D-Serine was purchased from Bachem Biosciences Inc, horse radish peroxidase from Worthington Biochemical Corporation and o-phenylenediamine from Pierce Biotechnology, Inc. All other chemicals were obtained from Sigma-Aldrich. A reliable 96-well plate D-amino acid oxidase (DAO/DAAO) assay was developed based on previously published methods [32 (link)]. Briefly,D-serine (5 mM) was oxidatively deaminated by human DAO in the presence of molecular oxygen and flavin adenosine dinucleotide (FAD; 10 µM), to yield the corresponding α–keto acid, ammonia and hydrogen peroxide. The resulting hydrogen peroxide was quantified using horseradish peroxidase (0.01 mg/mL) and o-phenylenediamine (180 µg/mL), which turns yellowish-brown upon oxidation. DAO activity was correlated to the rate formation of the colored product, i.e., rate of change of absorbance at 411 nm. All reactions were carried out for 20 min at room temperature in a 100-µL volume in Tris buffer (50 mM, Ph 8.5). Additionally, stock solutions and serial dilutions of potential DAO inhibitors were made in 20:80 DMSO:buffer with a final assay DMSO concentration of 2%.
Horse radish peroxidase
Manufactured by Worthington
Horse radish peroxidase is an enzyme that catalyzes the oxidation of various organic compounds in the presence of hydrogen peroxide. It is commonly used as a label or marker in various analytical and diagnostic techniques.
Automatically generated - may contain errors
3 protocols using horse radish peroxidase
1
Quantifying D-Serine Oxidation by DAO
2
Peroxidase-coupled Assay for KDM1A and KDM1B Inhibition
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For
inhibition studies
on KDM1A and -B, a peroxidase-coupled assay monitoring hydrogen peroxide
production was performed as previously described.32 (link) KDM1A was purchased from BPSBioscience (Cat. No. 50097);
KDM1B cloning, expression, and purification.33 (link) The time courses of the reaction were measured under aerobic conditions
using a Beckman Instruments DU series 600 spectrophotometer equipped
with a thermostat-controlled cell holder (T = 25
°C). The 100 μL reactions were initiated by addition of
enzyme (100–200 nM KDM1A, or 480 nM KDM1B) to reaction mixtures
consisting of 50 mM HEPES buffer (pH 7.5), 0.1 mM 4-aminoantipyrine,
1 mM 3,5-dichloro-2-hydroxybenzene-sulfonic acid, 0.76 μM horseradish
peroxidase (Worthington Biochemical Corp.), 100 μM inhibitor,
and 24 μM or 100 μM H3K4me2 for KDM1A and KDM1B, respectively.
Absorbance changes were monitored at 515 nm, and an extinction coefficient
of 26 000 M–1 cm–1 was
used to calculate product formation. For curve fitting and data analysis,
GraphPad Prism 6.0 was used.
inhibition studies
on KDM1A and -B, a peroxidase-coupled assay monitoring hydrogen peroxide
production was performed as previously described.32 (link) KDM1A was purchased from BPSBioscience (Cat. No. 50097);
KDM1B cloning, expression, and purification.33 (link) The time courses of the reaction were measured under aerobic conditions
using a Beckman Instruments DU series 600 spectrophotometer equipped
with a thermostat-controlled cell holder (T = 25
°C). The 100 μL reactions were initiated by addition of
enzyme (100–200 nM KDM1A, or 480 nM KDM1B) to reaction mixtures
consisting of 50 mM HEPES buffer (pH 7.5), 0.1 mM 4-aminoantipyrine,
1 mM 3,5-dichloro-2-hydroxybenzene-sulfonic acid, 0.76 μM horseradish
peroxidase (Worthington Biochemical Corp.), 100 μM inhibitor,
and 24 μM or 100 μM H3K4me2 for KDM1A and KDM1B, respectively.
Absorbance changes were monitored at 515 nm, and an extinction coefficient
of 26 000 M–1 cm–1 was
used to calculate product formation. For curve fitting and data analysis,
GraphPad Prism 6.0 was used.
3
Kinetic Assay for KDM1A Inhibition
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Peptide
1 was purchased
from GenScript USA Inc. (NJ, USA); the purity was stated by the manufacturer
to be >95%. Peptides 2 and 3 were synthesized, purified, and characterized
as described elsewhere.17 (link) The peptide sequences
are shown in Table1 . For inhibition studies
on KDM1A, a peroxidase-coupled assay monitoring hydrogen peroxide
production was performed as previously described.18 (link) KDM1A expression and purification was carried out as described
elsewhere;13 (link) non-GST-tagged KDM1A was purchased
from BPS Biosciences (#50097). The time courses of the reaction were
measured under aerobic conditions using a Beckman Instruments DU series
600 spectrophotometer equipped with a thermostat-controlled cell holder
(T = 25 °C). The 100 μL reactions were
initiated by addition of enzyme (100–200 nM) to the reaction
mixture (HEPES buffer (50 mM, pH 7.5), 4-aminoantipyrine (0.1 mM),
3,5-dichloro-2-hydroxybenzenesulfonic acid (1 mM), horseradish peroxidase
(0.76 μM, Worthington Biochemical Corp.), peptide 1 (100 μM),
and H3K4me2 histone peptide substrate (24 μM)). Absorbance changes
were monitored at 515 nm, and an extinction coefficient of 26 000
M–1·cm–1 was used to calculate
product formation. For curve fitting and data analysis, GraphPad Prism
6.0 was used.
1 was purchased
from GenScript USA Inc. (NJ, USA); the purity was stated by the manufacturer
to be >95%. Peptides 2 and 3 were synthesized, purified, and characterized
as described elsewhere.17 (link) The peptide sequences
are shown in Table
on KDM1A, a peroxidase-coupled assay monitoring hydrogen peroxide
production was performed as previously described.18 (link) KDM1A expression and purification was carried out as described
elsewhere;13 (link) non-GST-tagged KDM1A was purchased
from BPS Biosciences (#50097). The time courses of the reaction were
measured under aerobic conditions using a Beckman Instruments DU series
600 spectrophotometer equipped with a thermostat-controlled cell holder
(T = 25 °C). The 100 μL reactions were
initiated by addition of enzyme (100–200 nM) to the reaction
mixture (HEPES buffer (50 mM, pH 7.5), 4-aminoantipyrine (0.1 mM),
3,5-dichloro-2-hydroxybenzenesulfonic acid (1 mM), horseradish peroxidase
(0.76 μM, Worthington Biochemical Corp.), peptide 1 (100 μM),
and H3K4me2 histone peptide substrate (24 μM)). Absorbance changes
were monitored at 515 nm, and an extinction coefficient of 26 000
M–1·cm–1 was used to calculate
product formation. For curve fitting and data analysis, GraphPad Prism
6.0 was used.
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