493
T[ HAKAMATSUKA et al[
099) bu}er F in bu}er E and subsequently with
Dehydratase assay
bu}er F only "149 ml# at a ~ow rate of 2 ml:min[
Fractions of 8 ml were collected[
The puri_ed substrate\ 1\6\3?!trihydroxyiso!
~avanone\ in a micro!tube "ca[ 29 nmol# was dissolved
in Me1CO "099 ml# and then mixed with bu}er L "2
to 3 ml# which had been chilled on ice[ The substrate
solution "169 ml# was added to protein samples "29 ml#
which were diluted with adequate volume of bu}er L
prior to incubation[ The mixture was incubated for
19 min at 29>C and the reaction was terminated by
rapid chilling on ice[ The reaction products were
extracted with EtOAc "1 ml# and 0[4 ml of organic
layer was dried under N1 gas[ The residue was dis!
solved in 59) MeOH "199 ml# and 19 ml of this solu!
tion was analyzed by reversed phase HPLC column
"TSK gel ODS!019A\ 3[5×149 min#[ The column was
eluted with 59) MeOH at a ~ow rate of 9[7 ml:min
and the eluent was monitored at 143 nm[ Under these
conditions\ the retention times of 1\6\3?!trih!
ydroxyiso~avanone and daidzein were 4[1 and
01[4 min\ respectively[ The control incubation mixture
containing 29 ml bu}er instead of protein sample was
also assayed to measure the non!enzymatic conver!
sion[ The amount of daidzein formed was calculated
from the peak area using a calibration curve obtained
with the standard sample[
Anion!exchan`e chromato`raphy on mono Q[ The
pooled enzyme from phenyl sepharose "15 ml# was
applied to a PD!09 column pre!equilibrated with
bu}er G and proteins were eluted with bu}er G[ Using
a sample applicator ðSuperloop 49 "Pharmacia#Ł\ the
protein fractions were applied to a Mono Q column
"HR 4:4# which had been equilibrated with bu}er G[
After washing the column with 6 ml of bu}er G\ the
dehydratase was eluted with a linear gradient "31 ml#
of 9Ð39) bu}er H in bu}er G at 9[6 ml:min and
fractions of 9[24 ml were collected[
Chromatofocusin` on mono P[ Using a PD!09
column\ the bu}er of the pooled fractions from Mono
Q "0[3 ml# was changed to bu}er I\ and the protein
fractions were applied to a Mono P column "HR 4:4#
which had been equilibrated with bu}er I[ The column
was washed with 4 ml of bu}er I and the proteins
were eluted with 29 ml of bu}er J at 0 ml:min which
generated a pH gradient from 6[0 to 3[9\ and fractions
of 9[4 ml were collected and measured for their pH
value[
Gel_ltration chromato`raphy on G1999SWXL and
G2999SWXL[ An aliquot "9[14 ml# of the dehydratase
containing fractions from Mono P was applied in
tandem to G1999SWXL and G2999SWXL columns
which had been equilibrated with bu}er K[ The pro!
Puri_cation of the dehydratase
Hydroxyapatite chromato`raphy[ The desalted teins were eluted with bu}er K at 9[3 ml:min and
crude enzyme preparation "195 ml# was applied to a 9[1 ml fractions were collected[ Furthermore\ a mix!
column "4×6[5 cm# of hydroxyapatite "Bio Gel#\ ture of molecular weight standard proteins "189 999\
which had been equilibrated with bu}er B[ Unbound glutamate dehydrogenase^ 031 999\ lactate dehydro!
protein fractions eluted with bu}er B were pooled genase^ 56 999\ enolase^ 21 999\ adenylate kinase^
"249 ml# and precipitated with "NH3#1SO3 "79) satu! 01 399\ cytochrome C# were chromatographed under
ration# the pellet homogenized in bu}er B and the same condition to calibrate the column system[
desalted on PD!09 columns pre!equilibrated with
bu}er B[ The desalted protein was applied to a second
Analytical methods
hydroxyapatite column "4×3[5 cm# which had been
equilibrated with bu}er B\ and unbound fractions
eluted with the same bu}er were pooled "049 ml#[
Anion!exchan`e chromato`raphy on DEAE
sepharose[ The pooled dehydratase!containing frac!
tions from the hydroxyapatite column were adjusted
to pH 5[4 with 0 N HCl\ and loaded onto a column of
DEAE sepharose CL!5B "2[3×05 cm# which had been
equilibrated with bu}er C[ The column was washed
with bu}er C "259 ml# and the dehydratase was eluted
by a linear gradient "0199 ml# of 09Ð049 mM Pi in
Bu}er C at a ~ow rate of 0[1 ml:min[ Fractions of
09 ml were collected and assayed for dehydratase
activity[
Hydrophobic interaction chromato`raphy on phenyl
sepharose[ The pooled fractions from the DEAE
sepharose "043 ml# were brought to 19) saturation
with "NH3#1SO3\ adjusted to pH 6[9 "1 N KOH#\ and
applied to a HiLoad column 15:09 of phenyl
sepharose pre!equilibrated with bu}er E and the col!
umn was washed with 49 ml of bu}er E[ The dehy!
dratase was eluted by a linear gradient "499 ml# of 9Ð
Protein concentration was estimated by the Bio!
Rad protein assay using bovine serum albumin as
standard[ SDSÐpolyacrylamide gel electrophoresis
was performed according to Laemmli ð09Ł with 01)
gel and proteins were silver stained[ Molecular weight
standards for SDSÐPAGE "Bio!Rad# "rabbit muscle
phosphorylase b\ 86 399^ bovine serum albumin\
55 199^ hen egg white ovalbumin\ 34 999^ bovine car!
bonic anhydrase\ 20 999^ soybean trypsin inhibitor\
10 499^ hen egg white lysozyme\ 03 399#\ were used for
molecular weight calibration[
Acknowled`ements*A part of this work was sup!
ported by Grant!in!Aids of Ministry of Education\
Science\ Culture and Sports\ Japan[
REFERENCES
0[ Dewick\ P[ M[\ In The Flavonoids\ Advances in
Research since 0879\ eds J[ B[ Harborne and T[ J[
Hakamatsuka, Takashi
