Low-temperature EPR spectra of ferricyt <i>c</i> treated with AA.

<p>(A) EPR spectra of (300 µM) ferricyt <i>c</i> after a 12-h incubation with (1.0–5.0 mM) AA. (B) Time-response ERP spectra of ferricyt <i>c</i> treated with (1.0 mM) AA for 30 min. Incubation conditions: 50 mM phosphate buffer, pH 7.4, at 37°C. (C) MCD spectra of ferricyt <i>c</i> in the presence of AA. The conditions are ferricyt <i>c</i> (40 µM) MCD spectrum before treatment with AA (thick line) and MCD ferricyt <i>c</i> spectrum after 12 h of (5.0 mM) AA treatment (thin line). (D) CD spectrum of ferricyt <i>c</i> treated with 5.0 mM AA. Incubation conditions: 50 mM phosphate buffer, pH 7.4, at 37°C.</p

Proposed mechanism of AA oxidation catalyzed by iron and copper ions (Adapted from Dutra et al.[14]).

<p>Proposed mechanism of AA oxidation catalyzed by iron and copper ions (Adapted from Dutra et al.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057790#pone.0057790-Dutra1" target="_blank">[14]</a>).</p

EPR spin-trapping studies and computer simulation of the AA system in the presence and absence of ferricyt <i>c</i>, under aerobic conditions.

<p>EPR spectra of DMPO-radical adducts were obtained after a 4-min incubation of (15 mM) AA at 25°C with (150 µM) cyt <i>c</i> in 50 mM phosphate buffer (pH 7.4) with (400 mM) DMPO. (A) Experimental spectrum (trace a) and computer simulations (traces b-d) of the DMPO/AA system, (B) Experimental spectrum (trace a) and computer simulations of the DMPO/AA/cyt <i>c</i> system (traces b-e). Trace <i>c</i> in panels A and B represents the DMPO-<b>^•</b>OH adduct spectrum, and trace d can attributable to the DMPO-AA<b>^•</b> adduct. Trace e in panel B represents an unknown DMPO adduct. Instrumental conditions: microwave power, 20.2 mW; modulation amplitude, 1.0; time constant, 1.63 s; scan rate 0.1 G/s; and receiver gain, 1.12×106.</p

EPR spin-trapping studies of the ferricyt <i>c</i>/AA system under aerobic conditions.

<p>EPR spectra of DMPO-radical adducts were obtained after a 4-min incubation of 15 mM AA at 25°C in 50 mM phosphate buffer (pH 7.4) with (25 mM) DMPO: (A) DMPO experiments, (B) DMPO in the presence of DMSO 30% v/v, (C) DMPO in the presence of ethanol 30% v/v. For all of the figures: (a) control with ferricyt <i>c</i> (150 µM); (b) AA (15mM); (c) AA (15 mM)+desferoxamine (100 µM); (d) ferricyt <i>c</i> (150 µM)+AA (15 mM); (e) system d+CuZnSOD (50 U/mL); (f) system d+catalase (15 µM) for Fig. 2A and 2C only. Instrumental conditions: microwave power, 20.2 mW; modulation amplitude, 1.0; time constant, 1.63 s; scan rate 0.1 G/s; and receiver gain, 1.12×106.</p

Oxygen uptake by AA in the presence of ferricyt <i>c</i>.

<p>Experimental conditions: (50 µM) ferricyt <i>c</i> in the presence or absence of (5.0 mM) AA in 50 mM phosphate buffer, pH 7.4, at 37°C for 30 min. Experiments were performed in the absence or presence of catalase (5.0 µM) or CuZnSOD (50 U/mL). Data are representative of five independent runs. *p<0.05 relative to the system containing only AA and #p<0.05 relative to the AA/ferricyt <i>c</i> system.</p

The effect of heparin on steady-state FRET-peptide hydrolysis by HNE.

<p>The influence of heparin concentration upon steady-state HNE kinetics parameters <i>k</i>_cat [A] and <i>K</i>_M [B] was determined spectrofluometricallyas described under “Experimental Procedures”. The fluorescence increase of FRET-peptide hydrolysis (0.2 - 10 µM) by 12.6 nM HNE were performed in 10 mM Hepes, pH 7.4, containing 140 mM NaCl and 0,05% Triton X-100 at 37°C.</p

Representation of the complex of both FRET-peptide and heparin with HNE.

<p><i>A</i>, Docking of HNE with the substrate AMESVMGYFHRSQ,the secondary structure elements of the HNE are represented by <i>indigo blue schematics</i> (<i>arrows</i> for extended strands, and <i>cylinders</i> for helices).The carbon atoms of the substrate are indicated by a <i>green sticks</i>, view of the minimum energy conformation from docking, showing the substrate labeled at P1Val and P1'Met completely engulfed inside the HNE active site cavity: S195, H57 and D103 (dark blue for nitrogen atoms, light yellow for carbon atoms, red for oxygen atoms, grey for hydrogen atoms and dark yellow for sulfur). HNE active site residues are labeled following the numbering of chymotrypsin. <i>B</i>, Docking of HNE with heparin,the secondary structure elements of the HNE are represented by <i>green schematics</i> (<i>arrows</i> for extended strands, and <i>cylinders</i> for helices) and the catalytic residues of the HNE are labeled.The carbon atoms of heparin chain are indicated by a <i>cyan sticks</i> and its sulfur atoms are indicated by dark yellow. <i>C</i>, Enlarged view of the HNE catalytic residues S195, H57 and D103 with substrate labeled at P1Val and P1'Met. <i>D</i>, Stereo view of the ternary complex between HNE•Heparin•Substrate, the secondary structure elements of the HNE are represented by <i>green schematics</i>. The carbon atoms of the substrate are indicated by a <i>cyan sticks</i> and the carbon atoms of heparin are indicated by <i>magenta sticks</i>.</p

Stopped-flow time-tracesofFRET-peptidehydrolysis by HNE.

<p><i>A</i><b>,</b> Stopped-flow fluorescence kinetic recording of 3.8 µMFRET-peptide hydrolysis by 12.6 nM HNEat 25°C in 10 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl. The progress of the reaction was monitored by the fluorescence increase of the released product recorded on 2 adjacent time regions with distinct sampling periods: 0.5 ms from 0 to 2 s, 2 ms from 2 to 6 s. Gray solid line represents the best fit obtained from the mechanism depicted in Scheme I in the absence of heparin with the aid of DynaFit IV® software (see Experimental Procedures). The insert graphic represents the associate residual errors from the best fit curve with experimental data. <i>B</i>, the HNE species as a function of time reaction: free enzyme, E (<b>–</b>); complex enzyme-substrate, ES <b>(–</b> •<b>–</b>) and acyl-enzyme, ES' (<b>- - -</b>).</p

Steady-state kinetic parameters for hydrolysis of fluorogenic substrates by human neutrophil elastase in the presence of heparin.

a<p>Reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021525#pone.0021525-Spencer1" target="_blank">[4]</a>.</p

Stopped-flow time-traces of FRET-peptidehydrolysis by HNE in the presence of heparin.

<p><i>A</i>, the stopped-Flow fluorescence kinetic recording of 3.8 µMFRET-peptide hydrolysis by 12.6 nM HNEperformed at 25°C in 10 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl supplemented with 50 µM heparin. The progress of the reaction was monitored by the fluorescence increase of the released product recorded on two adjacent time regions with distinct sampling periods: 0.5 ms from 0 to 2 s, 2 ms from 2 to 4 s. Gray solid line represents the best fit deduced from the mechanism depicted in Scheme I in the presence of heparin using DynaFit IV® Software [see Experimental Procedures]. The insert graphic represents the associate residual errors from the best fit curve with experimental data. <i>B</i>, the HNE species in function of time reaction in presence of heparin: complex enzyme-heparin, EH (<b>–</b>); complex enzyme-substrate-heparin, ESH <b>(–</b> •<b>–</b>) and complex acyl-enzyme-heparin, ES'H (<b>- - -</b>).</p