Article
J. Agric. Food Chem., Vol. 58, No. 9, 2010 5221
Thioglo1 could convert the end-point assay of Collazo et al. (26)
to a continuous assay for COMT and one that could be readily
adapted for plate-reading spectrofluorimeters.
As a routine protocol, 10 μL of a stock substrate solution was first
added to the appropriate wells of a black 96 well microplate (Microfluor 2
black, flat well, ThermoFisher Scientific, Waltham, MA), and enzyme
assays were initiated by the addition of 140 μL of the reaction mixture
described above to each well. Well contents were thoroughly mixed by
pipet action, and the plate was placed in a Bio Tek Synergy HT fluore-
scence plate reader (Bio Tek Instruments, Winooski, VT) set at 37 °C with
a gain set to 80. Fluorescence was determined using an excitation filter 360
( 40 nm and an emission filter 528 ( 20 nm. Fluorescence signal was
measured every minute for 45 min with an instrument predesignated
“moderate shake” for 5 s between each read. Raw fluorescence data was
imported into Microsoft Excel (MS Office 2007) and analyzed. Fluore-
scence data were converted into picomoles of -SH groups released based
on a standard curve using glutathione as a standard.
For kinetic analyses, substrate concentrations were varied, but the total
volume added per well was maintained at 10 μL. Generally 30 μL of plant
extracts were assayed. All enzymatic analyses were performed in triplicate,
and these experiments have been repeated at least thrice. All chemicals
were reagent grade or better. All solutions, especially buffers and sub-
strates and plant extracts, were prepared fresh prior to an assay. Recombi-
nant enzymes were stored as aliquots at -80 °C and thawed prior to use.
During the development and validation of our assay for COMT, we
also found that adenosine deaminase was not required because of the rapid
reaction between Thioglo1 and homocysteine. The reaction rate could be
manipulated by varying the levels of both COMT at fixed SAHH levels or
increasing SAHH at fixed levels of COMT in the assay well. We have
optimized conditions that result in reproducible increase in fluorescence
for a range of substrates and one that permitted the analyses of COMT
activities in total plant homogenates.
MATERIALS AND METHODS
Recombinant Proteins. Sorghum COMT (Bmr12). The coding
region for sorghum COMT (Bmr12) was amplified by PCR using the
0
0
following primers forward 5 -CCCAGATCTGATGGGGTCGACGGC-
0
GGAGGACGTGGCGG-3 , reverse 5 -GAGTGCGGCCGCTTACTT-
0
GATGAACTCGATGGCCC-3 ; sorghum EST clone (ANR1_8_C01_A002;
GenBank numbers CX607344 and CX607415) as the template . The
amplicon was digested with BglII and NotI and ligated into pET30a vector
according to manufacturer’s protocol (Novagen, Madison, WI). Auto-
mated sequencing was performed by University of Nebraska;Lincoln
sequencing facility to verify sequence fidelity of the construct. An error
introduced into ANR1_8_C01_A002 through cloning, which changed
the codon for amino acid 282 from tyrosine to cysteine, was corrected
using site-directed mutagenesis. Recombinant protein was produced in
Escherichia coli Rosetta 2 cells (Novagen, Madison, WI), and purified
essentially as described earlier (27). Purity of the final enzyme preparation
was checked by gel electrophoresis under denaturing conditions (28).
Purified recombinant sorghum COMT was dialyzed against 50 mM Tris,
pH 7.5, and stored in aliquots at -80 °C.
Sulfolobus SAHH. The Sulfolobus SAHH clone was obtained through
a Materials Transfer Agreement with Dr. Raymond C. Trievel, Depart-
ment of Biological Chemistry, University of Michigan, Medical School
and was recombinantly produced and purified essentially as described by
Collazo et al. (26).
Protein concentrationsweredeterminedusing a colorimetricmicroplate
assay using Coomassie blue and bovine serum albumin as a standard, as
described by the manufacturer (Thermo Scientific, Rockford, IL).
Plant Materials. Switchgrass plants were grown in the field as described
earlier (29). Flowering tillers were hand separated into internodes, with
internode 1 being closest to the peduncle. Internodes were flash frozen in
GCMS Analyses. GCMS analyses were performed on assay solutions
containing caffeic acid to determine if ferulic acid (the product derived
from methylation of caffeic acid) could be detected in the assays, and to
serve as an independent estimator of the effect of substrate concentration
on product formation under our standardized assay conditions (see
above). Briefly, assays were performed as described above and at the
end of the fluorescence assay (∼45 min), all assay wells in the microplate
were acidified by the addition of 50 μL of 6 M HCl and the entire well
contents (∼200 μL) were transferred to a 1.7 mL microfuge tube. The
acidified solutions were back extracted thrice with 0.25 mL aliquots of
ethyl acetate, and the organic solutions were pooled. A few crystals of
anhydrous sodium sulfate were added to the combined organic phase,
followed by centrifugation at 13,500 rpm for 5 min. The supernatant was
transferred to a fresh tube and dried by centrifugal evaporation. The dried
residue was silylated and analyzed using an Agilent G2570A integrated
GCMS system equipped with a G2913A autoinjector module, 6850 series
II GC, and a 5973 network mass spectrometer (Agilent, Palo Alto, CA) as
described previously (29).
liquid N , and stored frozen at -80 °C until used.
2
Internodes were first ground with chucks of dry ice in a coffee grinder.
This powdered material was placed at -80 °C until the dry ice had
sublimated (approximately 48 h), then poured into 50 mL plastic tubes for
storage. When needed ∼0.7 g of internode material was weighed, trans-
ferred to a precooled mortar and pestle and extracted with 1.4 mL of
5
0 mM Tris buffer, pH 7.5, containing 0.35 M sucrose. Homogenates were
filtered through 1 layer of MiraCloth, and the solution was placed in
.0 mL microfuge tubes and centrifuged at 13,500 rpm for 15 min in a
2
refrigerated centrifuge (MicroGPR, Thermo Scientific, Rockford, IL).
Clarified supernatants were used as a source of native COMT. Protein
concentrations in extracts were determined as described above. Switch-
grass COMT was partially purified by binding 250 μL of clarified,
internode homogenates to a strong anion exchange membrane (Vivapure
Q, MiniH, Sartoriusstedim Biotech., Goettingen, Germany) equilibrated
with 50 mM Tris buffer, pH 7.5 containing 0.35 M sucrose. The membrane
filter was washed with 2 ꢀ 250 μL of equilibrating buffer. Bound proteins
were sequentially eluted with 1 ꢀ 250 μL of equilibrating buffer containing
Data Analyses and Parameter Estimates. Individual reaction rates were
calculated by fitting a 2-parameter line to the linear portion of the
fluorescence data using linear least-squares. To identify this portion,
regressions were performed sequentially every 5 min on the observed
linear portion of the fluorescence curve to obtain predicted maximal velo-
city of reaction. The slope of this line was taken as an estimate of product
formation over time. Reaction rates were then plotted vs substrate
concentration. The Michaelis-Menten kinetic model was fit to this data
using nonlinear least-squares in SigmaPlot 11.0 to provide estimates for
0.25 and 0.6 M NaCl. The wash fractions were pooled. All three fractions
were assayed for COMT activity using the standard assay solution (see
above).
m
the parameters K and Vmax.
Enzyme Assays. Routine COMT activity analyses were performed
under the following conditions in a final well volume of 150 μL: 100 mM
Tris-Cl, pH 7.5, 1 μM SAHH, 150 nM recombinant sorghum COMT,
RESULTS AND DISCUSSION
8
5 μM ado-met and ∼15 μM Thioglo1 (Covalent Associates Inc.,
Fluorescent COMT Assay. The essential features of our assay
are shown in Figure 1, and the assay is based on the original
method developed by Collazo et al. (26) to assay histone methyl-
transferases. We have modified this original method in two
important ways: (1) we have discontinued the use of adenosine
deaminase, and (2) we have utilized the fast reaction kinetics
between Thioglo1 with free sulfhydryls to convert a discrete end-
point assay into a reproducible real-time, continuous method to
assay plant COMTs involved in cell wall lignification.
Corvallis, OR) final concentrations were prepared. Thioglo1 was obtained
as its maleimide derivative. Neither the maleimido form nor the hydro-
lyzed unconjugated Thioglo1 exhibits intrinsic fluorescence (http://www.
covalentassociates.com/thioglo.htm). Thioglo1 was prepared as a stock
solution in dimethylformamide, divided into aliquots, and kept in foil
covered tubes at -80 °C. Phenolic substrates were prepared as 0.5 to 5 mM
stock solutions in 10% dimethylformamide in water (v/v) and used at the
indicated concentrations. For routine assays, the final concentration of a
phenolic substrate in each well was 250 μM. All of the components, except
for the phenolic substrate, were mixed together to yield a bulk reaction
mixture that was subsequently used for assays.
Reaction of Thioglo1 with Reduced Glutathione. Thioglo1
displayed a large dynamic fluorescence range when directly