Chem. Res. Toxicol. 2005, 18, 1669-1677
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The Role of Aromatic Amino Acid Oxidation, Protein Unfolding, and Aggregation in the Hypobromous Acid-Induced Inactivation of Trypsin Inhibitor and Lysozyme Clare L. Hawkins* and Michael J. Davies The Heart Research Institute, 114 Pyrmont Bridge Road, Camperdown, Sydney, NSW 2050, Australia Received July 27, 2005
Hypobromous acid (HOBr) generated by activated eosinophils has been implicated in tissue injury observed in asthma, allergic reactions, and some infections. Proteins are major targets for this oxidant, but the mechanisms by which HOBr induces loss of function are not wellestablished. In this study, we have examined the effect of HOBr on protein structure (as assessed by amino acid loss, side chain oxidation, fragmentation, aggregation, and unfolding) and activity of a model protease inhibitor, soybean trypsin inhibitor (STI), and the protective enzyme lysozyme. Exposure of both proteins to low oxidant concentrations (e5-fold molar excess) results in loss of function. In each case, loss of activity is associated with the selective oxidation of His, Trp, and Tyr residues, which results in protein unfolding (with lysozyme) and protein aggregation (with STI). Reaction with these residues accounts for 25 and 50% of the HOBr with STI (25-fold excess) and lysozyme (4-fold excess), respectively. These processes are believed to lead to changes in the structure of the proteins, which disrupts substrate binding. With both proteins, the oxidation of other residues, including Met, does not appear to play a major role. Bromamines, formed by reaction with amine groups, are major products, which account for 45 and 35% of the HOBr with STI (25-fold excess) and lysozyme (4-fold excess), respectively. Decomposition of these species correlates with secondary oxidation reactions, and with lysozyme, a time-dependent loss in activity. Overall, 70% of the HOBr can be accounted for with STI and 95% with lysozyme.
Introduction Hypobromous acid (HOBr)1 is produced at sites of inflammation via the release of eosinophil peroxidase from activated eosinophils and subsequent enzymecatalyzed oxidation of bromide ions (Br-) by H2O2 (1). At physiological pH, HOBr is in equilibrium with its ionized form -OBr [pKa ) 8.7 (2)]; HOBr is employed below to designate this mixture. There is also some evidence for the generation of HOBr by the enzyme myeloperoxidase (MPO), which is released by activated neutrophils, monocytes, and some macrophages (3, 4). Because of the magnitude of the specificity constants of MPO and the plasma levels of halide ions (Cl-, 100 mM; Br-, 20-100 µM; I- < 1 µM), it has been predicted that 10-fold molar excess HOBr resulted in the formation of an insoluble protein precipitate. In each case, the enzyme activity decreased on exposure to increasing concentrations of HOBr when compared to the native protein (Figure 1A,B). Significant decreases in activity were detected with a g5-fold molar excess of oxidant with STI and a g4-fold molar excess of oxidant with lysozyme. Loss of activity was rapid in both cases. A further small, but significant, decrease in activity was observed on incubation of STI and lysozyme (at 20 °C) with 25- and 4-fold molar excesses of HOBr, respectively, over 60 min (Figure 1C,D). As kinetic modeling calculations [based on the rate of the reaction of HOBr with protein components (22) and the amino acid composition of each
HOBr-Induced Enzyme Inactivation
Figure 1. Activity of STI and lysozyme after treatment with HOBr. Graphs represent the activity expressed as a percentage of the activity in the untreated control samples of (A) STI (23 µM) with 0-50-fold molar excess of HOBr and (B) lysozyme (850 nM) with 0-10-fold molar excess of HOBr for 15 min at 20 °C. Graphs of (C) STI (23 µM) treated with 25-fold molar excess HOBr and (D) lysozyme (850 nM) treated with 4-fold molar excess HOBr show the activity of the enzymes on incubation for 60 min at 20 °C. In all cases, the reaction was quenched at the required time by the addition of N-acetyl-Cys (50 mM). Statistically significant change *p < 0.05, **p < 0.01, and ***p < 0.001 as determined by one-way ANOVA in panels A and B, which compare samples to the untreated control with Dunnett’s posthoc testing and panels C and D, which compare different incubation times to the 10 s time point with Tukey’s multiple comparison test. Data are the means ( standard error of the mean of g4 experiments.
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protein] predict complete consumption of the added HOBr in 5- and 2-fold molar excesses of HOBr, respectively, for 15 min before quenching the reaction with N-acetyl-Cys (50 mM) resulted in a significant increase in the fluorescence (λex ) 387 nm and λem ) 479 nm) of the hydrophobic probe, ANS (Figure 2A,B). This increase in fluorescence is attributed to greater ANS binding to the protein as a result of the exposure of hydrophobic residues on protein unfolding. This unfolding was rapid for both proteins when 25- (for STI) and 4-fold (for lysozyme) molar excesses of oxidant were employed (Figure 2C,D), with changes complete by 5 min. No significant changes in ANS binding were observed in the absence of oxidant (Figure 2C,D). Protein fragmentation and aggregation were assessed by SDS-PAGE under reducing conditions. The reaction of STI and lysozyme (46.5 and 68.5 µM, respectively) for 15 min with increasing concentrations of HOBr (0-100fold molar excess for STI and 0-10-fold for lysozyme) resulted in fragmentation as evidenced by a loss in staining intensity of the parent protein band and the detection of additional bands with lower molecular mass at ∼6 and 14 kDa for STI and ∼4 kDa for lysozyme (Figure 3A,B). Aggregates were also observed with STI at ∼40 kDa and lysozyme at ∼30 kDa, consistent with dimer formation (Figure 3A,B). Both fragmentation and aggregation occurred to a much greater extent with STI than lysozyme. The nature of protein cross-links formed with STI was investigated further using protein where the His residues had been modified by DEP. A reduced extent of dimer formation was observed with the modified protein on reaction with HOBr (e25-fold molar excess) as compared to the native protein, as assessed by the density of the band observed at ∼40 kDa (Figure 4), consistent with His residues playing a role in cross-link formation. The time course of the changes observed with the native proteins was investigated using STI and lysozyme treated with a 25- or 4-fold molar excess of HOBr, respectively, for varying periods before examination by SDS-PAGE. Aggregate formation was observed at the shortest time point that could be examined after addition of the oxidant (10 s) (data not shown). With increasing incubation time (up to 180 min), a greater extent of smearing of all bands was observed (data not shown), consistent with secondary reactions playing a role in structural modification on extended incubation. Quantification and Stability of Protein Bromamines. N-Bromo species generated on reaction of HOBr with amine/amide groups on each protein were quantified using TNB after treatment of STI (20 µM) and lysozyme (125 µM) with a 25- and 4-fold molar excess of HOBr (500 µM), respectively (Figure 5). These species accounted for ca. 45 and 35% of the initial HOBr added with STI and lysozyme, respectively, after 2 min of incubation (Table 1). These species were unstable and decomposed rapidly with both proteins, with 75-80% of the bromamines lost after 30 min at 20 °C (Figure 5); the above yields are therefore likely to be underestimates as a result of this rapid decomposition.
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Figure 3. SDS-PAGE gels of STI and lysozyme treated with HOBr. Reaction of (A) STI (46.5 µM) and (B) lysozyme (68.5 µM) with increasing amounts of HOBr for 15 min before quenching with N-acetyl-Cys (20 mM). (A) Lane 1, STI only; lanes 2-7, STI treated with 5-, 10-, 25-, 50-, 75-, and 100-fold molar excess of HOBr, respectively. (B) Lane 1, lysozyme only; lanes 2-7, lysozyme treated with 1-, 2-, 4-, 6-, 8-, and 10-fold molar excess of HOBr, respectively.
Figure 2. Protein unfolding observed by ANS binding studies. Graphs represent the extent of protein unfolding as assessed by an increase in ANS fluorescence (λex ) 387 nm and λem ) 479 nm) on treating (A) STI (10 µM) with 0-50-fold molar excess of HOBr and (B) lysozyme (10 µM) with 0-10-fold molar excess of HOBr for 15 min at 20 °C. The effect of incubation time at 20 °C on the extent of protein unfolding is shown in the presence (closed circles) and absence (open circles) of oxidant. (C) STI treated with 25-fold molar excess HOBr and (D) lysozyme treated with 4-fold molar excess HOBr. In each case, the reaction was quenched at the required time by the addition of N-acetyl-Cys (5 mM). In (A), (B), statistically significant change as compared to the untreated control with **p < 0.01 as determined by one-way ANOVA with Dunnett’s posthoc testing. Data are the means ( standard deviation of the mean of six experiments.
Figure 4. Effect of His modification on dimer formation in HOBr-treated STI. The graph shows the density of the dimer band (in arbitrary units) at ca. 40 kDa observed on reaction of native STI (hatched bars) and STI pretreated with DEP (see Materials and Methods) to modify the His residues (dark bars) with a 0-25-fold molar excess of HOBr for 15 min before quenching with N-acetyl-Cys (20 mM). Statistically significant reduction in dimer formation comparing native to DEP-treated STI *p < 0.05 and **p < 0.01 as determined by two-way ANOVA with Bonferroni posthoc testing. Data are the means ( standard error of the mean of three experiments.
Quantification Side Chains and amino acid targets assessed by HPLC
of Loss of Specific Amino Acid the Formation of Products. The of HOBr on STI and lysozyme were analysis of total amino acids. Each
HOBr-Induced Enzyme Inactivation
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Figure 5. Stability of protein bromamines. The graph shows the concentration of bromamines observed on reaction of STI (20 µM) (marked with closed squares) and lysozyme (125 µM) (marked with open squares) with HOBr (500 µM) and subsequent incubation at 20 °C. Data are the means ( standard deviation of the mean of four experiments. Table 1. Comparison of the % Consumption of Oxidant after Reaction of STI and Lysozyme with a 25- and 4-Fold Molar Excess of HOBr, Respectively, Based on the Concentration of Amino Acid Residues Lost and Products Formed % consumption of oxidant amino acid/product
STI
loss of Met 3 loss of His 8 loss of Trp 4 formation of Br-Tyr 10 and diBr-Tyr formation of di-Tyr
