Arsenicals, iAs (Sigma S7400, NaAsO2, purity equal or more than 90%) prepared as a 10 mM stock in ddH2O was further diluted in H2O to the desired concentrations. Methyl arsonous acid (MAs III; 0.37 mg/L) synthesis was performed as previously described (Negro Silva et al. 2021 (link)) and its stock concentration was 25 mM. DMA (Sigma C4945, Sodium cacodylate trihydrate, (CH3)2AsO2Na 3H2O) stock was 10 mM in ddH2O. Rat liver cofactors thioredoxin (TRX, Sigma T0910) and thioredoxin reductase were purchased from Sigma (TRR, Sigma T9698), and L-glutathione reduced (GSH, Sigma G6529), NADPH (Sigma 10107824001), DTT, Adenosyl-L-Methionine (SAM), and S-[methyl-3H] (SAM [3H], PerkinElmer). Human recombinant histones H2A, H2B, H3, and H4 were purchased from New England Biolabs.
Thioredoxin
Manufactured by Merck Group
Sourced in Germany, Switzerland, United Kingdom
Thioredoxin is a small, ubiquitous protein found in both prokaryotic and eukaryotic cells. Its primary function is to act as a redox regulator, facilitating the reduction of other proteins by cysteine thiol-disulfide exchange.
Automatically generated - may contain errors
Lab products found in correlation
L glutathione reduced gsh, by Merck Group (1 mentions)
Dithiothreitol (dtt), by PerkinElmer (1 mentions)
Butylated hydroxytoluene bht, by Merck Group (1 mentions)
Pentobarbital sodium, by Biowet (1 mentions)
L glutathione oxidized, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Catalase, by Merck Group (1 mentions)
Xanthine oxidase, by Merck Group (1 mentions)
Superoxide dismutase, by Merck Group (1 mentions)
Xanthine, by Merck Group (1 mentions)
Retinol, by Merck Group (1 mentions)
L glutathione reduced, by Merck Group (1 mentions)
β nicotinamide adenine dinucleotide 2 phosphate reduced tetrasodium salt hydrate, by Merck Group (1 mentions)
L ascrobic acid, by Merck Group (1 mentions)
Chloro 2 4 dinitro benzene cdnb, by Merck Group (1 mentions)
5 5 dithiobis 2 dinitrobenzoic acid, by Merck Group (1 mentions)
8 hydroxy 2 deoxyguanosine 8 ohdg, by Cayman Chemical (1 mentions)
Benzaldehyde d6, by Merck Group (1 mentions)
6 protocols using thioredoxin
1
Arsenicals Methylation Reaction Assay
2
Oxidative Stress Biomarker Quantification
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Drugs and reagents were obtained from the following sources: 3-(3-carbamoylphenyl)phenyl N-cyclohexylcarbamate (URB597), 4-hyroxynonenal (4-HNE), 8-iso Prostaglandin F2α-d4 (8-isoPGF2α–d4), 8-iso Prostaglandin F2α (8-isoPGF2α) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) from Cayman Chemical Company (Ann Arbor, MI, USA); 11-deoxycorticosterone acetate (DOCA), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), xanthine, xanthine oxidase (XO), 9,9′-Bis(N-methylacridinium nitrate) (lucigenin), superoxide dismutase (SOD), catalase (CAT) thioredoxin (Trx), l -ascrobic acid, retinol, l -glutathione reduced (GSH), l -glutathione oxidized (GSSG), benzaldehyde-d6 and Tween 80 were acquired from Sigma-Aldrich (Steinheim, Germany); pentobarbital sodium was purchased from Biowet (Puławy, Poland); chloro-2,4-dinitro benzene (CDNB), butylated hydroxytoluene (BHT), and 5,5′-dithiobis (2-dinitrobenzoic acid) (DTNB) were acquired from Sigma-Aldrich (Steinheim, Germany). DOCA was dissolved in DMF, whereas URB597 was dissolved in an URB597 solvent: a mixture of DMSO, Tween 80, and saline (0.9% NaCl) [1:2:7; v/v/v].
3
Calibration and Preparation of Proteins for MALDI-TOF/TOF
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The rapifleX MALDI-TOF/TOF
instrument (Bruker) was used with a high mass acquisition method with
dedicated high mass calibrants. Calibrant proteins: insulin (no. I5500),
ubiquitin (no. U6253), and thioredoxin (no. T0910) were ordered from
Sigma-Aldrich and prepared as stock solutions at 50 pmol/μL
in TA30 (70% water, 30% acetonitrile (ACN), and supplemented with
0.1% trifluoroacetic acid (TFA)). Asialofetuin (no. A4781, Sigma)
was reduced (dithiothreitol), alkylated (iodoacetamide), and digested
using trypsin (Promega) according to standard vendor protocols. Each
protein was mixed with Super-DHB (sDHB, Bruker) matrix solution (50
mg/mL) and 1 μL directly spotted on a ground steel MALDI target
plate (Bruker).
instrument (Bruker) was used with a high mass acquisition method with
dedicated high mass calibrants. Calibrant proteins: insulin (no. I5500),
ubiquitin (no. U6253), and thioredoxin (no. T0910) were ordered from
Sigma-Aldrich and prepared as stock solutions at 50 pmol/μL
in TA30 (70% water, 30% acetonitrile (ACN), and supplemented with
0.1% trifluoroacetic acid (TFA)). Asialofetuin (no. A4781, Sigma)
was reduced (dithiothreitol), alkylated (iodoacetamide), and digested
using trypsin (Promega) according to standard vendor protocols. Each
protein was mixed with Super-DHB (sDHB, Bruker) matrix solution (50
mg/mL) and 1 μL directly spotted on a ground steel MALDI target
plate (Bruker).
4
Purification of DnaK, DnaJ and GrpE
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DnaK, DnaJ, and ΔDnaJ purification was according to Gur et al. (2004 (link)). GrpE was a gift from H.-J. Schönfeld, F. Hoffmann-La Roche, Basel, Switzerland. Glucose-6-phosphate Dehydrogenase (G6PDH) from Leuconostoc mesenteroides, Bovine rhodanese, Bovine Insulin and Thioredoxin from Spirulina sp. was purchased from Sigma-Aldrich. Protein concentrations were estimated by the Bradford Assay and protein concentrations were always expressed in protomer.
5
Cloning and Expression of PfPMV Gene
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The chemical and reagents including casein, thioredoxin, chloramphenicol, kanamycin, isopropyl-D-thiogalactopyranoside (IPTG), Tris-HCl, NaCl, imidazole, Triton X, mercaptoethanol, HisTrap column, and glycerol used in this study were purchased from Sigma-Aldrich, United Kingdom. All other chemicals and solvents used were of analytical grade.
We extracted the PMV gene that spans the whole length (identified as PlasmoDB ID: PF3D7 1323500) from the genomic DNA of the 3D7 strain of P. falciparum. The process of cloning the PfPMV gene and creating plasmid DNA was performed at the Faculty of Life Sciences, School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, located in Liverpool, United Kingdom.
We extracted the PMV gene that spans the whole length (identified as PlasmoDB ID: PF3D7 1323500) from the genomic DNA of the 3D7 strain of P. falciparum. The process of cloning the PfPMV gene and creating plasmid DNA was performed at the Faculty of Life Sciences, School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, located in Liverpool, United Kingdom.
6
Fluorescent Replication Fork Assay
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Oligodeoxynucleotides labeled with fluorescein (FAM) on the 3′-end and BHQ1 on the 5′-end were purchased from Biosearch Technologies and RP-HPLC purified (Novato, CA). Oligodeoxynucleotides labeled with fluorescein on the 5′-end, 2-AP labeled and unlabeled oligodeoxynucleotides were purchased from Integrated DNA Technologies (Coralville, IA). These DNAs were gel-purified and extracted from the gel by electroelution (Whatman Schleicher & Schuell). Replication fork substrates were created by heating the appropriate DNA strands to 95°C for 5 min and slowly cooling to room temperature. The DNA sequences are provided in Supplementary file 1 .
T7 helicase (gp4A′), T7 gp5 exo-, and E. coli SSB were purified as described (Lohman et al., 1986 (link); Patel et al., 1991 (link), 1992 (link); Kim et al., 1992 (link)). Thioredoxin was purchased from Sigma (St. Louis, MO).
T7 helicase (gp4A′), T7 gp5 exo-, and E. coli SSB were purified as described (Lohman et al., 1986 (link); Patel et al., 1991 (link), 1992 (link); Kim et al., 1992 (link)). Thioredoxin was purchased from Sigma (St. Louis, MO).
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