Supplementary Figure 1. Characterization of Vn. (a) UV-Vis spectrum showing a band at ~420 nm corresponding to the band gap of nano-v 2

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1 Supplementary Figure 1. Characterization of Vn. (a) UV-Vis spectrum showing a band at ~420 nm corresponding to the band gap of nano-v 2 O 5.(b) Electron dispersive X-ray (EDX) spectra of Vn showing the peaks of V and O. Since Au wassputtered on Vn to make it conducting, peaks due to Au are also seen in the spectra.

2 Supplementary Figure 2. The GPx-like activity of Vn at various assay conditions. (a) Plot of absorbance g versus time (min) revealing the activity of Vn in the presence of Vn (0.02 mg ml -1 ), GSH (2 mm), NADPH (0.4 mm), GR (1.7 units), H 2 O 2 (240 µm) in phosphate buffer (100 mm, ph 7.4) at 25 o C. It should be noted that in the absence of at least one of the components in the reaction mixture, Vn failed to show GPx activity. (b) Supernatant solution obtained after incubation of Vn in phosphate buffer ph 7.4 for 10 min did not show any noticeable GPx activity, indicating that the reaction occurs on the surface of Vn. (c,d) Effect of GR (0-3.4 units) on the reduction of H 2 O 2 (240 µm) in the presence of NADPH (0.4 mm) and in the absence of GSH. No significant activity was observed as the initial rates are very similar to that of the control reaction (control reaction, fig. S2d).

3 Supplementary Figure 3. Michaelis-MentenMenten kinetics of Vn. (a-b) Lineweaver-Burk plots of GPx activity of Vn corresponding to the variation of H 2 O 2 concentration and GSH concentration, respectively. (c-d) Michaelis-Menten kinetic plots corresponding to the variation of GSH at the different H 2 O 2 concentrations (75 and 200 µm). (e) Lineweaver-Burk plot corresponding to c and d, indicating ping-pong mechanism and binding of one molecule of GSH to active site per catalytic cycle. Data is represented as mean ± s.e.m, P (T-test) <

4 * * * * Supplementary Figure 4. Reaction of Vn with peroxynitrite (PN). (a) PN scavenging activity of Vn in PN- mediated BSA nitration was studied by gel electrophoresis. Conditions: PN (1 mm) to BSA ( µm) in 0.5 M phosphate buffer of ph 7.4 at 20 C. The reaction mixture was then incubated for 20 min at 22 C. The reactions of BSA with PN were performed at different concentrations of Vn/Vn-GSH mixture. After the reactions, the mixture was denatured by boiling at 100 C for 5 min in the presence of sample loading dye and subjected to polyacrylamide gel electrophoresis and immunoblot analyses. The bardiagram corresponding to the density of the blots indicate that Vn alone does not have any effect on the PN- mediated nitration. The decrease in the nitration is mainly due GSH. (Note: The gel electrophoresis and immunoblotting experiments were carried out by following literature procedures: Bhabak, K.P.; Mugesh, G. Chem. Eur. J.16, (2010)). Data is represented as mean ± s.e.m, P < , 0001 P* < (b) The effect of Vn on PN-mediated nitration of free L-tyrosine was carried out by UV-Vis spectroscopy. Conditions: L-tyrosine (1 mm) and PN (1.5 mm) in a phosphate buffer (100 mm) of ph 7.4 with increasing concentration of Vn (0-40 µg). The mixture was incubated in a cuvette for 5 min before recording absorbance. The formation of 3-nitro-L-tyrosine was monitored at the wavelength 428 nm. The % nitration was calculated by comparing the absorbance of 3-nitro-L-tyrosine formed in the absence of Vn/Vn-GSH mixture. This study also confirms that Vn alone have no effect on nitration reaction. Data is represented as mean ± s.e.m, P < , P* < (c) Effect of Vn on PN-mediated nitration reaction in the presence of GSH. These experiments suggest that Vn alone have no reactivity with PN even in the presence of GSH, as GSH alone provides significant protection against PN-mediated reaction. Conditions: PN = 1mM, Tyr = 1 mm, Vn = 20 µg, GSH (100 µm (lane 2) or 200 µm (lane 3)). Data is represented as mean ± s.e.m, P < , P* < 0.01.

5 UT Vn (0 h) Supplementary Figure 5. ESEM images of Vn internalization in mammalian cells. HEK293T cells were either left untreated (upper panel), treated with Vn prior to fixation, partial dehydration d and imagingi by environmental scanning electron microscope (Scale bar: 5 µm).

6 a b c d e Supplementary Figure 6. Effect of variable concentration ti of Vn and H 2 O 2 on cellular l peroxide levelsl and redox recycling of Vc. (a) Comparison of GPx-like activity of Vn and Vb. (b) Dose response analysis of H 2 O 2 treatment on HEK293T cells. HEK293T cells were either kept untreated (UT) or exposed to increasing concentrations of H 2 O 2 as mentioned for 15 min. The cellular viability was flow cytometricaly analyzed by PI staining. (c) Analysis of dose dependent response of Vn was carried out by pretreating HEK293T cells with increasing concentrations of Vn for 15 min, followed by H 2 O 2 treatment for 15 min. (d) The cells were treated as mentioned in (c) and viability analysis was done through MTT assay. Data is represented as mean ± s.e.m, P (T-test) < with reference to UT for all the depicted charts. (e) 51 V NMR spectra of Vc showing redox recycling without affecting the ligand environment.

7 a b c d e f g h i Vc 2 Vc 3 Supplementary Figure 7. Comparative account of Vn and vanadium complexes in cellular ROS reduction and protection of cell viability. (a,b) ) Relative mean fluorescence intensity of HEK293T cells left untreated or pre- treated with nano-v 2 O 5 (Vn) for 15 min prior exposure to H 2 O 2 or CuSO 4 or 3-AT alone (for 15 min) or in combination with allyl alcohol (AA), buthionine sulfoximine (BSO), vanadium complex (Vc) and glutathione (GSH). N-acetyl cysteine treated cells (NAC) were used as positive control. The cells were stained with DCFDA dye and analyzed by flow cytometry. Data is represented as fold mean fluorescence intensity (MFI) over unstained cells. Bars denote mean ± s.e.m. n=3, P (T-test) < (c, d) The viability of cells exposed to different combinations of treatment were stained with propidium iodide followed by FACS analysis. Bars represent mean ± s.e.m. n=3, P (T-test) < (e) The beneficial effects of Vn was measured by monitoring the relative cell viability over different time intervals, when the cells were exposed to 200 µm H 2 O 2 after pretreatment with 50 ng/µl Vn for 15 min. Bars represent mean ± s.e.m. n=3, P (T-test) < (f, f g,h) The overall ROS levels of cells or relative viability in cells treated for 15 min with bulk vanadium (Vb) or vanadium complexes (Vc) in absence and presence of H 2 O 2 /CuSO 4,wasmeasured using DCFDA staining i or MTT assay. Data is represented as mean ± s.e.m. n=8, P (T-test) <0.0001, P (T-test) < (i) Structure of Vc 2 and Vc 3 complexes.

8 a b c DCFDA Staining of SH-SY5Y cells UT Vn d LNCaP e SH-SY5Y H 2 O 2 Vn + H 2 O 2 H 2 O 2 + Vc f SH-SY5Y Supplementary Figure 8. Role of Vn in reduction of intracellular ROS in other cell species. (a,b) Image quantification of DCFDA staining of HeLa cells (shown in fig 4e-f). Images were quantified by selecting uniform area from each panel using ImageJ software. The intensity of untreated cells was set as 1. (c-e) The universality of the ROS modulatory function of Vn was tested on cell lines from different origins such as prostate carcinoma cell line LNCaP and neuroblastoma cell line SHSY-5Y 5Y. The cells were treated as indicated, and level of H 2 O 2 was measured by using HyPer probe or DCFDA staining and analyzed by flow cytometry or microscopy. (f) Cell viability of SHSY-5Y under treated condition was analyzed by MTT assay. Dataisrepresented as mean ± s.e.m. n=8, P (T-test) <0.0001, P (T-test) <0.001.

9 a b c d e Catalase GPx1 SOD2 β-actin Prx3 FTH1 FLT1 TrxR1 Trx f GCLM Catalase GCLC HO-1 SOD2 β-actin Prx3 FTH1 Supplementary Figure 9. Intracellular retention of Vn and its effect on the antioxidant machinery of the cell. (a) The level of cellular GSH or FLT1 GSSG was measured in HEK293T cells after treatment with Allyl alcohol (AA) alone or in combination with Vn. (b) The effect of prolonged Vn TrxR1 treatment on cell viability and maintenance of ROS levels. Cell exposed to Vn were incubated for indicated time and the percent cell viability was Trx calculated by MTT assay. (c) The retention of Vn ROS regulatory activity GCLM after prolonged time period was adjudged by peroxide treatment of cells pre-exposed exposed to Vn for indicated time periods. Bars represent mean ± GCLC s.e.m. n=8, P (T-test) <0.0001, P (T-test) < (d, e) The effect of Vn treatment on the antioxidant machinery and Nrf2 responsive genes HO-1 was analyzed by western blotting using specific antibodies against the β-actin indicated antioxidant enzymes and stress-markers. Blots were probed with anti-β-actin antibody as a loading control. (f) To co-relate the effect of Vn with GPx, cells overexpressing GPx were either left untreated or treated with H 2 O 2 and immunoblotted using antibodies as described above (EV: empty vector control).

10 Supplementary Figure 10. In vitro DNA damage prevention assay (Calf Thymus type 1 DNA). In a typical assay (0.5 ml), the reactants were added in the following order in phosphate buffer (100 mm, 7.4 ph, 28 o Cindark),DNA (10 µg ml -1 ), CuSO 4.5H 2 O (450µM), Vn (0.02mg ml -1 ), GSH (2 mm), H 2 O 2 (1mM).

11 Supplementary Figure 11. ProbingtheinteractionsofGSHonVnsurface.(a) and (b) Scanning transmission electron microscopy (STEM) image and Electron dispersive X-ray (EDX) spectra of GSH- treated Vn, respectively. A small peak due to sulfur is seen in EDX spectra. (c) X-ray mapping images of sulfur and vanadium, indicating GSH is bound to Vn surface. (d) Appearance of a peak for sulfur (S2p) in X-ray photoelectron spectra (XPS) of GSH-treated Vn further indicates the interaction between GSH molecules and Vn surface. Inatypical experiment, 2mg ml -1 Vn was treated with 0.5 ml of 12.5 mm GSH. After 5minof yp p, g incubation, Vn was washed five times with ultrapure water to remove unbound GSH and then with acetone. The Vn was allowed to settle down during each washing. The material was dried and redispersed in ethanol under sonication. 10 µl of the dispersion was drop-casted on formvar-coated copper TEM grid. The dispersion on the grid was dried for 24 h in air and analyzed by TEM under STEM mode for X-ray mapping analysis. For X-ray photoelectron spectroscopic studies, the GSHtreated Vn (dried) was coated on carbon tape attached to a piece of silicon wafer and then analyzed.

12 Supplementary Figure 12. In situ monitoring of vanadium peroxido species in the presence of H 2 O 2. (a) FTIR spectra showing the disappearance of band at 1000 cm -1 (V=O) upon addition of requisite reaction components, suggesting the formation of vanadium peroxo species on the surface of Vn. Each spectrum was acquired at the time interval of 20 sec during the progress of GPx activity. (b) Raman spectra of Vn and other reactions with Vn using various hydroperoxides. Treatement of Vn with H -1 2 O 2 resulted in appearance of peak at 591 cm and its corresponding overtone at ~1200 cm -1, confirming the formation of peroxide vanadium species on the surface of Vn. Similar reactions of Vn with t-buooh and CumOOH did not result in the production of peroxide vanadium species, indicating the selectivity towards the reduction of H 2 O 2.

13 Supplementary Figure 13. Mechanism of the formation of vanadium peroxido species. (a) Electron paramagnetic resonance (EPR) spectra of the reaction mixture containing Vn and H 2 O 2, accounting for the formation of OOH and few V(IV) species at liquid nitrogen temperature. However, at room temperature such a species rapidly recombine to produce peroxide intermediate t as discussedd mechanistic details in manuscript text. t (b) Detailed mechanism showing the formation of V(IV) and OOH radical species at liquid nitrogen temperature. These species rapidly undergo recombination at room temperaturere to produce vanadium hydroperoxido species, which eliminates water and forms vanadium peroxide species. The interaction of sulphydryl proton of GSH with the negatively charged oxygen further increases the nucleophilic affinity of thiolate (GS ) towards the positively charged oxygen of peroxide species, resulting in reaction progress.

14 Supplementary Figure 14. Analysis of oxidation state of Vn. (a) V2p and O1s XPS spectra of Vn and GSH-treated t Vn. Appearance of peaks at ev and ev accounting for V2p 3/2 and V2p 1/2, respectively, in both the cases indicate that the Vanadium ions in Vn are stable over reduction by GSH. (b) Comparison of the EPR spectra of the reaction mixture containing i Vn + GSH and Vb + GSH. It should be noted that GSH-treated Vn show negligible amount of the formation of V(IV) species, whereas GSH-treated Vb resulted in significant amount of V(IV) species. (c) Cyclic voltammogram of Vn and Vb showing a significant difference in their redox potential. The lithiation- delithiation induced redox peaks (V 5+ to V 4+ ) at potential V and 3.15 V and (4+ to 5+) at the potential3.34vand3.46v,respectively,areshowntobeshiftedinthecaseofvnat2.82vand2.97 V for reduction and oxidation, respectively. (d) Visual color changes of Vn dispersion in the presence of different reducing agents (2 mm). The dispersion of GSH-treated Vn shows a very slight change in color, probably indicating an interaction between Vn surface and GSH. However, the reaction of Vn with dithiothreitol (DTT) and sodium borohydride (NaBH 4 ) resulted in the reduction of V(V) to V(IV) in Vn. tris(2-carboxyethyl)phosphine (TCEP) showed no reaction with Vn.

15 Supplementary Figure 15. Analysis of GSOH formation. (a) HPLC chromatogram showing the peak at retention time 2.85 min corresponding to the formation of GSOH (3) as an intermediate during the progress of reaction. (b) Mass spectra of GSOH (3) showing peak at m/z

16 Supplementary Figure 16. The mechanism of the formation of peroxido species form vanadium dihydroxy species upon reaction with H 2 O 2. This mechanism is similar to the mechanism proposed for vanadium chloroperoxidase enzyme.the protonation of one of the hydroxyl units at vanadium center by H 2 O 2 produces a water molecule and HOO. The weakly bound water molecule then dissociates from the vanadium ion, which facilitates the attack of HOO at vanadium, leading to the formation of a hydroperoxide species. A spontaneous elimination of another molecule then generates the vanadium peroxide species.

17 Supplementary Figure 17. Analysis of GSO 2 Hformation.(a) HPLC chromatogram showing the peak at retention time 1.25 min corresponding to the formation of GSO 2 H (5) due to rapid over oxidation of GSOH (3) at the higher concentration of H 2 O 2 (1.6 mm). (b) Mass spectra showing the peak at m/z accounting for the formation of GSO 2 H (5) at the higher concentration of H 2 O 2.

18 Supplementary Figure 18. Thiol peroxidase activity of other thiol small molecules. Plots showing the thiol peroxidase activity of Vninthepresenceof H 2 O 2 and various thiol substrates such as (a) cysteine, (c) cysteamine hydrochloride, (e) mercaptoethanol. (b, d, f) Decreasing intensity of yellow color represents decreasing amount of the formation of thiolate from 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB, 5mM) after its reduction with available cysteine, cysteamine hydrochloride and mercaptoethanol during the courseof thiol peroxidase reaction, respectively. Data is represented as mean ± s.e.m, P (T-test) < Thiol peroxidase activity was carried out in the presence of H 2 O 2 (1.6 mm), Vn (0.04 mg ml -1 ), cysteine/cysteamine hydrochloride/mercaptoethanol (2 mm) in phosphate buffer (100 mm, ph 7.4). After every 0.5 min interval, 100 µl of reaction mixture was added to 100 µl methanolic solution of DTNB (5mM) and incubated for 2 min and then finally diluted to 1 ml using phosphate buffer (100 mm, ph 7.4). The resulting thiolate from the reduction of DTNB by the above mentioned thiols was monitored by UV/Vis spectroscopy at 412 nm. As the activity was found to be lower in the presence of mercaptoethanol, it was monitored at every 1 min interval.

19 a b CuSO Anti-Flag 25 kda Vn AA GSH Vc NAC Anti-GPx kda kd Anti-β-actin 50 kda β-actin 50 kda Supplementary Figure 19. Uncropped western blot films for (a) Fig. 5g and (b) Fig. 6a