protein structure and function; methionine sulfoxide formation can however be reversed in some circumstances. The major fate of oxidised proteins is catabolism by proteosomal and lysosomal pathways, but some materials appear to be poorly degraded and accumulate within cells. The accumulation of such damaged material may contribute to a range of human pathologies.

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^☆: A part of this report has been presented at the 10th International Dresden Symposium on Lipoproteins and Atherosclerosis, Dresden, Germany, 9–11 December 1999, and has been published in abstract form: Pietzsch, J., and Julius, U. (1999) Atherosclerosis147(Suppl. 2), S28.

²: Address correspondence to author. Fax: +49-351-4585374. E-mail: [email protected]..

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Regular ArticleMeasurement of 5-Hydroxy-2-aminovaleric Acid as a Specific Marker of Iron-Mediated Oxidation of Proline and Arginine Side-Chain Residues of Low-Density Lipoprotein Apolipoprotein B-100☆

Abstract

Free Radical Biol. Med.

Free Radical Biol. Med.

FEBS Lett.

Arch. Biochem. Biophys.

Methods Enzymol.

Free Radical Biol. Med.

Free Radical Biol. Med.

J. Chromatogr.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Atherosclerosis

Anal. Biochem.

J. Surg. Res.

J. Lipid Res.

Methods Enzymol.

J. Chromatogr.

J. Biol. Chem.

Regular Article
Measurement of 5-Hydroxy-2-aminovaleric Acid as a Specific Marker of Iron-Mediated Oxidation of Proline and Arginine Side-Chain Residues of Low-Density Lipoprotein Apolipoprotein B-100☆