Materials. Met-HemoCD-P and met-hemoCD-I were synthesized in our laboratory as described in previous reports.25 We used sodium hydrogen sulfide (NaSH) as a hydrogen sulfide source. NaSH was purchased from Stream Chem. Inc. Before the use of NaSH, its purity was determined by iodometric titration.30 All other reagents were purchased and used as received.
Preparation of the NaSH solution. Since NaSH is easily oxidized in water, we prepared a stock solution of NaSH before each application. Milli-Q water was deoxidized by nitrogen gas bubbling for 30 min. Then, NaSH was solubilized in deoxidized water and used as a stock solution. Once a NaSH solution was prepared, it was used within 3 h. The concentration of NaSH in solution, C [mM], was determined as follows:
$$C \left[\text{m}\text{M}\right]=\left\{\left(x \left[\text{m}\text{g}\right]\times y\left[\%\right]\times {10}^{3}\right)/\left(56.063\left[\text{g}\bullet {\text{m}\text{o}\text{l}}^{-1}\right]\times z\left[\text{m}\text{L}\right]\right)\right\}$$
where x is the weight of NaSH, y is the purity of NaSH determined by iodometric titration as described above, and z is the volume of the stock solution.
Instruments. UV‒vis spectra were recorded on a Shimadzu UV-2450 spectrophotometer with a thermostatic cell holder. Kinetic studies were carried out using a JASCO FS-110 Fast Scan Spectrometer with a thermostatic cell holder. EPR measurements were obtained with a Bruker E500 spectrometer at the Institute for Molecular Science in continuous-wave (CW) mode operating at ~ 9.66 GHz and equipped with an Oxford Instruments ESR900 continuous helium flow cryostat. The experimental parameters were 5 mW microwave power, 100 kHz field modulation, and 10 G modulation amplitude. MALDI-TOF mass spectra were measured on Bruker Daltonics Autoflex speed spectrometers in linear mode. a-Cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix. ESI-TOF mass spectra were taken on a JEOL JMS-T100CS spectrometer.
Spectroscopic measurements. The binding constants (K) of hydrogen sulfide to met-hemoCD-P and met-hemoCD-I were determined by UV‒vis photometric titration. NaSH (up to 50 mM) was added to an aerobic solution of met-hemoCD-I or met-hemoCD-P (5 µM) at 25°C in 0.05 M phosphate buffer at pH 6.0, 7.0, 7.4 and 8.0. The absorbance changes versus [NaSH] were plotted, and the data were fitted to a theoretical curve of an equation for 1:1 complex formation to obtain K.
The association rate constants (kon) for the binding of hydrogen sulfide to met-hemoCD-P and met-hemoCD-I were obtained under pseudo-first-order conditions with excess NaSH at 25°C. An aerobic solution of met-hemoCD-P or met-hemoCD-I (5 µM) was mixed rapidly with various concentrations of NaSH (100–1400 µM before mixing) in 0.05 M phosphate buffer at pH 7.0, 7.4 or 8.0. The change in absorbance at 410 nm was monitored, and the data were fitted to a single or double exponential function to obtain the observed rates kobs. When the reaction between met-hemoCD and hydrogen sulfide was too fast (kobs > 0.5), a double exponential fitting was required due to an unknown slow reaction component. In that case, the component of kfast occupied more than 80% of the total. In addition, that ratio was independent of pH. Therefore, we adopted kfast as the actual kobs. (reaction between met-hemoCDs and hydrogen sulfide), while kslow is possibly due to the reaction between met-hemoCDs and impurities derived from NaSH, such as polysulfides.45 Finally, the kon values were obtained from linear regression of kobs as a function of the hydrogen sulfide concentration.
Quantification of hydrogen sulfide. An aqueous solution of NaSH (1.0 mM) was mixed with met-hemoCD or met-Hb (50 µM) and incubated at 25°C. The residual hydrogen sulfide concentrations were determined using the reported “methylene blue” method with some modifications.46,47 The solution containing hydrogen sulfide and its scavenger was diluted to 2.5 mL with 0.05 M phosphate buffer at pH 7.4 to adjust the total hydrogen sulfide concentration to less than 50 µM. To the solution was added trifluoroacetic acid (0.5 mL). Then, solutions of N,N-dimethyl-p-phenylenediamine sulfate (200 mM stock solution, 30 µL) dissolved in HClaq (7.2 mM) and FeCl3 (300 mM stock solution, 30 µL) dissolved in 1.2 mM HCl were added successively. The solutions were allowed to stand for 20 min at ambient room temperature. Finally, the absorbance at 663 nm was read, and the hydrogen sulfide concentration was determined based on the standard curve of methylene blue.
Quantification of sulfate and sulfite. The relative production rate of sulfate and sulfite ions from hydrogen sulfide was determined by a simple turbidimetric method, in which the amount of sulfate (SO42–) was quantified by turbidity through the following reaction:48
SO42– + BaCl2 → BaSO4↓ + 2Cl–
The average absorbance between 700 nm and 800 nm was used as turbidity, where met-hemoCD-I and other scavengers showed no absorbance in the area. The turbidity showed a linear correlation with the sulfate concentration from 0.5 mM to 4.0 mM upon the addition of 1.5 equivalents of BaCl2. We also confirmed that sulfite (SO32–) ions form a similar insoluble precipitate of BaSO3 with BaCl2 as follows:49
SO32– + BaCl2 → BaSO3↓ + 2Cl–
Therefore, the relative production of sulfate and sulfite ions in solution was simply determined by reading the average absorbance between 700 nm and 800 nm after treatment of the solution with BaCl2.
Animal experiments. All animal studies were performed under the approval of Doshisha University and carried out in accordance with the Guidelines for Animal Experiments of Doshisha University. We used female BALB/cCrSlc mice (Shimizu Laboratory Supplies, Co., Ltd.) weighing 20–22 g. The study is reported in accordance with ARRIVE guidelines. For acclimatization, the mice were housed under a 12 h/12 h light/dark cycle with free feedings under specific-pathogen-free (SPF) conditions for one week before the day in the experiments.
For survival analysis of met-hemoCD-I in lethal hydrogen sulfide intoxication model mice, a solution of NaSH (21 mg/kg) in PBS (0.1 mL) was injected intraperitoneally. One minute after the injection of NaSH, a solution of met-hemoCD-I (7 mM) in PBS (0.2 mL) was injected intraperitoneally. For the predosing experiments, a solution of met-hemoCD-I (7 mM) in PBS (0.2 mL) was injected intraperitoneally. After 10 min, a solution of NaSH (21 mg/kg) in PBS (0.1 mL) was injected intraperitoneally. Survival rates were then monitored for one hour after hydrogen sulfide intoxication was induced.
We determined the CcO activity and the concentration of lactate in the blood of lethal hydrogen sulfide intoxication model mice. Tissue samples (brain, heart, and liver) were collected from met-hemoCD-I-treated surviving mice one hour after hydrogen sulfide intoxication was induced. As a control, we collected tissue samples (brain, heart, and liver) from untreated mice immediately after death. The CcO activity in these organs was determined by the following method according to the literature.50 Each tissue sample (~ 20 mg) was homogenized in 0.5 mL of sucrose muscle homogenization buffer (250 mM). Then, the suspension was centrifuged at 600 g for 10 min at 4°C. The supernatant (5 µL) was added to a 1 mL cuvette which contains 400 µL of Milli-Q water, 500 µL of potassium phosphate buffer (0.1 M, pH 7.0) and 50 µL of reduced cytochrome c (Product name; ab109911, abcam, UK). The absorbance at 550 nm was read for 3 min. The rate of activity (OD/min) was determined by calculating the slope between two points within the linear region. Finally, the CcO activity was determined by normalizing the rate activity by its protein amount in tissue using BCA assay (Thermo Fisher Scientific, Japan). The concentration of lactate in the blood was measured using LT-1730 Lactate Pro2 (Arkray).
Statistical analysis. Statistical analyses were performed using GraphPad Prism, version 10.2.3 (GraphPad Software). All the data are presented as the means ± standard errors from at least three different experiments and were analyzed by Student's t test. Survival curves were analyzed using Kaplan‒Meier curves and the log-rank test. Differences with P values less than 0.05 were considered significant.