Rapid Reaction of Hydrogen Sulfide with the Neutrophil Oxidant

Rapid Reaction of Hydrogen Sulfide with the Neutrophil

Oxidant Hypochlorous Acid to Generate Polysulfides

Supporting Information

Péter Nagy and Christine C. Winterbourn

Department of Pathology, University of Otago Christchurch, P.O. Box 4345,

Christchurch, New Zealand.

Materials and Methods

Reagents. All chemicals were A.C.S. certified grade or better. Water was purified by running through a Milli-Q system (Millipore) so that its resistivity was greater than 18 M Ω-cm. All reagents were purchased from Sigma-Aldrich (St Louis) unless otherwise indicated. The buffer solutions were prepared from NaH 2PO 4·2H 2O and Na 2HPO 4, the ionic strength was adjusted with NaCl, and the pH 2- was adjusted with ~6M HCl or freshly prepared ~6M NaOH (mostly free of CO 3 contamination). Unless stated otherwise, reactions were carried out at 25 oC and a constant ionic strength of 1.0 M. Diethylenetriamine-penta-acetic acid (DTPA; 50 µM) was added to the HS - solutions to chelate contaminating trace metal ions that can catalyze the otherwise slow oxidation of HS - by oxygen. HS - stock solutions were prepared fresh daily from the solid Na 2S·9H 2O. Na 2S·9H 2O crystals were washed and dissolved in doubly distilled H 2O. The concentration of the stock solution was determined colorimetrically by the reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to 5-thio-2-nitrobenzoic - - acid (TNB) as reported previously as well as by the absorbance of HS at 230 nm using ε230nm = 7700 M 1 -1 cm at pH > 8.5 (1). The purity of the solid Na 2S·9H 2O was investigated by comparing the measured and calculated (based on the mass) HS - concentrations. OCl - stock solutions were prepared from - commercially available Janola. Its concentration was measured spectrophotometrically ( ε292nm = 350 M 1cm -1). Solutions of OBr - were prepared by reacting OCl - with a large excess of Br -. Solutions of OBr- -1 -1 were also standardized spectrophotometrically at 329 nm ( ε329nm = 332 M cm ). The solutions of OBr- were used within an hour of preparation.

pH and [OH -] Measurements. The pH of the solutions were measured by a Metrohm pH-meter with an Ag/AgCl combination pH electrode, calibrated using potassium-borate/carbonate (pH 10.00) 2- and potassium-phosphate (pH 7.00). Sodium hydroxide solutions (mostly free of CO 3 contamination) that were used when measuring the pH-dependency of the pseudo first-order rate constants were quantified by titration with oxalic acid using phenolphthalein as an indicator.

UV/vis Spectroscopy. Electronic spectra were measured using an Agilent 8453 diode array spectrophotometer using quartz cells with calibrated 1 cm path lengths, or the PDA detector of the Applied Photophysics SX20 stopped-flow instrument equipped with a 150W Xe lamp.

Stopped-Flow Studies. Kinetic measurements were completed with an Applied Photophysics SX20 stopped-flow spectrophotometer using a Xe arc lamp. Monochromatic kinetic traces were collected using a photomultiplier detector, and polychromatic data with a photo diode array spectrophotometer attached to the observation cell. All kinetic data were collected at 1 M ionic strength (NaOH and NaCl) and at 25 oC. The temperature was maintained at 25 oC in the observation cell with a Haake Model DC10-K10 Refrigerated Circulator thermostat during the kinetic runs. In all kinetic experiments at least a 5 fold excess of HS - over OCl - was used to ensure pseudo first-order conditions. In the stopped-flow experiments error bars represent the standard deviation of the average of at least 6 kinetic measurements on the same solution. The concentration and pH dependencies of the pseudo first-order rate constants were obtained by linear least-squares fits of the data using Sigmaplot. [HS -] dependency: The concentration of HS - was varied in the range of 0.53 – 5.3 mM at a constant 100 µM [OCl -]. The experiment was carried out at 25 oC and at pH 13, which was achieved by adding 0.2M NaOH to the OCl - stock solution before mixing. The ionic strength was adjusted to I = 1M by NaCl. Kinetic traces were collected at 290 nm. [OH -]-1 dependency: The concentration of OH - was varied in the range of 10 – 250 mM. The final OH - concentrations were achieved by adding the appropriate amount of NaOH to the OCl - stock solutions before mixing. The data are from two series of experiments where [OCl -] was kept constant at 50 µM and [HS -] was either 1.6 or 3.4 mM. The experiments were carried out at 25 oC and I = 1M (NaOH + NaCl). Kinetic traces were collected at 290 nm.

One complication in the kinetic analysis is that because the product(s) had a larger absorbance at all wavelengths we could not follow the consumption of the reactants. In such a situation it is important to make sure that the reaction of interest is rate limiting. Therefore we have used HOBr as an alternative oxidant. The reaction of HS - with HOBr also resulted in the formation of polysulfides based on the similar absorbance spectra when the two different oxidants were used (not shown). However the reaction of HS - with OBr - was much faster under the same conditions and occurred during the mixing time of the stopped-flow instrument. This is in agreement with the previously reported analogous - reaction of these oxidants with other nucleophiles, such as SCN , and is due to the larger pK a of HOBr compared to HOCl (2, 3 ). In theory, this does not exclude the possibility that we were following the reaction of the SCl - intermediate with HS -. However, the absorbance change upon mixing OCl - with HS - (see Figure S1, dashed arrow) was similar to the absorbance change during the course of the reaction in an [OCl -] dependent manner and the initial absorbance on Figure S2a was consistent with the absorbance of the reactants, (i.e. that of OCl -'s, because HS - has negligible absorbance at 290 nm). Therefore it would imply that, unless the SCl - intermediate exhibit identical absorbance as OCl - (which is unlikely), we can exclude this possibility. Also our previous study had shown that the analogous sulfenyl chloride and sulfenyl bromide intermediates of Cys hydrolyze instantaneously (4). Therefore we believe that we are following the rate determining bimolecular reaction of HOCl with HS -.

Changes in the Uv-spectra upon the reaction of HS - with OCl -. Equal volumes of HS - and OCl - stock solutions were mixed under vigorous mixing condition to result in a final HS - = 30 mM and OCl - = 100 – 1200 µM OCl -. The HS - solutions contained 100 mM phosphate buffer and 100 µM DTPA and the final pH after mixing was either 7.4 or 11.4. Uv-spectra were recorded within 1 minute after mixing.

Qualitative and Quantitative Analysis of S 8. Polysulfides were generated by mixing 500 µL of HS - with 500 µL of OCl - stock solutions under vigorous stirring conditions. The reaction mixtures were acidified by the addition of 40 µL of 43% H 3PO 4 (final pH ~ 2) and was incubated in the dark at room temperature for 2h. The yellow precipitate was extracted into CHCl 3. To ensure the efficiency of the extraction the volume of the CHCl 3 phase was 5 times the volume of the aqueous phase. The organic phase was separated from the aqueous phase and its Uv-spectrum was recorded in a sealed cuvette. The obtained spectra were consistent with the formation of S 8 (see Figure 2). The experiment was repeated - - at several [OCl ]0 = 0.4 - 2.4 mM concentrations (using a control sample with no [OCl ]0) at a large - excess of HS = 60 – 180 mM and at pH = 7.4 or 13. The S8 concentration of the CHCl 3 phase after -1 -1 extraction was determined using ε280 = 811 M cm (that was determined previously by dissolving different amounts of the authentic sample of S 8 in CHCl 3) (5). In all cases > 85 % of the oxidizing equivalents were converted to S 8.

References (1) Nashef, A. S., Osuga, D. T. and Feeney, R. E. (1977) Determination of Hydrogen-Sulfide with 5,5'-Dithiobis-(2-Nitrobenzoic Acid), N-Ethylmaleimide, and Parachloromercuribenzoate. Anal. Biochem. 79 , 394-405. (2) Ashby, M. T., Carlson, A. C. and Scott, M. J. (2004) Redox Buffering of Hypochlorous Acid by Thiocyanate in Physiologic Fluids. J. Am. Chem. Soc. 126 , 15976-15977. (3) Nagy, P., Beal, J. L. and Ashby, M. T. (2006) Thiocyanate is an efficient endogenous scavenger of the phagocytic killing agent hypobromous acid. Chem. Res. Toxicol. 19 , 587-593. (4) Nagy, P. and Ashby, M. T. (2007) Reactive sulfur species: kinetics and mechanisms of the oxidation of cysteine by hypohalous acid to give cysteine sulfenic acid. J. Am. Chem. Soc. 129 , 14082-14091. (5) Nagy, P., Wang, X., Lemma, K. and Ashby, M. T. (2007) Reactive sulfur species: Hydrolysis of hypothiocyanite to give thiocarbamate-S-oxide. J. Am. Chem. Soc. 129 , 15756-15757.

Figure S1. Change in absorbance upon the reaction of OCl - with HS -. Uv-spectrum of 1.14 mM OCl - (solid line), 30 mM HS - (dotted line) and the reaction mixture of 1.14 mM OCl - with 30 mM HS - at pH = 13 (dashed line). The arrow represents the change in absorbance at 290 during the course of the reaction.

1.4 1.2 1.0 0.8 0.6 Absorbance 0.4 0.2 0.0 250 300 350 400 450 500 Wavelength (nm)

Figure S2. Concentration dependencies of the rate law for the reaction of HS - with HOCl. (a) Representative stopped-flow kinetic trace for the reaction of HS - with HOCl (black dots) and the corresponding exponential fit (red line). Conditions: 1.5 mM HS - was reacted with 54 µM OCl - at 125 mM NaOH and T = 25 oC at I = 1M (NaCl + NaOH). (b) HS - concentration dependency of the observed pseudo first-order rate constants and the corresponding linear fit. For conditions see Materials and Methods.

( a)

0.08

0.06

0.04

0.02 Expected absorbance Absorbance (290 nm) (290 Absorbance of 54 µM OCl - 0.00 0.00 0.04 0.08 0.12 t (s) (b)

300

250

200 ) -1

(s 150 obs k 100

0 0.0 2.0 4.0 6.0 - [HS ] (mM)

Figure S3. Spectral titration of HS - with OCl -. (a) Changes in the spectrum of the reaction mixture upon the addition of increasing amounts of HOCl/OCl- (0, 117, 234, 351, 468, 585 µM) to an excess of HS - (30 mM) at pH = 7.4. The inset shows a linear correlation between the increase in absorbance at 290 nm and [HOCl] tot (b) Change in absorbance at 290 nm and the corresponding linear fit upon the addition of increasing amounts of OCl - to 30 mM HS - at pH = 11.4 in 50 mM phosphate buffer, 50 µM DTPA at 25 oC. For conditions see Materials and Methods.

( a)

1.4 - 0.6 1.2 [OCl ] 0.4 1.0 0.2 0.8 (290nm) Absorbance

0.0 0.6 0 100 200 300 400 500 600

Absorbance [HOCl] ( µM) 0.4 tot 0.2 0.0 250 300 350 400 450 500 Wavelength (nm)

(b)

0.8

0.6

0.4

0.2 Absorbance (290nm) Absorbance ∆

0.0 0 400 800 1200 [HOCl] ( µM) tot