Du series 600 spectrophotometer

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 Table 1. 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.