Original articleSerum electrolytes can promote hydroxyl radical-initiated biomolecular damage from inflammation
Graphical abstract
Introduction
Chronic inflammatory disorders [1], including atherosclerosis [2], arthritis [3], and several neurodegenerative diseases [4], are associated with damage to biomolecules (e.g., proteins) resulting from Reactive Oxygen Species (ROS), chlorine, and proteases emitted by leukocytes [[1], [2], [3], [4], [5], [6], [7]]. ROS include superoxide (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), although subsequent interactions produce Reactive Nitrogen Species (e.g., peroxynitrite) and chlorine [2]. High ROS levels are associated with oxidative stress, including damage to proteins [[1], [2], [3], [4], [5], [6], [7]]. Because •OH is far more reactive than O2•- and H2O2, •OH is believed to play an important role in damaging biomolecules [[1], [2], [3],6,7], but demonstrating the contribution of specific ROS is difficult.
ROS-associated biomolecular damage could result from intracellular ROS production by mitochondria or from ROS emitted to serum by leukocytes [[1], [2], [3]] or glial cells [4]. The impact of serum electrolytes on ROS reactivity with biomolecules has received little attention. Here, we demonstrate that •OH reactions with the high levels of carbonates and halides typical of serum can fundamentally alter the degradation of amino acids in proteins and enhance the loss of enzymatic activity. Accounting for serum electrolytes is critical for developing a fundamental understanding of inflammation-associated damage by ROS.
Hydroxyl radical reactions with carbonates and halides produce carbonate radical (CO3•-) and Reactive Halogen Species (RHS; e.g., •OH + Br− + Cl− → ClBr•- + OH−). Rate constants for amino acid reactions with these daughter radicals vary by orders of magnitude (for CO3•-, <103 M−1 s−1 for alanine and 4.5 × 107 M−1 s−1 for tyrosine) while those for •OH reactions approach the diffusion limit (4.3 × 108 M−1 s−1 for alanine and 1.3 × 1010 M−1 s−1 for tyrosine) [8,9]. While •OH would target amino acids approximately equally, its conversion to these daughter radicals focuses their oxidizing power on the more reactive amino acids, thereby increasing their observed degradation rates (Fig. 1A).
Previous studies have demonstrated that, relative to •OH, CO3•- can selectively degrade methionine, tryptophan and tyrosine within polypeptides [10,11] or alter protein degradation pathways (e.g., promote lysozyme dimerization) [11]. However, these studies generated CO3•- under conditions clearly favoring CO3•- over •OH (e.g., reaction of •OH with 700 mM carbonates at pH 10) [10]. The importance to protein degradation of CO3•- and RHS generated from •OH under serum-relevant conditions was unclear. Davies et al. demonstrated increased tryptophan losses in bovine serum albumin during γ-radiolysis in the presence of 100 mM carbonate at pH 7 [6]. An elegant γ-radiolysis study by Wolcott et al. demonstrated that synthetic solutions containing chloride (150 mM) and carbonate (100 mM) concentrations relevant to the leukocyte phagosome increased bacterial inactivation, due to the generation from •OH of long-lived oxidants, particularly CO3•- [12]. While •OH dominated the oxidation of dissolved fluorescein (an oxidation probe), reactions of longer-lived oxidants dominated for particle-bound fluorescein [12]. These results suggested that short-lived •OH would dominate the oxidation of proteins dissolved in serum, but protein sequestration in bacteria or tissue cell membranes could restrict reactivity to oxidants with sufficient lifetimes to permit transport to the particle-bound target. We demonstrate the importance of these daughter oxidants for selective targeting of amino acids and loss of enzymatic activity even for aqueous proteins under serum conditions.
Section snippets
Chemical reagents
Hydrogen peroxide (30% solution), ammonium formate (≥99%, Optima™ LC/MS grade) and 6-aminoquinoline-N-hydroxy-succinimidyl ester (AQC) were purchased from Fisher Scientific (Waltham, MA, USA). l-Tyrosine (99.5%) was purchased from Chem Service Inc. Tryptamine (98%), γ-aminobutyric acid (≥99%), sodium phosphate monobasic dihydrate (99%), and sodium phosphate dibasic (99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-Acetylated amino acids included: Sigma-Aldrich (St. Louis, MO, USA)
Effect of serum electrolytes on N-acetyl amino acid degradation
A mixture of 50 μM each of the N-acetylated analogues of the 20 common amino acids was exposed to •OH generated from water by steady-state γ-radiolysis at pH 7.4 (10 mM phosphate buffer) with or without serum levels of chloride (100 mM), bromide (60 μM) and carbonates (20 mM) [25]. The N-acetyl group mimicked the peptide bond. These electrolytes were evaluated because of their high serum concentrations and reaction rate constants with •OH relative to other serum electrolytes [9]. Serum
Conclusions
Understanding how amino acid interactions translate into protein structure and function is a complex problem, even without accounting for oxidatively generated modifications to these amino acids. Our results highlight the need for research on oxidative stress associated with chronic inflammatory disorders to account for the role of daughter radicals produced from interactions of •OH with serum electrolytes. Serum electrolytes altered the degradation of amino acids in all four proteins and of
Declarations of interest
None.
Acknowledgements
We thank the members of the Mitch and Herschlag labs for sharing protocols and scientific discussions. Y.K. was supported by the Japan Society for the Promotion of Science Overseas Research Fellowships. A.S. was supported by a Stanford Graduate Fellowship. J.C. was partially supported by the Creative-Pioneering Researchers Program at Seoul National University. M.M.P. was supported by an NSF Graduate Research Fellowship and KSI work was supported by NSF Grant MCB-1714723.
References (29)
- et al.
Protein damage and degradation by oxygen radicals
J. Biol. Chem.
(1987) What really happens in the neutrophil phagosome?
Free Radic. Biol. Med.
(2012)- et al.
A fast photochemical oxidation of proteins (FPOP) platform for free-radical reactions: the carbon radical anion with peptides and proteins
Free Radic. Biol. Med.
(2019) - et al.
Production of lysoozyme and lysozyme-superoxide dismutase dimers bound by a ditryptophan cross-link in carbonate radical-treated lysozyme
Free Radic. Biol. Med.
(2015) - et al.
Bactericidal potency of hydroxyl radical in physiological environments
J. Biol. Chem.
(1994) - et al.
A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase
J. Biol. Chem.
(1952) - et al.
Electrometric and colorimetric determination of carbonic anhydrase
J. Biol. Chem.
(1948) - et al.
The active center of catalase
J. Mol. Biol.
(1985) - et al.
Reactive oxygen species in inflammation and tissue injury
Antioxidants Redox Signal.
(2013) - et al.
Role of oxidative modifications in atherosclerosis
Physiol. Rev.
(2004)
Free Radicals and Inflammation: protection of synovial fluid by superoxide dismutase
Science
Oxidative stress in neurodegeneration: cause or consequence?
Nat. Med.
Neutrophil recruitment and function in health and inflammation
Nat. Rev. Immunol.
Rate constants for the reaction of carbonate radical with compounds of biochemical interest in neutral aqueous solution
Radiat. Res.
Cited by (6)
The Changes of Antioxidant Activity of Dendrobium huoshanense Jiaosu
2023, Journal of Chinese Institute of Food Science and TechnologyDominant Dissolved Oxygen-Independent Pathway to Form Hydroxyl Radicals and the Generation of Reactive Chlorine and Nitrogen Species in Breakpoint Chlorination
2023, Environmental Science and TechnologyMisconceptions about the chemistry of aqueous chlorine atoms and HClOH˙(aq), and a revised mechanism for the photochemical peroxydisulfate/chloride reaction
2022, Physical Chemistry Chemical PhysicsDefective Ag-In-S/ZnS quantum dots: an oxygen-derived free radical scavenger for mitigating macrophage inflammation
2021, Journal of Materials Chemistry B
- 1
These authors contributed equally to this work.