Measurement of the H2S production capacity in tissues was performed as previously described.17 (link),27 (link) In brief, 80 μg of protein was incubated in 10 mM L-cysteine and 1 mM pyridoxal 5′-phosphate hydrate (Sigma-Aldrich, St Louis, MO), or 20 μL of plasma was incubated in 100 mM L-cysteine and 10 mM pyridoxal 5′-phosphate hydrate. This mixture was sealed under lead acetate paper and incubated until black lead sulfide precipitate was detected, but not saturated. The lead sulfide presence was quantified using Fiji software, version 1.53f51 (available at: http://fiji.sc/Fiji ).
Pyridoxal 5 phosphate hydrate
Manufactured by Merck Group
Sourced in United States
Pyridoxal 5'-phosphate hydrate is a chemical compound that serves as a coenzyme in various enzymatic reactions. It is the active form of vitamin B6 and plays a crucial role in protein metabolism, amino acid synthesis, and neurotransmitter production.
Automatically generated - may contain errors
Lab products found in correlation
L cysteine, by Merck Group (3 mentions)
Acetone, by Merck Group (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
L homocysteine, by Merck Group (2 mentions)
Polydiallyldimethylammonium chloride, by Merck Group (1 mentions)
Bradford protein assay dye reagent, by Bio-Rad (1 mentions)
Iminodiacetic acid, by Merck Group (1 mentions)
Polyallylamine, by Merck Group (1 mentions)
Rhodamineb isothiocyanate mixed isomers, by Merck Group (1 mentions)
2 phenylethylamine, by Merck Group (1 mentions)
Lead acetate, by Merck Group (1 mentions)
Whatman, by Cytiva (1 mentions)
L valine, by Merck Group (1 mentions)
2 ketobutyric acid, by Merck Group (1 mentions)
L threonine, by Merck Group (1 mentions)
L isoleucine, by Merck Group (1 mentions)
Isopropyl β d thiogalactoside, by Promega (1 mentions)
Anhydrous calcium chloride, by Merck Group (1 mentions)
15 protocols using pyridoxal 5 phosphate hydrate
1
Quantifying Tissue H2S Production Capacity
2
Agarose Microbead-Based Enzyme Assay
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Agarose microbeads (50–150 μm
diameter) were purchased from Agarose Bead Technologies (Madrid, Spain).
Polyethyleneimine (PEI) solution in H2O (Mw ∼ 60 000, 50 wt %), polyallylamine (PAH)
solution in H2O (Mw ∼
65 000, 10 wt %), polydiallyldimethylammonium chloride (PDADMAC)
solution in H2O (Mw < 100 000,
35 wt %), pyridoxal 5′-phosphate hydrate (PLP, 98%), rhodamine
B isothiocyanate mixed isomers (RhB), acetone, 2-phenylethylamine
(PEA, 98%), iminodiacetic acid (IDA), albumin bovine serum standard
(BSA), and other reagents and solvents of analytical grade were purchased
from Sigma-Aldrich (St. Louis, IL). Nicotinamide adenine dinucleotide-reduced
sodium salt (NADH) and flavin mononucleotide sodium salt (FMN) were
purchased from GERBU Biotechnik GmbH (Heidelberg, Germany). Flavin
Adenine dinucleotide disodium salt hydrate (FAD, 94%) was purchased
from Cymit Quimica S.L. (Barcelona, Spain). Riboflavin (Rf, 98%) was
purchased from Acros Organics B.V.B.A. (Fair Lawn, New Jersey, United
States). Isopropyl-β-d -thiogalactopiranoside (IPTG,
100%) was purchased from Fisher Bioreagents. The Bradford protein
assay dye reagent was purchased from BIORAD (Biorad. Hercules, CA).
Clear bottom black and white microplates (96-well) were purchased
from Avantor (2021 VWR International, LLC). μ-Slides 8 well
glass bottom was purchased from Ibidi (Planegg, Germany).
diameter) were purchased from Agarose Bead Technologies (Madrid, Spain).
Polyethyleneimine (PEI) solution in H2O (Mw ∼ 60 000, 50 wt %), polyallylamine (PAH)
solution in H2O (Mw ∼
65 000, 10 wt %), polydiallyldimethylammonium chloride (PDADMAC)
solution in H2O (Mw < 100 000,
35 wt %), pyridoxal 5′-phosphate hydrate (PLP, 98%), rhodamine
B isothiocyanate mixed isomers (RhB), acetone, 2-phenylethylamine
(PEA, 98%), iminodiacetic acid (IDA), albumin bovine serum standard
(BSA), and other reagents and solvents of analytical grade were purchased
from Sigma-Aldrich (St. Louis, IL). Nicotinamide adenine dinucleotide-reduced
sodium salt (NADH) and flavin mononucleotide sodium salt (FMN) were
purchased from GERBU Biotechnik GmbH (Heidelberg, Germany). Flavin
Adenine dinucleotide disodium salt hydrate (FAD, 94%) was purchased
from Cymit Quimica S.L. (Barcelona, Spain). Riboflavin (Rf, 98%) was
purchased from Acros Organics B.V.B.A. (Fair Lawn, New Jersey, United
States). Isopropyl-β-
100%) was purchased from Fisher Bioreagents. The Bradford protein
assay dye reagent was purchased from BIORAD (Biorad. Hercules, CA).
Clear bottom black and white microplates (96-well) were purchased
from Avantor (2021 VWR International, LLC). μ-Slides 8 well
glass bottom was purchased from Ibidi (Planegg, Germany).
3
H₂S Production in HepG2 and SNU449 Cells
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H2S production of HepG2 and SNU449 cells was detected by the lead sulfide method described in Hines et al.69 (link). Briefly, cells were cultured in 96 well plates in growth media supplemented with 10 mM L-cysteine and 10 μM pyridoxal 5-phosphate hydrate (Sigma). A piece of 703 style Whatman filter paper (VWR), soaked in 20 mM lead acetate (Sigma) and dried, was placed over the culture wells and covered with the plate lid with a heavy object on the top. The cells were cultured in a CO2 incubator at 37 °C for 24 h. The formation of lead sulfide indicated by the dark circles on the filter paper was recorded with a digital camera.
4
Biosynthesis of Branched-Chain Amino Acids
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l -Threonine, l -isoleucine, l -valine, 2-ketobutyric acid, and pyridoxal-5-phosphate hydrate
(PLP) were purchased from Sigma-Aldrich. Isopropyl β-d -thiogalactoside (IPTG) was supplied by Promega (Madison). All of
the primers were from Genewiz (Suzhou, China). All of the enzymes
and other bioreagents were purchased from Takara (Dalian, China).
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(PLP) were purchased from Sigma-Aldrich. Isopropyl β-
the primers were from Genewiz (Suzhou, China). All of the enzymes
and other bioreagents were purchased from Takara (Dalian, China).
5
Kynurenine Pathway Regulation by NSAIDs
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L-Kynurenine sulfate salt (K3750), acetaminophen (A7085), acetylsalicylic acid (A5376), diclofenac sodium salt (D6899), ibuprofen (I4883), indomethacin (I7378), meloxicam sodium salt hydrate (M3935), naproxen sodium (M1275), nimesulide (N1016), piroxicam (P5654), reagents for Krebs Ringer buffer preparation: sodium chloride (S7653), potassium chloride (P9333), magnesium sulfate heptahydrate (M7506), calcium chloride anhydrous (C1016), sodium phosphate monobasic dihydrate (71,505), sodium phosphate dibasic (S0876), glucose (G8270); dimethyl sulfoxide (DMSO) (D1435); reagents for KATs analysis: Trizma base (T1503), acetic acid (A6283), pyridoxal 5′-phosphate hydrate (P9255), 2-mercaptoethanol (M3148), sodium pyruvate (P2256), and D-glutamine (D9003) were obtained from Sigma-Aldrich. Substances needed to perform high-performance liquid chromatography (HPLC) were purchased from J.T. Baker Chemicals and from Sigma-Aldrich. Most tested drugs were dissolved in DMSO, whereas naproxen was administered in an aqueous solution. DMSO was given to adequate control samples and its concentration was not higher than 5% [25 (link)].
6
H2S Production Assay in Tissues
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H2S production capacity from tissues and serum was performed via the lead acetate/lead sulfide method [32 (link)]. Briefly, tissues were homogenized and lysed in Passive Lysis Buffer (Promega) using flash freezing/thawing in liquid nitrogen to crack the cellular membranes. After clearing debris via centrifugation, protein concentrations were determined and normalized via the Pierce BCA assay (ThermoFisher Scientific, Waltham, MA, USA). Equal amounts of protein per sample were added to a 150 µL reaction in 96-well plate format containing 10 mM L-cysteine (Sigma) and 1 mM Pyridoxal 5′-phosphate hydrate (Sigma) in PBS. A lead acetate embedded filter paper was placed above the wells and incubated at 37 °C until detectable, but not saturated, and lead sulfide circles appeared above the wells. The lead sulfide circles were analyzed for integrated density using the IntDen function in ImageJ as previously described [26 (link)].
7
Garlic Alliin Extraction and Quantification
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The following chemicals were purchased from Sigma-Aldrich: (±)-L-alliin (primary reference standard), Ethylenediaminetetraacetic acid (EDTA), Pyridoxal 5′-phosphate hydrate (PLP), Dithiothreitol (DTT), N[Tris(hydroxymethyl)methyl]glycine (Tricine), Polyethylene glycol (PEG) 6000, Bovine serum albumin (BSA), Nutrient Broth (NB), Kanamycin sulfate, Fluorescein diacetate, Propidium iodide (PI), Polyethylenimine (PEI). Glycerol, Sodium phosphate dibasic heptahydrate, Sodium phosphate monobasic decahydrate, Potassium dihydrogen phosphate, Phosphate-buffered saline (phosphate buffer, potassium chloride and sodium chloride), citric acid, hydrochloric acid, ascorbic acid, tryptone, yeast extract, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, lithium chloride, and potassium hydroxide were purchased from Penta (Czech Republic). Magnesium nitrate hexahydrate was purchased from Fisher Scientific. L-Lactate dehydrogenase (LDH) from rabbit muscle and β-Nicotinamide adenine dinucleotide (NADH) disodium salt grade II (approx. 98%) were purchased from Roche Diagnostic (Germany). Garlic was purchased from Farmers’ Market, Zahradnictví Riegelová, country of origin Czech Republic.
8
Synthesis and Immobilization of Transaminase Biocatalyst
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Acetone (≥99.9%), (3-aminopropyl)triethoxysilane (APTES; ≥ 98%), 4′-bromoacetophenone (BAP; ≥ 98%), n-dodecane (≥90%), hydrochloric acid (37% wt, aqueous solution), pyridoxal 5′-phosphate hydrate (PLP; ≥ 98%), sodium cyanoborohydride solution (5.0 M in 1 M NaOH; NaBH3CN), sodium hydroxide aqueous solution (50%), sodium pyruvate (≥99%), tetraethyl orthosilicate (TEOS; ≥ 98%), toluene (≥99.8%, anhydrous), trimethyltetradecylammonium bromide (TTAB; ≥ 99%) were purchased from Sigma-Aldrich. 4-Bromo-α-methylbenzylamine (BMBA; ≥ 99%; racemate), dibasic potassium phosphate (≥99%), dimethylsulfoxide (DMSO; ≥ 99.8%) and monobasic potassium phosphate (≥99%) were purchased from Acros Organics. d -Alanine (≥99%) was purchased from CarlRoth. Tetrahydrofuran (THF; ≥ 99.6%) was purchased from VWR Chemicals. Heat-shrinkable polytetrafluoroethylene (PTFE) tube was purchased from RS Components. Codexis generously supplied ATA-117 transaminase. Distilled water was applied for all synthesis and treatment processes.
9
Synthesis and Characterization of Phenylbutan-2-one Derivatives
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The ketones 4-phenylbutan-2-one and (E)-4-phenylbut-3-en-2-one, titanium (IV) isopropoxide, ammonia solution 2.0 M in ethanol, pyridoxal 5′-phosphate hydrate, and isopropylamine are commercially available from Sigma–Aldrich®, Saint Louis, MO, USA.
Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker Fourier 300 (B0 7.1 T) spectrometer, at operating frequencies of 75 MHz for 13C NMR- and 300 MHz for 1H NMR-spectroscopy. The chemical shifts (δ) are reported in parts per million (ppm), using, as an internal reference, tetramethylsilane (TMS, δ = 0.0) and, in the scale relative to CDCl3, 7.24 ppm for 1H NMR and 77.23 for 13C NMR. The coupling constants (J) were measured in Hertz (Hz) and were characterized as doublet (d), double doublet (dd), multiplet (m), singlet (s), broad singlet (bs), sextet (st), and triplet (t).
Infrared (IR) spectra were recorded on an FTIR spectrophotometer (Vertex 70—Bruker), equipped with attenuated total reflectance (ATR), operating at 400–4000 cm−1.
Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker Fourier 300 (B0 7.1 T) spectrometer, at operating frequencies of 75 MHz for 13C NMR- and 300 MHz for 1H NMR-spectroscopy. The chemical shifts (δ) are reported in parts per million (ppm), using, as an internal reference, tetramethylsilane (TMS, δ = 0.0) and, in the scale relative to CDCl3, 7.24 ppm for 1H NMR and 77.23 for 13C NMR. The coupling constants (J) were measured in Hertz (Hz) and were characterized as doublet (d), double doublet (dd), multiplet (m), singlet (s), broad singlet (bs), sextet (st), and triplet (t).
Infrared (IR) spectra were recorded on an FTIR spectrophotometer (Vertex 70—Bruker), equipped with attenuated total reflectance (ATR), operating at 400–4000 cm−1.
10
Fluorometric Assay for H2S Production
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H2S production was measured using 7-azido-4-methylcoumarin (AzMC) fluorescent dye (Sigma, #802409). The protocol was adapted from Szabo Laboratory with minor modifications (Szabo et al., 2014 (link)). AzMC reaction master mix consisted of 200 mM Tris-HCl pH 8, 20 mM L-cysteine (Sigma, #30089), 1 mM L-homocysteine (Sigma, #69453), 100 μM pyridoxal 5’-phosphate hydrate (Sigma, #P9255), 20 μM AzMC in H2O was prepared and kept on ice. Cells were washed with cool PBS and then harvested in cell lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% v/v IGEPAL CA-630(Sigma-Aldrich-I3021), 1% v/v Triton-X100). Samples were kept on ice for 1 hr and then centrifuged at 20,000 g for 10 min at 4°C before protein quantification by DC protein assay; 400 µg proteins were mixed with 100 μl AzMC reaction master mix and incubated at 37°C in dark for 2 hr. Samples were read at 340 nm excitation and 445 nm emission wavelength using Cytation 3 cell imaging multi-mode reader (BioTek).
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