Reaction mechanisms in the radiolysis of peptides, polypeptides and proteins

Garrison, Warren M.

doi:10.2172/5415209

Cited by 105 publications

(169 citation statements)

References 44 publications

(76 reference statements)

Supporting

Mentioning

162

Contrasting

Unclassified

Order By: Relevance

“…To our knowledge, for the first time, the participation of residues other than aromatic in oxidative processes is demonstrated. Tryptophanyl radicals might reduce the peptide bond which would then undergo fragmentation, in agreement with [34]. Indeed, we found that Trp108 and/or Trp111, which are the closest to the Asn103-Gly104 sequence, are oxidized.…”

Section: Discussionsupporting

confidence: 86%

Re‐evaluation of intramolecular long‐range electron transfer between tyrosine and tryptophan in lysozymes

Stuart-Audette¹,

Blouquit

Faraggi

et al. 2003

European Journal of Biochemistry

View full text Add to dashboard Cite

One-electron oxidation of six different c-type lysozymes from hen egg white, turkey egg white, human milk, horse milk, camel stomach and tortoise was studied by gammaand pulse-radiolysis. In the first step, one tryptophan side chain is oxidized to indolyl free radical, which is produced quantitatively. As shown already, the indolyl radical subsequently oxidizes a tyrosine side chain to the phenoxy radical in an intramolecular reaction. However this reaction is not total and its stoichiometry depends on the protein. Rate constants also vary between proteins, from 120AEs )1 to 1000AEs )1 at pH 7.0 and room temperature [extremes are hen and turkey egg white (120AEs) and human milk (1000AEsIn hen and turkey egg white lysozymes we show that another reactive site is the Asn103-Gly104 peptidic bond, which gets broken radiolytically. Tryptic digestion followed by HPLC separation and identification of the peptides was performed for nonirradiated and irradiated hen lysozyme. Fluorescence spectra of the peptides indicate that Trp108 and/or 111 remain oxidized and that Tyr20 and 53 give bityrosine. Tyr23 appears not to be involved in the process. Thus new features of long-range intramolecular electron transfer in proteins appear: it is only partial and other groups are involved which are silent in pulse radiolysis.Keywords: gamma and pulse radiolysis; intramolecular longrange electron transfer; lysozyme; one-electron oxidation.The early suggestions [1,2] of intramolecular long-range electron transfer (LRET) have now been verified by numerous observations of LRET between donor/acceptor redox centres with known separation distances in proteins, peptides and other small rigid organic molecules. This property exists in polymers and especially in biopolymers (proteins and DNA). Thus these macromolecules are considered as candidates for nanoelectronics components. In this view, several studies were devoted to a better understanding of the factors leading to modulation of LRET (for a review, see [3] and references therein [4-6]). As part of a program to unravel the mechanistic basis of LRET in proteins, systematic investigations on electron transfer between tyrosine and tryptophan in proteins and peptides have been conducted [7][8][9][10]. In this experimental system, developed first by Pru¨tz et al. [11], pulse radiolytically generated azide radical and dibromide radical anion preferentially oxidize the side chain of tryptophan (Trp) to the indolyl radical (Trp AE ) in molecules that also contain tyrosine (Tyr).The initiating oxidation (1) can be effected by other radiolytically generated species such as the dibromide (Br 2 -) and dichloride (Cl 2 -) radical anions [12]. The tryptophan neutral radical, with a midpoint reduction potential that is near 1 V at pH 7.0 [13], rapidly oxidizes the tyrosine side chain to the phenoxy radical (TyrO AE ) in the intramolecular equilibration:Both TyrO AE and Trp AE absorb strongly in different parts of the visible region. One of the first observations of reaction (2) in proteins was obtained ...

show abstract

Section: Discussionsupporting

confidence: 86%

Re‐evaluation of intramolecular long‐range electron transfer between tyrosine and tryptophan in lysozymes

Stuart-Audette¹,

Blouquit

Faraggi

et al. 2003

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Oxidation of tryptophan is initiated by hydroxyl radical insertion either at the aromatic ring generating hydroxyphenyl or at the indole group that generates formylkinurenine [35]. The radiolytic products corresponding to hydroxy-phenyl have masses that are 116u or 1 32u (formylkinurenine [35]) greater than the unmodified side chain [32]. In the case of doubly oxidized peptide 1-16 ( Fig.…”

Section: Identification Of Oxidation Site(s) By Tandem Mass Spectromementioning

confidence: 99%

“…The similarity of the thermodynamic data (midpoint urea concentration, m values, and DG8 N -U ) derived from fluorescence analysis to data obtained based on oxidative modification of tryptophan side chains of helix A (Table 1) support the hypothesis that the structure of apomyoglobin is not altered during millisecond exposure to X-rays. This implies that reactions of oxygen-containing radicals are sufficiently fast [31,35,36] such that the oxidation of aminoacid side chains does not perturb the protein structure during radiolysis. Therefore, the method can be used to study protein folding and protein/ligand interactions that occur on timescales longer than several milliseconds.…”

Section: Extent Of Protein Oxidation As a Function Of Structurementioning

confidence: 99%

“…In these types of experiments, exposure times or chemical reaction times have been limited by single-hit kinetics requirements (fraction of cleaved molecules < 10 -30%) to assure that cleaved products are not further cleaved that could bias the analysis. Side chain oxidation of nucleic acid bases or amino acids occurs on similar timescales, and on a faster timescale than cleavage of backbone structure [35,36]. When electrophoresis methods are applied to quantify the extent of backbone cleavage products, the side chain oxidation products are not accounted for due to the resolution limits of the gel.…”

Section: Extent Of Protein Oxidation As a Function Of Structurementioning

confidence: 99%

“…The reactions of oxygenated radicals with proteins induce the modification of amino-acid side chains at rates of between 5 Â 10 9 and 1 Â 10 10 M 21 : s 21 . Amide bonds of the protein backbone are cleaved at a slower rate of 1 Â 10 9 M 21 : s 21 [35,36]. Previous radical-based oxidation studies have utilized the Fenton iron-EDTA/H 2 O 2 [37 -40] or peroxonitrous acid [41] reagents to generate radicals over several seconds to minutes.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Unfolding of apomyoglobin helices by synchrotron radiolysis and mass spectrometry

Maleknia¹,

Downard²

2001

Eur J Biochem

View full text Add to dashboard Cite

The synchrotron X-ray protein radiolysis technique is based on a quantitative determination of the extent and the site of millisecond radiolytic oxidation of amino-acid side chains by mass spectrometry. The amino acids most susceptible to radiolytic oxidation are cysteine, methionine, phenylalanine, tyrosine, tryptophan, proline, histidine, and leucine. These residues serve as reactive markers within a protein structure that can be used to monitor changes in solvent accessibility during folding or as part of macromolecular interactions. To monitor the unfolding, the extent of radiolytic products of side chains of reactive amino acids is quantitatively measured by mass spectrometry as a function of the denaturant concentration following proteolysis. This approach provides site-specific unfolding isotherms for various segments of a protein without the use of mutation or labeling techniques. Application of this technique to the equilibrium urea unfolding of apomyoglobin at pH 7.8 has demonstrated the cooperative unfolding of helices A to C consistent with midpoints, DG, and m values derived from fluorescence data. The G helix, in contrast, showed a local unfolding behavior. The similarity of the thermodynamic data derived by this synchrotron-based method for helix A (containing two oxidizable tryptophan residues) to that of the fluorescence data indicates that the limited oxidation of proteins by exposure to X-rays on millisecond timescales does not alter the structure of apomyglobin. This supports the viability of the method for the study of protein folding and the mapping of protein interaction sites.Keywords: protein folding; X-ray radiolysis; synchrotron; mass spectrometry; apomyoglobin.The interactions between amino-acid side chains play an important role in influencing protein structure and are significant for the stability of the hydrophobic core of the protein. Amino-acid side chains have well known propensities for secondary structure formation and influence the formation of helices and sheets [1 -6]. The specific nature and thermodynamic stability of these tertiary contacts are very important in understanding protein folding, protein structure, and formation of macromolecular complexes [4,[7][8][9][10][11], and have been shown to be useful in predicting misfolding or aggregation events in relation to disease [12][13][14][15][16][17].A common method for probing the nature and energetic contributions of these tertiary contacts has been the use of site-directed mutagenesis combined with chemical denaturation, where CD or fluorescence spectroscopy is commonly used as a probe, to generate isotherms allowing the global free energy change to be measured [4,7,8,18]. The small, single domain proteins usually analyzed by this method have a highly cooperative, two-state unfolding transition. Thus, for each mutation, the change in DG value (analyzed as a DDG ) can be interpreted as the specific sidechain contribution to the overall global stability. This analysis must be carefully applied because any particular terti...

show abstract

Reactivity of hypervalent iron with biological compounds

Bielski

1992

Ann Neurol.

View full text Add to dashboard Cite

Although it is well established that the ferryl (Fe==O2+) and perferryl (Fe==O3+) oxidation states of iron have a beneficial role in enzymatic oxidation and hydroxylation processes, as small complexes that are suspected to be formed in Fenton reactions, they may have deleterious effects on living cells. I discuss the basic chemistry of small hypervalent iron complexes. Their reactivity with biological compounds is illustrated by considering ascorbic acid and essential amino acids. A method is proposed for the determination of the ratio of [.OH]/[ferryl] in Fenton-type reactions.

show abstract

Reaction mechanisms in the radiolysis of peptides, polypeptides and proteins

Cited by 105 publications

References 44 publications

Re‐evaluation of intramolecular long‐range electron transfer between tyrosine and tryptophan in lysozymes

Re‐evaluation of intramolecular long‐range electron transfer between tyrosine and tryptophan in lysozymes

Unfolding of apomyoglobin helices by synchrotron radiolysis and mass spectrometry

Reactivity of hypervalent iron with biological compounds

Contact Info

Product

Resources

About