554 D. M. Tehrani et al.
enzyme assay and buffer preparation were of reagent
grade and obtained from Merck Company.
continued to reach the plateau (substrate depletion). e
Michaelis–Menten analysis was applied to determine the
reaction rate of the pure enzyme (Figure 1). Since sucrase
is more stable in pH 7, further kinetics experiments
were done at pH 7. Edie–Hofstee plot was employed
to determine the kind of inhibition in the presence
of different concentrations of ranitidine (Figure 2). A
noncompetitive inhibition in sucrase was observed after
different concentrations of ranitidine were added to the
enzyme and substrate solution.
e Km (11.5 mM) of enzyme was constant in the
presence of various concentrations of ranitidine
(Figure2),whileitsVmax decreasedbyincreasingranitidine
concentration (Figure 3). e concentration of ranitidine
producing 50% inhibition of maximal activity (IC50) was
determined to be about 2.2 mM (Figure 3). According to
Dixon plot, the inhibition constant (Ki) of ranitidine was
obtained to be 2.3 mM (Figure 4).
Enzyme activity
Enzyme activity was measured according to the method
reported in Ref. 9. Sucrase solution (0.5mg/ml) was pre-
pared by dissolving the enzyme in distilled water. e
optimum pH for enzyme activity was determined to be
4.5; however, the enzyme is more stable in pH 710. As a
matter of fact, ranitidine has been reported to be unstable
in acidic or basic solutions11; therefore, the enzyme assay
was performed in 0.1 M phosphate buffer at pH 7.
e reaction was started by adding 100 μl of sucrase
solution to the test tubes containing different concentra-
tions of sucrose (2.5–75 mM). For the assays done in the
presence of ranitidine, appropriate amounts of ranitidine
were added to the test tubes from the stock solution
(50 mM). e final volume in each test tube was always
2 ml and the concentration of ranitidine varied from 0.5
to 3 mM.
Structural changes
Sucrose hydrolyzing activity of sucrase was deter-
mined by measuring the rate of liberation of glucose and
fructose from sucrose and measured by the ferricyanide
method for determination of reducing sugars.
e enzyme was incubated with sucrose for 1, 3, 5, 10,
and 15 min in the presence and the absence of ranitidine.
e activity of enzyme was stopped at the mentioned
times by incubating the solution at 80°C. e ferricya-
nide colorimetric method was performed for the enzyme
activity12 using Perkin-Elmer visible spectrophotometer.
e results were the average of at least three separate
experiments and were expressed as mean standard
deviation.
To ensure the purity of the enzyme, sodium dodecyl sul-
fate polyacrylamide gel electrophoresis (SDS-PAGE) was
performed which showed only a single protein bond.
Fluorescence measurements were done for determination
of conformational changes occurred after binding of sub-
strate and inhibitor to the enzyme. e excitation was per-
formed at 285nm for exciting tryptophan residues and the
emission was recorded at a region between 310 and 400nm.
Excitation of pure enzyme showed a peak at 340nm (Figure
5, graph A). Binding of substrate to the enzyme reduced the
peakintensitywithoutanyshift(Figure5,graphB).Addition
of inhibitor to pure enzyme decreased the intensity of peak,
which was accompanied by a red shift to 344nm (Figure 5,
graph C). Addition of inhibitor to enzyme–substrate com-
plex also decreased the peak intensity and induced a red
shift to 344nm (Figure 5, graph D).
Fluorescence measurements
e fluorescence spectra were taken on a Perkin-Elmer
SL45 fluorometer. e excitation wavelength was 285nm
andtheemissionspectrawererecordedinarangebetween
310 and 400nm. e enzyme solution was prepared with
a concentration of 0.04mg/ml in 0.1M phosphate buffer
(pH 7). Sucrose was used with a concentration of 30mM,
and ranitidine concentration was 6.5 μM.
Discussion
e yeast sucrase has been proposed to be applied for
treatment of patients with CSID6. Sacrosidase (a liquid
Results
Inhibition of sucrase activity by ranitidine
e effect of ranitidine and cimetidine on sucrose activity
was monitored. Although both these drugs have nearly
the similar structure and effect, no change in the enzyme
activity was observed after using different concentrations
of cimetidine. In contrast, ranitidine affected the sucrase
activity significantly. So our measurements were focused
on ranitidine and its effects on sucrase activity.
e reaction rate of enzyme on sucrose (substrate)
was determined in the absence and the presence of
ranitidine by detecting the formation of products (glucose
and fructose) at 410 nm. e incubation of enzyme with
sucrose continued to pass into the steady state and
Figure 1. Michaelis–Menten plot shows that above 30 mM sucrose
concentration, the enzyme reaches to zero-order reaction.
Journal of Enzyme Inhibition and Medicinal Chemistry
Minai-Tehrani, Dariush
