and
2372
EXPERIMENTAL
Material. The flowers (inflorescence) of Daphne
picked in March and April were stored frozen
10 g of the acetone powder of the flowers were suspended in 300 ml of
enzyme. Step 1.
until used.
Purification
M phosphate buffer
6.4, the suspension stirred for 10 min in the cold and centrifuged at 15,000
for 30 min. Three volumes of cold acetone at -20” were added to the supernatant, and after 30 min stand-
ing, the precipitate collected by centrifugation. It was taken in 50 ml of
M buffer, the viscous brownish
suspension centrifuged to discard insoluble materials and the supernatant passed through Sephadex G-50
(fine) column (76 x 200 mm).
Step 2. To the effluent
being free from D-8-G,
was added to 30
saturation
saturation, the
The
and after 1 hr the precipitate was discarded. The supernatant was further made up to
protein collected by centrifugation and dissolved in
was dialysed overnight against the same buffer.
10 ml of
M phosphate buffer
3. The dialvsate (Fraction was aoolied on a DEAE cellulose column
x 60 mm) which had
been equilibrated
washed with 10 ml of
buffer (Fraction 3).
M sodium phosphate buffer
M and 50 ml of
6.4. The discharged‘column was
M buffer, and the enzyme then extracted with 50 ml of
M
Step 4. The protein collected between 35-55 saturation of
was dissolved in water (10 ml).
The solution was dialysed against cold water and the precipitated protein was taken in
tion 4).
buffer (Frac-
Step 5. The above solution was further applied on a DEAE cellulose column (equilibrated with
M
buffer) and a fraction was obtained bv extraction with
(Fraction 5).
M buffer following
with
M buffer
The estimation of the enzyme activity. The hydrolase and transglucosylase activities were principally
estimated from decrease and increase, respectively, of the fluorescence intensity of D-8-G. It was deter-
mined with a spectrophotometer using the 350nmsetting for activation and measuring the output of 497
Linear relationship to the intensity can be observed only with
less than
M and
D-8-G up to
M can be estimated. Because the fluorescence intensity depends upon
the results in
M.
Fig. 2 were obtained from a
relation curve at D-8-G concentration of 5 x
Hydrolase. Mixtures consisting of 1 m-mole of buffer
total volume of 10 ml were incubated at room temp.,
recorded every 30 sec.
of D-8-G and enzyme in a
and the decrease of fluorescence at 493 nm was
The reaction system is composed of 1 m-mole of buffer
(glucose acceptor) and 2 of daphnin in a volume of ml. The reaction was proceeded as
above and the increase of fluorescence was estimated.
1
of daphnetin
One enzyme unit is defined as the amount of enzyme which transformed 1
under the qualified conditions.
of D-8-G for 1 min
Starch gel electrophoresis. Starch gel electrophoresis (Fig. 2) was conducted in
M phosphate buffer
6.4, using 10 V/cm at
block, a paper strip sprayed with
and running for
hr at 5”. For detecting the hydrolase band on the
M D-8-G solution was placed for several minutes on the one side
(HD in Fig. 2) and the area where the fluorescence disappeared was located. The transglucosylase band was
detected by placing another strip sprayed with a saturated daphnin solution on the other side of the block (TG
in Fig. 2) and by observing the appearance of fluorescence.
Substrate specificity. Hydrolase. In place of
of D-S-G in standard reaction mixture, 20
each of glucoside were used. The reaction was run for 10 min and stopped by adding 1 ml of
M
A part of the solution was chromatographed on
No. 1 filter paper with
and glucose was
determined by the method of
which was concentrated to a definite volume. The amount of glucose was
Transglucosylase. Daphnin in standard reaction solution was replaced, if necessary, by 2
glucoside and the formation of D-8-G was examined by fluorometry and paper chromatography.
Measurement of protein. This was carried out by the method of Lowry et
each of
Chemicals. Cichoriin was isolated from the flowers of Cichorium
obtained. andp-glucosyloxycinnamic were synthesized according to the literature.
was readily obtained in a large amount from mature Daphne flowers in which only a small amount of
daphnin is present. After the evaporation of ethanol extract of the flowers, the residue was extracted
and esculin commercially
D-8-G
with hot ethanol. Ethanol was replaced by water, daphnetin removed by extraction with ethyl acetate,
and the aqueous phase concentrated to a moderate volume. Crude crystals, obtained by standing overnight,
were recrystallized from hot water, m.p. 223-224”.
M. SOMOGHI, J. Biol. Chem. 195, 19 (1952).
0. H. LOWRY, N. J. ROSENBROLJGH , A. L. FARR and R. J. RANDALL,
Chem. 193,265 (1951).
J. B.
E.
Biochem. J. 74, 270 (1959).
Rec. Chim. 57, 535 (1938).
4, 245 (1965).
and 0.
D. J. AUSTIN and M. B. MEYERS,