To analyze CO consumption rate, a myoglobin assay was conducted by modifying a previously reported protocol48 (link)49 (link). To prepare cell suspensions, cells were cultured on MM1-CO medium and then harvested by centrifugation at 7500 × g for 20 min at room temperature under anaerobic conditions. Cell pellets were washed twice and resuspended in MM1 medium. Final cell density (expressed as optical density at 600 nm) was measured with a BioPhotometer plus UV-Vis spectrophotometer (Eppendorf, Hamburg, Germany) and adjusted to 0.3. Aliquots of 100 μl of the cell suspension were mixed with 0.9 ml of CO-saturated MM1 medium and incubated at 80 °C. During the reaction, 10 μl of the reaction solution was collected every 3 mins and immediately added to a quartz cuvette containing a myoglobin reaction mixture that included 56 μM myoglobin (Sigma-Aldrich, St. Louis, UAS) from equine heart dissolved in 10 mM sodium thionite in 0.1 M potassium phosphate buffer (pH 7). After a 3 min incubation, the absorbance at 423 nm was measured to analyze consumed CO.
Myoglobin
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe
Myoglobin is a globular protein found in the muscle tissue of vertebrates, including humans. Its primary function is to store and transport oxygen within muscle cells. Myoglobin has a high affinity for oxygen, allowing it to efficiently bind and release oxygen as needed to support muscle function and metabolism.
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Lab products found in correlation
Cytochrome c, by Merck Group (25 mentions)
Bovine serum albumin (bsa), by Merck Group (16 mentions)
Lysozyme, by Merck Group (13 mentions)
Transferrin, by Merck Group (9 mentions)
Formic acid, by Thermo Fisher Scientific (8 mentions)
Carbonic anhydrase, by Merck Group (7 mentions)
Ubiquitin, by Merck Group (7 mentions)
Human serum albumin (hsa), by Merck Group (7 mentions)
Acetonitrile, by Thermo Fisher Scientific (7 mentions)
Hemoglobin, by Merck Group (6 mentions)
Catalase, by Merck Group (6 mentions)
Insulin, by Merck Group (5 mentions)
Trypsin, by Merck Group (5 mentions)
Sequencing grade modified trypsin, by Promega (4 mentions)
β lactoglobulin, by Merck Group (4 mentions)
Alcohol dehydrogenase, by Merck Group (4 mentions)
Concanavalin a (cona), by Merck Group (4 mentions)
Trifluoroacetic acid, by Merck Group (4 mentions)
β casein, by Merck Group (4 mentions)
Methanol, by Thermo Fisher Scientific (4 mentions)
87 protocols using myoglobin
1
Myoglobin-based CO Consumption Assay
2
Determination of IrGST1 Kinetic Constants
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The determination of IrGST1 apparent Michaelis constants to CDNB and GSH as substrates were performed in triplicates, with varying concentrations of CDNB and constant GSH (1 mM), or constant CDNB (1 mM) and varying concentrations of GSH, respectively. Kinetic constants were calculated by non-linear regression analysis of the experimentally measured activities. Data were fitted to the Michaelis-Menten equation using GraphPad Prism 6.0 software. The inhibition of IrGST1 by haem-related
compounds was investigated by CDNB activity assay with Haemin (haem-chloride), haematin (haemhydroxide, Sigma H3281), protoporphyrin IX (PPIX) (Sigma P8293), and myoglobin (Sigma M0630).
Haemin and haematin were dissolved in 100 mM NaOH, PPIX in DMSO, and myoglobin in H 2 O.
compounds was investigated by CDNB activity assay with Haemin (haem-chloride), haematin (haemhydroxide, Sigma H3281), protoporphyrin IX (PPIX) (Sigma P8293), and myoglobin (Sigma M0630).
Haemin and haematin were dissolved in 100 mM NaOH, PPIX in DMSO, and myoglobin in H 2 O.
3
Quantification Technique Validation Protocol
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An experiment was conducted to validate the quantification techniques. Additional bulk samples containing 15 µg of yeast proteins (four independent replicates) were supplemented with purified enzymes at different concentrations: insulin (Sigma, ref. I5500, Saint Louis, MO, USA) at 51.9 fmol; alpha-lactalbumin (Sigma, ref. L5385, Saint Louis, MO, USA) at 108.6 fmol; myoglobin (Sigma, ref. M0630, Saint Louis, MO, USA) at 181.8 fmol; and ribonuclease A (Sigma, ref. R5500, Saint Louis, MO, USA) at 342.6 fmol. The samples underwent in-gel digestion as described above. The extracted tryptic peptides were vacuum dried and resuspended in 75 μL of loading buffer. Then, 1.5 µL of digested UPS2 (424 ng/µL) was spiked into 7.5 µL of the digested peptides (from the yeast proteins and purified enzymes). The expected and observed protein abundances for the samples were compared for the different quantification techniques.
4
MALDI-TOF Mass Spectrometry of Desalted Samples
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Crude samples desalted by ultrafiltration were analyzed using a MALDI Micro MX mass spectrometer (Waters). All measurements were performed in the m/z range of 1,000–10,000 in linear ion mode. The lyophilized samples were reconstituted in 10 μL aqueous 0.1% TFA. One μL of sample was mixed with 1 μL aliquot of α-cyano-4-hydroxycinnamic acid (α-matrix, 10 mg/mL in ethanol/ACN, 1:1, v/v), and 1 μL of the solution was spotted onto a metal 96-spot MALDI target plate. The instrument was operated in positive ion mode, with 3.5 kV set on the sample plate, and 12 kV on the extraction grid. A nitrogen laser (337 nm, 5 Hz) was used for ionization/desorption and the extraction of ions was delayed by 500 ns. The pulse voltage was 1100 V and the detector voltage was set to 2.15 kV. MassLynx v4.1 software was used for data acquisition (Waters). Each spectrum was combined from 15 laser pulses. Angiotensin II, bradykinin, ACTH, insulin, cytochrome C, and myoglobin (all Sigma) at 1 to 10 pmol on target were used to calibrate the mass spectrometer.
5
Analytical Scale Protein Characterization
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Water was obtained
from a direct-QTM Millipore system Millipore (Millipore, Billerica,
MA, U.S.A.). MS grade acetonitrile (ACN) and TFA were purchased from
Biosolve B.V. (Valkenswaard, The Netherlands). Escherichia
coli lyophilized protein lysate was purchased from Bio-Rad
(Veenendaal, The Netherlands). Ubiquitin (human >95%), ribonuclease
A (bovine pancreas Type X-A, ≥90%), ribonuclease B (bovine
pancreas, ≥80%), myoglobin (equine heart >90%), lysozyme
(chicken
egg white, >90%), carbonic anhydrase (bovine erythrocytes, ≥95%),
cytochrome c (equine heart, >95%), transferrin (human, >98%),
trypsinogen
(bovine pancreas, lyophilized powder) as well as other reagents were
acquired from Sigma–Aldrich (Zwijndrecht, The Netherlands).
Standard proteins were used as received without additional purification
and were solubilized in Milli-Q grade water at 2 mg/mL. The injection
volume on analytical scale columns was 2 μL of the protein standard.
For capillary LC-MS experiments, the sample was diluted to 0.2 mg/mL,
and the injection volume was 1.0 μL. The lyophilized lysate
of E. coli was diluted to a final concentration of 2.5 mg/mL, and
5.0 μL was injected.
from a direct-QTM Millipore system Millipore (Millipore, Billerica,
MA, U.S.A.). MS grade acetonitrile (ACN) and TFA were purchased from
Biosolve B.V. (Valkenswaard, The Netherlands). Escherichia
coli lyophilized protein lysate was purchased from Bio-Rad
(Veenendaal, The Netherlands). Ubiquitin (human >95%), ribonuclease
A (bovine pancreas Type X-A, ≥90%), ribonuclease B (bovine
pancreas, ≥80%), myoglobin (equine heart >90%), lysozyme
(chicken
egg white, >90%), carbonic anhydrase (bovine erythrocytes, ≥95%),
cytochrome c (equine heart, >95%), transferrin (human, >98%),
trypsinogen
(bovine pancreas, lyophilized powder) as well as other reagents were
acquired from Sigma–Aldrich (Zwijndrecht, The Netherlands).
Standard proteins were used as received without additional purification
and were solubilized in Milli-Q grade water at 2 mg/mL. The injection
volume on analytical scale columns was 2 μL of the protein standard.
For capillary LC-MS experiments, the sample was diluted to 0.2 mg/mL,
and the injection volume was 1.0 μL. The lyophilized lysate
of E. coli was diluted to a final concentration of 2.5 mg/mL, and
5.0 μL was injected.
6
Antioxidant Capacity Assessment after TBI
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Within 1 h after the last triggered SD with (n = 12) or without (n = 9) prior TBI, rats were euthanized, and their brains were dissected for antioxidant capacity assays as previously described [76 (link)]. Brain tissue of healthy rats was collected as control (n = 13). The injured right cortical hemisphere was isolated and homogenized (100 mg/mL) in 1.55 M KCl. Brain homogenates were added to a reaction mix with myoglobin (Sigma-Aldrich, St. Louis, MO, USA, ref. M0630) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (Roche, Mannheim, Germany, ref. 10102946001), and ABTS oxidation by H2O2 was measured spectrophotometrically (SpectraMax M3, Molecular Devices, San Jose, CA, USA) at an absorbance of 734 nm (Figure S2 ). Time to absorbance shift was measured and compared to a Trolox (an artificial antioxidant) (EMD Millipore, Temecula, CA, USA, ref. 648471) standard line.
7
Zinc Oxide Nanoparticle Assays
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Suspensions of n-ZnO (mean particle size 35 nm), reduced glutathione (GSH), bovine serum albumin, phenylmethylsulfonyl fluoride (PMSF), 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), nicotinamide adenine dinucleotides (NADH, NAD, NADPH), EDTA, dihydrorhodamine, Hoescht 33342 dye, 2,4-Dinitrophenylhydrazine, tyrosin, hemoglobin, chymotrypsinogen, cytohrome c, myoglobin, ubiquitin, Sephadex G-50, β-mercaptoethanol, ethoxyresorufin, certified reference material ERM-BB422 and Lactobacillus leichmannii D-Lactate dehydrogenase were purchased from Sigma Chem. Co. (St. Louis, USA). All other chemicals were obtained from the Synbias (Kyiv, Ukraine), Bayer (Kyiv, Ukraine) and Balkanpharma-Dupnitsa (Dupnitsa, Bulgaria) commercial suppliers. All reagents were of the analytical grade or higher.
8
Analytical Gel Filtration of Protein Complex
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Analytical gel filtration experiments were conducted using a Superdex 200 10/300 GL column (GE Healthcare Life Sciences) equilibrated in Buffer A (20 mM HEPES–NaOH (pH 7.0), 50 mM NaCl, 10% β-mercaptoethanol). Calibration of the Superdex 200 10/300 GL column was performed by running molecular ruler standards consisting of Thyroglobulin (165 kDa, Sigma), ConAlbumin (75 kDa, GE Healthcare Life Sciences), Albumin (43 kDa, Sigma), Myoglobin (17.6 kDa, Sigma) and Vitamin B12 (1.4 kDa, Sigma). The standard calibration curve was created by plotting retention volume data against the logarithm of the molecular weights of the calibration proteins and was fitted by linear least squares. Five hundred microliters samples consisting of 5 μM of each indicated component (PolB1, PolY, PCNA123, RFC, DNA21/31) and 1.6 mM ATP were mixed, nutated at room temperature for 10 min, and injected in the Superdex column (4°C). Protein elution was monitored at 280 nm and fractions collected at regular intervals.
9
Preparation of Biochemical Reagents
10
Protein Purification and Characterization
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