Elsevier

Free Radical Biology and Medicine

Volume 141, September 2019, Pages 475-482
Free Radical Biology and Medicine

Original article
Serum electrolytes can promote hydroxyl radical-initiated biomolecular damage from inflammation

https://doi.org/10.1016/j.freeradbiomed.2019.07.023Get rights and content

Highlights

  • Serum-level carbonate and halide concentrations alter *OH-mediated protein decay.

  • Conversion of *OH to carbonate radical altered the targeting of amino acids.

  • Serum electrolytes promoted tyrosine degradation in 3 of 4 proteins.

  • Serum electrolytes promoted activity loss in 2 of 3 enzymes.

Abstract

Chronic inflammatory disorders are associated with biomolecular damage attributed partly to reactions with Reactive Oxygen Species (ROS), particularly hydroxyl radicals (OH). However, the impacts of serum electrolytes on ROS-associated damage has received little attention. We demonstrate that the conversion of OH to carbonate and halogen radicals via reactions with serum-relevant carbonate and halide concentrations fundamentally alters the targeting of amino acids and loss of enzymatic activity in catalase, albumin and carbonic anhydrase, three important blood proteins. Chemical kinetic modeling indicated that carbonate and halogen radical concentrations should exceed OH concentrations by 6 and 2 orders of magnitude, respectively. Steady-state γ-radiolysis experiments demonstrated that serum-level carbonates and halides increased tyrosine, tryptophan and enzymatic activity losses in catalase up to 6-fold. These outcomes were specific to carbonates and halides, not general ionic strength effects. Serum carbonates and halides increased the degradation of tyrosines and methionines in albumin, and increased the degradation of histidines while decreasing enzymatic activity loss in carbonic anhydrase. Serum electrolytes increased the degradation of tyrosines, tryptophans and enzymatic activity in the model enzyme, ketosteroid isomerase, predominantly due to carbonate radical reactions. Treatment of a mutant ketosteroid isomerase indicated that preferential targeting of the active site tyrosine accounted for half of the total tyrosine loss. The results suggest that carbonate and halogen radicals may be more significant than OH as drivers for protein degradation in serum. Accounting for the selective targeting of biomolecules by these daughter radicals is important for developing a mechanistic understanding of the consequences of oxidative stress.

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.

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  • Cited by (6)

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    These authors contributed equally to this work.

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