Na2s2o4

BZP pellets were synthesized at the College of Chemistry and Molecular Engineering of Zhengzhou University. Each 100-mg pellets contain 10.83 mg BZP. The purity of BZP was 99.4%. BZP was dissolved in DMEM (purchased from Corning Co., Ltd.) to a final concentration of 100 mmol/L, then stored at 4°C until use. C8E4 was purchased from Shanghai Civic Chemical Technology Co., Ltd. It was dissolved DMSO at a concentration of 100 mmol/L and stored at 4°C until use. Stock solutions were diluted with 0.9% saline or DMEM prior to use. Triphenyl chloro tetrazole (2,3,5-triphenyl tetrazolium chloride, TTC) was purchased from Sigma Inc, and dissolved to final concentration of 2% in distilled water prior to use. Nylon thread was purchased from Beijing Shadong Biotechnology Co., Ltd. Paraformaldehyde (4%) was prepared by dissolving 40 g of paraformaldehyde powder in 1000 ml of PBS in a 40°C water bath, then allowed to cool prior to use. Na₂S₂O₄ was purchased from Sigma Co., Ltd. Phosphate buffer (PBS, pH 7.4) was sealed after high pressure sterilization and stored at 4°C. Trypsin was purchased from Solabel Biotechnology Co., Ltd. and stored at 4°C until use.

Na₂MoO₄·2H₂O, R-lactic acid, 1,2,4-triazole and Na₂S₂O₄ were purchased from Sigma. All solvents and chemicals were of commercially analytical grade. pH value was determined by PHB-8 digital pH meter. Elemental analyses (for C, H, and N) were performed with an Vario ELIII elemental analyzer. Spectra of vibrational circular dichroism for 1, 2 and {Na₂[Δ-Mo^*O₂(R-lact^*)₂]}₃ ∙ 13H₂O, {Na₂[Λ-Mo^*O₂(S-lact^*)₂]}₃ ∙ 13H₂O were recorded on a BioTools Chiral IR-2X spectrometer, where solid samples were mixed in KBr pellets at a mass ratio of 1:100, respectively. The VCD spectra of extracted FeMo-co and K₅[Λ,Λ,Λ,Λ-Mo^*₄O₁₁(R-Hhomocit^*)₂]Cl∙5H₂O were measured by Bruker VCD/IRRAS module PMA 37 with MCT detector. To minimize the NMF left in the FeMo-co film, the samples were pumped for more than 8 h before the measurement. More physical measurements, preparations of [Λ/Δ-Mo^*₂O₂(μ₂-S)(μ₂-O)(S-Hlact^*)₂(trz)₂(trz)] (2), [Mo₂O₂(μ₂-S)(μ₂-O)(Hglyc)₂(trz)₂(H₂O)] (3), {Na₂[Δ-Mo^*O₂(R-lact^*)₂]}₃ ∙ 13H₂O and K₅[Λ,Λ,Λ,Λ-Mo^*₄O₁₁(R-Hhomocit^*)₂]Cl∙5H₂O (21), gas adsorption and X-ray crystallography can be seen in the Part I of the Supplementary Methods of Supporting Information.

Human haemoglobin (Sigma Aldrich) was dissolved in water (20 mg/ml) and reduced by a 10-fold molar excess of sodium dithionite (Na₂S₂O₄; Sigma Aldrich). Excess reductant was removed by gel filtration over Sephadex G-25 (PD10 desalting column; GE Healthcare) according to the manufacturer’s instructions. Oxyhaemoglobin (OxyHb) was eluted with 3.5 ml of water, and only the middle run was collected. The concentration of OxyHb was determined spectrophotometrically, as described in.²⁶ Aliquots of the OxyHb stock solution were kept at −80°C, thawed on the day of experimentation and discarded after use.

CYP3A4 and CYP2C9 holo P450 protein content in the enriched protein samples was determined using carbon monoxide spectral assays^{39 (link)}. CYP450 protein samples were diluted in storage buffer to a concentration of 1 mg/ml and divided into two cuvettes (one reference and one sample cuvette). The cuvettes were placed into a Varian Cary 50 conc UV Visible spectrophotometer (Varian, Australia) and a baseline reading between 400 and 500 nm was recorded. Carbon monoxide (Speciality Gasses, SA) was bubble into the bottom of the sample cuvette (~60 bubbles at a rate of 1 bubble per second). A small spatula tip (~1 mg) of sodium dithionite (Na₂S₂O₄, Sigma-Aldrich, Germany) was added to each cuvette. Cuvettes were covered with parafilm and inverted several times to dissolve the sodium dithionite. The cuvettes were then placed back inside the spectrophotometer and spectra between 400 and 500 nm were measured several times until the peak at 450 nm stopped increasing.
Readings from the final spectra were used to calculate the amount of P450 and P420 protein present in the sample using the following equations: $$[{({\mathrm{\Delta }\mathrm{A}}_{450}-{\mathrm{\Delta }\mathrm{A}}_{420})}_{\mathrm{observed}}-{({\mathrm{\Delta }\mathrm{A}}_{450}-{\mathrm{\Delta }\mathrm{A}}_{420})}_{\mathrm{baseline}}]/0.091=\mathrm{nmol}\mathrm{P}450\mathrm{per}\mathrm{ml}$$ $$\begin{array}{l}[{({\mathrm{\Delta }\mathrm{A}}_{420}-{\mathrm{\Delta }\mathrm{A}}_{490})}_{\mathrm{observed}}-{({\mathrm{\Delta }\mathrm{A}}_{420}-{\mathrm{\Delta }\mathrm{A}}_{490})}_{\mathrm{theoretical}}\\ –[{({\mathrm{\Delta }\mathrm{A}}_{420}-{\mathrm{\Delta }\mathrm{A}}_{490})}_{\mathrm{baseline}}]/0.110=\mathrm{nmol}\mathrm{of}\mathrm{P}420\mathrm{per}\mathrm{ml}\end{array}$$ where $${({\mathrm{\Delta }\mathrm{A}}_{420}-{\mathrm{\Delta }\mathrm{A}}_{490})}_{\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}}=(\mathrm{n}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{P}450\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{l}\mathrm{f}\mathrm{r}\mathrm{o}\mathrm{m}[1.1])\times (-0.041)$$

Glutaraldehyde (50–70%), NaCl (sodium chloride), KCl (potassium chloride), NaOH (sodium hydroxide), Na₂S₂O₄ (sodium dithionite), CaCl₂.2H₂O (calcium chloride), sodium lactate, N-acetyl-L-cysteine (NALC), NaCNBH₃ (sodium cyanoborohydride), NaBH₄ (sodium borohydride), Na₂HPO₄ (sodium phosphate dibasic), and NaH₂PO₄ (sodium phosphate monobasic) were procured from Sigma-Aldrich (St. Louis, MO). The HF tangential flow filtration (TFF) modules (rated pore sizes: 0.2 μm, 50 nm, 500 kDa, and 100 kDa) were purchased from Spectrum Laboratories (Rancho Dominguez, CA). K₃FeCN₆ (potassium ferricyanide), KCN (potassium cyanide) and all other chemicals were purchased from Fisher Scientific (Pittsburgh, PA). Expired human RBC units were generously donated by Transfusion Services, Wexner Medical Center, The Ohio State University, Columbus, Ohio.

PreOLs were divided into four groups as follows: control group (CTL), OGD-treated group (OGD), catalpol-treated group (CAT), and KB-R7943+catalpol-treated group (KB+CAT). The OGD model was established as described previously to mimic an in vitro model of cerebral ischemia [24 (link)]. Briefly, PreOL cultures were incubated in glucose-free DMEM medium (Gibco Life Technologies, Carlsbad, CA, USA) with 8 mM Na₂S₂O₄ (Sigma-Aldrich, St Louis, MO, USA) at 37 °C for 30 min to scavenge O₂ molecules in solution and to reduce the partial pressure of O₂ to zero. Following OGD, the cells were maintained in glucose-containing medium in a 5% CO₂-containing atmosphere at 37 °C for an additional 12 h. Cells in the CAT group were pretreated with 0.5 mM catalpol for 1 h prior to OGD. Cells in the KB+CAT group were simultaneously pretreated with 10 μM NCX inhibitor KB-R7943 (Sigma-Aldrich) and 0.5 mM catalpol for 1 h prior to OGD. The CTL group was maintained under a normoxic atmosphere in glucose-containing medium without catalpol or KB-R7943 treatment.

The human neuroblastoma SH‐SY5Y cell line was obtained from the Shanghai Saily Biotechologies Co., Ltd. The DNA of the cell lines found to perfectly match the type of cell lines in a cell line retrieval. DSMZ database shows that cells called SH‐SY5Y, corresponding to the cell number 209. No multiple alleles were discovered in this cell line. SH‐SY5Y cells were maintained in Dulbecco's modified Eagle's medium (DMEM, HyClone, USA) supplemented with 10% foetal bovine serum (FBS, HyClone, USA) in an atmosphere of 5% CO₂ at 37°C.
The OGD model was established with a solution of sodium dithionite (Na₂S₂O₄, Sigma‐Aldrich) at a concentration of 20 mmol/L in glucose‐free DMEM (Gibco), since sodium dithionite could react with oxygen dissolved in the culturing medium.