358
TARUN et al.
except for hesperidin (4 mg) dissolved in a mixture of and 46°C from the half-logarithmic plots of the kinetic
DMF (0.8 ml) and 50% EtOH (0.2 ml).
curves of the enzyme activity changes plotted versus
time, A/A0−time. Similarly, for the process of urease
Sonication of aqueous solutions of urease
(25 nM) at pH 5.4 was carried out at 36 and 46°C (in
the absence and presence of various flavonoid concen-
trations) in a polystyrene vial with an inner diameter of
3.3 cm and a height of 8 cm containing a total volume
of 30 ml of the enzyme solution. The temperature of the
sonicated solution was maintained with an accuracy of
0.5°C. The sonicated aqueous solutions of urease at
pH 5.4 were saturated with air, which provided practi-
cally time-independent concentration of the dissolved
oxygen, which was approximately ~10–4 M, according
to [25].
*
thermal inactivation, the effective rate constants, ki ,
were calculated from the half-logarithmic plots of the
curves in the A/A0−time coordinates. The rate constants
of the US inactivation of urease, kiUS in the presence of
flavonoids and in their absence, were calculated as the
*
difference ki – ki = ki(US). The error in the determina-
tion of the rate constants was about 13%.
ACKNOWLEDGMENTS
We thank Dr. Sci. (Chemistry) N.V. Kovganko
(IBCh, NAS of Belarus) for a gift of astragalin and
kaempferol.
This work was supported by the Belarusian Repub-
lic Foundation for Basic Research (project no. X05-
052).
An UZT-1.01F Apparatus for Ultrasonic Therapy
(EMA, Moscow, Russia) was used for the US treat-
ment. An US source with a piezoelectric converter
working at a frequency of 2.64 MHz was used. The
effective source area was 3.14 cm2, and the specific
power was 1 W/cm2. The waveguide was immersed into
the solution in a such way that the distance between its
butt-end and the bottom of the flask was 2.0 cm. The
sonication of solutions was carried out continuously for
1–2 h.
From the total solution volume of 30 ml, which con-
tained urease and flavonoids at various concentrations,
aliquots of the volume of 2.5–4.0 ml were taken to
study the thermal inactivation of urease, which was
studied at the same temperature as the sonication of the
solutions. The aliquots of 0.176 ml were taken every
5 min to determine the residual activity of urease (A).
Catalytic activity of the urease prior to its sonica-
tion (A0) and during the inactivation (A) were deter-
mined using pH-indicator according to the technique
earlier described in [6].
The preparation of substrate mixtures was achieved
using a 0.03 M urea solution. EDTA was added to
0.03 M urea aqueous solution (100 ml) to a final con-
centration of 0.05 mM, to bind impurities of heavy
metal ions. Bromocresol Purple was dissolved in a mix-
ture containing 0.2 ml of 0.05 M NaOH and 0.3 ml of
water. The final concentrations of urea, Bromocresol
Purple, and urease in the reaction mixture were, respec-
tively, 23 µM, 38.2 µM, and 5.5 nM.
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 4 2006