EXPERIMENT NO. 4 & 5
ENZYME KINETICS AND ELECTROPHORESIS (SDS-PAGE)
Background of the experiment
The experiment aims to extract Polyphenol oxidase from Lakatan banana; thus, inspect its kinetic activity on
catechol substrate by determining the Km and Vmax, and identify the type of inhibition that benzoic acid
exhibited. The experiment also aims to classify the molecular weight of Polyphenol oxidase from Lakatan banana
through electrophoresis (SDS-PAGE).
Results and Discussion
Enzymes are proteins that act as biological catalysts that speed up reactions. They work by forming a complex
with the reactants or substrates, resulting in transition-state species which become the product. An example of
enzymes is the Polyphenol oxidase which is present in tissues of many plants and fungi. Its enzymatic activity is
characterized by a discoloration, such as brown pigments that contribute to higher absorbance of light; hence,
UV-Vis Spectrophotometry can be used to measure its concentration.
In the experiment, Polyphenol oxidase was obtained from a Lakatan banana, a non-fully ripened fruit. This was
done to avoid the mentioned formation of brown spots that resulted from enzymatic oxidation. The banana was
then cut, and 100 g of it was weighed which was then added with 100 ml of 0.1 M phosphate buffer with pH 7 to
stabilize the target enzyme. The sample was then subjected to homogenization using a blender to disrupt the cells
and release the enzymes of interest. This was also to form a mixture with uniform consistency. The resulting
crude extract was then filtered using a cheesecloth into an Erlenmeyer flask immersed in an ice bath. This step in
the procedure was to isolate the enzymes, and keep it from denaturing. The filtrate was then centrifuged for 10
minutes or until the supernatant was clear. This was to further remove the undesired components in the sample. It
was maintained in an ice bath until use to avoid denaturation.
Catechol substrates were also prepared to assess the enzyme. When exposed to the polyphenol oxidase, the
catechol turns into reddish brown. A 50 mL stock solution with a concentration of 10 mM was made, and 5
working solutions with varying concentrations were prepared: 4.8 mM, 1.2 mM, 0.6 mM, and 0.3 mM. The
dilution was done using a phosphate buffer. Three trials of 160 µL of each solution were then pipetted into the
wells of 96-well plate, along with a blank consisting of the phosphate buffers. From the extracted sample, 80 µL
was added into each prepared well and the absorbance was read for 5 minutes at 420 nm at 10-second intervals.
Enzyme production or activities can be slowed down with interfering substances, resulting in slower rate of
reaction. There are three known reversible modes of inhibition namely competitive, non-competitive, and
uncompetitive. In a competitive inhibition, the inhibitor competes with the substrate to bind to the enzyme’s
active site, blocking the substrate. This is due to the high affinity of the inhibitor to the enzyme. However,
competitive inhibition can be overcome by increasing the concentration of the substrate. On the other hand,
non-competitive inhibitors bind to allosteric sites or other sites aside from the active site. This results in a change
of the active site’s shape which hinders the activity to proceed catalysis requires a high free of specificity. Lastly,
uncompetitive inhibitors bind with the enzyme-substrate complex, preventing product formation. Usually, this
type of inhibitor binds to an allosteric site, changing the conformation of the enzyme so that the affinity of the
substrate for the active site is reduced.
In the experiment, benzoic acid was used as an inhibitor and its type of inhibition was determined by computing
for the Km and Vmax values of the inhibited and uninhibited reactions. The average absorbance readings per
concentration obtained after using UV-VIS spectrophotometer were first plotted against time in order to determine
the respective initial velocity, Vo, of each. The Michaelis constant, represented by the Km, shows the amount of
the substrate present for a reaction to have half of its Vmax, suggesting that affinity of the enzyme for the
substrate. The mentioned Vmas can illustrate the efficiency of enzymes by showing how much of the substrate is
, needed to create the product given an interval of time. The following figures show the plots of the uninhibited
(Figure 1) and inhibited (Figure 2), wherein each figure of the plots of five working solutions are shown below.
Figure 1. Plot absorbance readings versus time for uninhibited solutions
Figure 2. Plot absorbance readings versus time for inhibited solutions
It can be observed in both figures that as the concentration of catechol in a phosphate buffer gets higher, the
absorbance readings obtained gets higher as well, thereby implying higher enzymatic activity. The slope for each
working solution, both inhibited and uninhibited, were determined as summarized in the table (Table 1) below.
The slope represents the initial velocity or Vo. The gathered initial velocities and concentration of catechol
solution ([S]) of each set-up were used to obtain the Michaelis-Menten and the linearized methods:
Lineweaver-Burk, Eadie-Hofstee and Hanes-Woolf plots. The Km and Vmax of the enzymatic reactions under
inhibited and uninhibited reactions were then computed using the four equations derived from the mentioned
plots.
Working Solution and Initial Velocity, Vo, Initial Velocity, Vo,
Concentration of Catechol (Uninhibited), min-1 (Inhibited), min-1
ENZYME KINETICS AND ELECTROPHORESIS (SDS-PAGE)
Background of the experiment
The experiment aims to extract Polyphenol oxidase from Lakatan banana; thus, inspect its kinetic activity on
catechol substrate by determining the Km and Vmax, and identify the type of inhibition that benzoic acid
exhibited. The experiment also aims to classify the molecular weight of Polyphenol oxidase from Lakatan banana
through electrophoresis (SDS-PAGE).
Results and Discussion
Enzymes are proteins that act as biological catalysts that speed up reactions. They work by forming a complex
with the reactants or substrates, resulting in transition-state species which become the product. An example of
enzymes is the Polyphenol oxidase which is present in tissues of many plants and fungi. Its enzymatic activity is
characterized by a discoloration, such as brown pigments that contribute to higher absorbance of light; hence,
UV-Vis Spectrophotometry can be used to measure its concentration.
In the experiment, Polyphenol oxidase was obtained from a Lakatan banana, a non-fully ripened fruit. This was
done to avoid the mentioned formation of brown spots that resulted from enzymatic oxidation. The banana was
then cut, and 100 g of it was weighed which was then added with 100 ml of 0.1 M phosphate buffer with pH 7 to
stabilize the target enzyme. The sample was then subjected to homogenization using a blender to disrupt the cells
and release the enzymes of interest. This was also to form a mixture with uniform consistency. The resulting
crude extract was then filtered using a cheesecloth into an Erlenmeyer flask immersed in an ice bath. This step in
the procedure was to isolate the enzymes, and keep it from denaturing. The filtrate was then centrifuged for 10
minutes or until the supernatant was clear. This was to further remove the undesired components in the sample. It
was maintained in an ice bath until use to avoid denaturation.
Catechol substrates were also prepared to assess the enzyme. When exposed to the polyphenol oxidase, the
catechol turns into reddish brown. A 50 mL stock solution with a concentration of 10 mM was made, and 5
working solutions with varying concentrations were prepared: 4.8 mM, 1.2 mM, 0.6 mM, and 0.3 mM. The
dilution was done using a phosphate buffer. Three trials of 160 µL of each solution were then pipetted into the
wells of 96-well plate, along with a blank consisting of the phosphate buffers. From the extracted sample, 80 µL
was added into each prepared well and the absorbance was read for 5 minutes at 420 nm at 10-second intervals.
Enzyme production or activities can be slowed down with interfering substances, resulting in slower rate of
reaction. There are three known reversible modes of inhibition namely competitive, non-competitive, and
uncompetitive. In a competitive inhibition, the inhibitor competes with the substrate to bind to the enzyme’s
active site, blocking the substrate. This is due to the high affinity of the inhibitor to the enzyme. However,
competitive inhibition can be overcome by increasing the concentration of the substrate. On the other hand,
non-competitive inhibitors bind to allosteric sites or other sites aside from the active site. This results in a change
of the active site’s shape which hinders the activity to proceed catalysis requires a high free of specificity. Lastly,
uncompetitive inhibitors bind with the enzyme-substrate complex, preventing product formation. Usually, this
type of inhibitor binds to an allosteric site, changing the conformation of the enzyme so that the affinity of the
substrate for the active site is reduced.
In the experiment, benzoic acid was used as an inhibitor and its type of inhibition was determined by computing
for the Km and Vmax values of the inhibited and uninhibited reactions. The average absorbance readings per
concentration obtained after using UV-VIS spectrophotometer were first plotted against time in order to determine
the respective initial velocity, Vo, of each. The Michaelis constant, represented by the Km, shows the amount of
the substrate present for a reaction to have half of its Vmax, suggesting that affinity of the enzyme for the
substrate. The mentioned Vmas can illustrate the efficiency of enzymes by showing how much of the substrate is
, needed to create the product given an interval of time. The following figures show the plots of the uninhibited
(Figure 1) and inhibited (Figure 2), wherein each figure of the plots of five working solutions are shown below.
Figure 1. Plot absorbance readings versus time for uninhibited solutions
Figure 2. Plot absorbance readings versus time for inhibited solutions
It can be observed in both figures that as the concentration of catechol in a phosphate buffer gets higher, the
absorbance readings obtained gets higher as well, thereby implying higher enzymatic activity. The slope for each
working solution, both inhibited and uninhibited, were determined as summarized in the table (Table 1) below.
The slope represents the initial velocity or Vo. The gathered initial velocities and concentration of catechol
solution ([S]) of each set-up were used to obtain the Michaelis-Menten and the linearized methods:
Lineweaver-Burk, Eadie-Hofstee and Hanes-Woolf plots. The Km and Vmax of the enzymatic reactions under
inhibited and uninhibited reactions were then computed using the four equations derived from the mentioned
plots.
Working Solution and Initial Velocity, Vo, Initial Velocity, Vo,
Concentration of Catechol (Uninhibited), min-1 (Inhibited), min-1
