Cellohexaose was
used as substrate. One hundred microliter reactions were set up with
750 μM cellohexaose, 1 mM ascorbic acid, 100 μM H2O2 (no H2O2 added in the
O2 turnover reactions), and 1 μM LsAA9 or purple LsAA9 in 5 mM MES at pH 7.0 and were
incubated at 40 °C for 2 h. The reaction was then quenched by
addition of 3 reaction volumes of ethanol (98% v/v). Reactions with
H2O2 were performed inside an N2 atmosphere
glovebox. A 1 μL amount of sample was then mixed with 2 μL
of 10 mg/mL 2,5-dihydroxybenzoic acid in 50% acetonitrile, 0.1% trifluoroacetic
acid on a Bruker SCOUT-MTP 384 target plate. The spotted samples were
then dried in air under a lamp before being analyzed by mass spectrometry
on a Ultraflex III matrix-assisted laser desorption ionization-time-of-flight/time-of-flight
(MALDI-TOF/TOF) instrument (Bruker), as described previously.9 (link) The purple species LsAA9 sample
used in the assay was incubated with EDTA overnight to remove Cu2+ from any LsAA9 that had not been converted
to the purple species. The resulting [Cu(EDTA)]2– complex was then removed from the solution by ultracentrifugation
through 10 kDa cutoff size-exclusion filters.
used as substrate. One hundred microliter reactions were set up with
750 μM cellohexaose, 1 mM ascorbic acid, 100 μM H2O2 (no H2O2 added in the
O2 turnover reactions), and 1 μM LsAA9 or purple LsAA9 in 5 mM MES at pH 7.0 and were
incubated at 40 °C for 2 h. The reaction was then quenched by
addition of 3 reaction volumes of ethanol (98% v/v). Reactions with
H2O2 were performed inside an N2 atmosphere
glovebox. A 1 μL amount of sample was then mixed with 2 μL
of 10 mg/mL 2,5-dihydroxybenzoic acid in 50% acetonitrile, 0.1% trifluoroacetic
acid on a Bruker SCOUT-MTP 384 target plate. The spotted samples were
then dried in air under a lamp before being analyzed by mass spectrometry
on a Ultraflex III matrix-assisted laser desorption ionization-time-of-flight/time-of-flight
(MALDI-TOF/TOF) instrument (Bruker), as described previously.9 (link) The purple species LsAA9 sample
used in the assay was incubated with EDTA overnight to remove Cu2+ from any LsAA9 that had not been converted
to the purple species. The resulting [Cu(EDTA)]2– complex was then removed from the solution by ultracentrifugation
through 10 kDa cutoff size-exclusion filters.
