Nadph

Manufactured by Biosynth

Sourced in United Kingdom

NADPH is a coenzyme that plays a crucial role in various biochemical reactions. It serves as an electron carrier, facilitating the transfer of electrons in metabolic processes. NADPH is essential for the activity of many enzymes involved in anabolic reactions, such as lipid and nucleic acid synthesis.

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Reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) (2 citations)

7 protocols using nadph

Extraction and Characterization of Zolfino Landrace Saponins

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Bovine serum albumin (BSA), d,l-dithiothreitol (DTT), d,l-glyceraldehyde (GAL), EDTA and DSC18 hydrophobic interaction cartridges Supelco Discovery were purchased from Sigma-Aldrich (Saint Louis, MO, USA). NADPH, l-idose, soyasaponin Ba (SSBa) and soyasaponin Bb (SSBb) were supplied by Carbosynth (Compton, England); YM10 ultrafiltration membranes were obtained from Merck-Millipore (Darmstad, Germany); soyasaponin Bd (SSBd) was obtained from ALB Technology Limited (Mongkok Kowloon, Hong Kong); PTFE filtration membranes 0.45 µm pore size were from Phenomenex Italy (Bologna, Italy); HPLC grade methanol, formic acid, and acetic acid were purchased from VWR (Milano, Italy). HPLC grade water (18 MΩ) was prepared by a Mill-Ω purification system (Millipore Corp., Bedford, MA). All other chemicals were of reagent grade.
Dry seeds of yellow Zolfino landrace were obtained from the farm Agostinelli Mario in Leccio-Reggello (Florence, Italy; 43° 42' N, 11° 27' E) and authenticity was confirmed by comparing their features with those registered in the “Regione Toscana” germplasm data bank (access VE_027): http://germoplasma.arsia.toscana.it/.

Balestri F., De Leo M., Sorce C., Cappiello M., Quattrini L., Moschini R., Pineschi C., Braca A., La Motta C., Da Settimo F., Del-Corso A, & Mura U. (2018). Soyasaponins from Zolfino bean as aldose reductase differential inhibitors. Journal of Enzyme Inhibition and Medicinal Chemistry, 34(1), 350-360.

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Purification and Quantification of LPO

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LPO from bovine milk (ε412 = 112,000 M⁻¹ cm⁻¹ (51 )), Hank’s balanced salt solution and PBS for cell culture, Cibacron Blue 3GA agarose (type 3000-CL), 1-chloro-2,4-dinitrobenzene, sodium thiocyanate, NADH, EDTA, IAM, EC-oxyrase, calcium chloride dihydrate, spectinomycin dihydrochloride pentahydrate, Vivaspin 500 (MWCO = 3 kDa), and Amicon Ultra 0.5 centrifugal filter units (MWCO = 10 kDa) were purchased from Sigma–Aldrich (Merck). Competence stimulating peptide-1 was purchased from AnaSpec. Bovine serum albumin was from Gibco (Thermo Fisher Scientific). Auranofin was from Santa Cruz Biotechnology. H₂O₂ (30%) (ε₂₄₀ = 43.6 M⁻¹ cm⁻¹ (52 (link))) was from LabServ. 2-Nitro-5-thiobenzoate (TNB) was prepared from 5,5′-dithiobis-(2-nitrobenzoic acid) (Sigma–Aldrich) through alkaline hydrolysis as described (53 (link)). NADPH was purchased from Carbosynth and trypsin (sequencing grade) from Promega. HOSCN was generated and quantified as described (21 (link)), kept on ice, and used within 30 min of quantification.

Shearer H.L., Pace P.E., Paton J.C., Hampton M.B, & Dickerhof N. (2022). A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid. The Journal of Biological Chemistry, 298(9), 102359.

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EGCG, NADPH, L-idose Synthesis

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EGCG, NADPH and L-idose were obtained from Carbosynth (Compton, England). HNE and 3-glutathionyl-4-hydroxynonanal (GSHNE) were synthesised as described²⁵ . All other chemicals were of a reagent grade.

Balestri F., Cappiello M., Moschini R., Mura U, & Del-Corso A. (2022). Models of enzyme inhibition and apparent dissociation constants from kinetic analysis to study the differential inhibition of aldose reductase. Journal of Enzyme Inhibition and Medicinal Chemistry, 37(1), 1426-1436.

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Preparation of Organic Reagents and Catalysts

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GSH, bovine serum albumin (BSA), D,L-glyceraldehyde (GAL), 2nd generation Grubbs catalysts, lipase acrylic resin from Candida antarctica, R/S-oct-1-en-3-ol, Met-Arg-Phe-Ala (MRFA) peptide, sequencing-grade trypsin and precoated silica gel polyethylene terephthalate (PET) foils were purchased from Sigma Aldrich (St. Louis, MO, USA). Bradford reagent was purchased from Bio-Rad (Hercules, CA, USA). YM10 membranes (10 kDa cut-off value) were purchased from Amicon Millipore (Darmstadt, Germany). All inorganic chemicals were of reagent grade from BDH (VWR International Ltd., Poole Dorset, UK). L-idose and NADPH were from Carbosynth (Compton, England). All solvents were of high performance liquid chromatography (HPLC)-grade from J.T. Baker Chemicals (Fisher Scientific, Thermo Fisher, Waltham, MA, USA). Anhydrous dichloromethane (DCM) was obtained from the commercial HPLC-grade DCM by distillation on CaH₂.

Balestri F., Barracco V., Renzone G., Tuccinardi T., Pomelli C.S., Cappiello M., Lessi M., Rotondo R., Bellina F., Scaloni A., Mura U., Del Corso A, & Moschini R. (2019). Stereoselectivity of Aldose Reductase in the Reduction of Glutathionyl-Hydroxynonanal Adduct. Antioxidants, 8(10), 502.

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Purification and Characterization of Polyphenol Compounds

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Gallo catechin gallate (GCG; HPLC purity > 98%), catechin gallate (CG; HPLC purity > 98%), epi catechin gallate (ECG; HPLC purity > 97.5%), catechin (C; HPLC purity > 99%), gallocatechin (GC; HPLC purity > 98%), and epicatechin (EC; HPLC purity > 99%) were from Extrasynthese (Genay Cedex, France); NADPH and L-idose were from Carbosynth (Compton, England). Bovine serum albumin (BSA), D,L-dithiothreitol (DTT), D,L-glyceraldehyde (GAL), EDTA, glycerol and glutathione (GSH) were purchased from Sigma-Aldrich (Saint Louis, MO, USA); ammonium sulfate, sodium phosphate monobasic were from J.T. Baker (Phillipsburg, NJ, USA); YM10 ultrafiltration membranes were from Merck-Millipore (Darmstadt, Germany). All other chemicals were of a reagent grade.

Balestri F., Poli G., Piazza L., Cappiello M., Moschini R., Signore G., Tuccinardi T., Mura U, & Del Corso A. (2022). Dissecting the Activity of Catechins as Incomplete Aldose Reductase Differential Inhibitors through Kinetic and Computational Approaches. Biology, 11(9), 1324.

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Enzymatic Synthesis of NAD Cofactors

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Nicotinamide adenine dinucleotides (NAD⁺, NADH, NADP⁺ and NADPH) were purchased from Carbosynth (Berkshire, UK). Ethyl acetoacetate (EAA), ethyl 3-hydroxybutyrate, ethyl 2-chloroacetoacetate and ethyl 4-chloroacetoacetate were purchased from Alfa Aesar (Karlsruhe, Germany). Alcohols, ketones and aldehydes were supplied by Sigma-Aldrich (St. Louis, IL). Polyethyleneimine (PEI) (MW. 25–60 kDa) and polyallylamine (PAA) (MW. 17 kDa) were purchased from Sigma-Aldrich (St. Louis, IL, USA). Plain 6BCL agarose was purchased from Agarose Beads Technologies (Madrid, Spain). Protein concentrations were determined using BCA Protein Assay Kit from Pierce (Rockford, IL, USA). Enzymatic assays were carried out on a V-730 spectrophotometer from JASCO Analitica Spain S.L. (Madrid, Spain). Buffers, mediums and other reagents were obtained from Sigma-Aldrich Co. (St. Louis, IL, USA). Single particle experiments were carried out on a Cytation 5 cell image multi-mode reader from Biotek Instruments Inc. (Winooski, VT, USA). The gene from formate dehydrogenase (FDH) from Candida boidinii were synthetized and cloned in pET28b by GenScript (Piscataway, USA).

Orrego A.H., Andrés-Sanz D., Velasco-Lozano S., Sanchez-Costa M., Berenguer J., Guisan J.M., Rocha-Martin J, & López-Gallego F. (2021). Self-sufficient asymmetric reduction of β-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase co-immobilised with NADH. Catalysis Science & Technology, 11(9), 3217-3230.

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Purification and Characterization of Aldehyde Metabolizing Enzymes

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2.1 Materials. 9,10-phenanthrenquinone (PQ), bovine serum albumin, dimethyl sulfoxide (DMSO), D,L-dithiotreitol (DTT), reduced glutathione (GSH), hexanal, NADP + , nonanal, propanal, protease inhibitors cocktail, sodium dodecyl sulphate (SDS), trans-2-hexenal, trans-2-nonenal, trans-2propenal (acrolein) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HHE was purchased from Cayman Chemicals (AnnArbor, MI, USA). 4(R)-HNE was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Whatman DEAE-cellulose (DE-52) and Sephacryl S200 were purchased from GE Healthcare (Little Chalfont, UK). Blue Sepharose and Bradford reagent were purchased from Bio-Rad (Hercules, CA, USA). YM10 membranes (10 kDa cut-off) were purchased from Amicon Millipore (Darmstadt, Germany). Dialysis tubing (10 kDa cut-off) was purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA). All inorganic chemicals were of reagent grade from BDH (VWR International, Poole, Dorset, UK). All solvents were HPLC grade from J.T. Baker Chemicals (Center Valley, PA, USA). NADPH was from Carbosynth (Compton, England).

Moschini R., Rotondo R., Renzone G., Balestri F., Cappiello M., Scaloni A., Mura U, & Del-Corso A. (2017). Kinetic features of carbonyl reductase 1 acting on glutathionylated aldehydes. Chemico-biological interactions, 276.

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