This will increase as the pH gets
This experiment was to test how different
pH levels affect lactase activity. Enzymes
help speed up reactions within the human body.
Lactase is one of those enzymes and helps break down milk protein. Using an enzyme concentration of 1/1000 and 2.5mM
ONPG, we observed the enzyme activity with 3 different pH levels. The result was that pH 4 had the highest average
activity compared to pH 7 and pH 10.
This shows that lactase increases its activity within acidic pH levels
and decreases within basic levels.
catalysts; this means without permanently chemically changing itself, they can
increase the rate of a reaction of another substance (Freeman
et al., 2017). Enzymes have an active site that allows them
to bind substrates together, which helps break old bonds and form new ones to
produce a product (Freeman et al., 2017). All reactions need some amount of initial
energy input to be completed. Enzymes
lower the activation energy (the amount of energy needed to reach the transit
state) which increases the speed of the reaction (Freeman et al., 2017).
Every enzyme has
its optimal functional conditions. Certain
factors can affect the function of an enzyme.
Things like the pH level, temperature level, interactions with different
molecules, and changes to its primary structure can alter the function of an
enzyme (Freeman et al., 2017). Temperature can affect an enzymes movement
and folding pattern (Freeman et al., 2017). The pH can affect the charge on amino and carboxyl
groups in side chains and the active site’s ability to react with the transfer
of protons and electrons (Freeman et al.,
2017). When properly used, these
factors can help regulate an enzyme’s activity.
The human body
uses enzymes to help with many functions.
Lactase is an enzyme located in the small intestine that helps break
down a milk sugar called lactose in the human body (Gerbault, 2014). Since this enzyme is located within the
digestive system, it will encounter fluids within the human digestive
tract. Gastric fluids are known to have
a high acidic pH of 1.5-3.5 (O’Connor and O’Morain, 2014). A common average pH level of the fluids in
the small intestine is 6-7 (Evans et al.,
1988). This brought up the question, if
the pH level in the small intestine was more acidic, would the lactase reaction
rate increase? We conducted an
experiment to find out if different pH levels affect the reaction rate of
that the pH level would affect the lactase reaction rate. Using different pH levels as the environmental
condition, we can test to see if an acidic, basic, or neutral pH produces the
optimal lactase reaction rate. Since the
enzyme is in the digestive system, lactase activity will increase as the pH
gets lower. Which in turn, the lactase
activity will decrease as the pH level gets higher.
We blanked the
spectrophotometer (or zeroed it out) at the wavelength 420 nm with 3 mL of
phosphate buffer in a cuvette. Next, we
made the enzyme concentration 1/1000 by crushing up a lactate pill with a
mortar and pestle. We then took that
powder and put it into a beaker containing 10 mL of phosphate buffer in
it. After we stirred the solution and
let it sit for 2 minutes, we filtered the solution into a different beaker with
a paper towel. The filtered solution
became our stock enzyme solution.
Next, we filled 3
large test tubes with 9 mL of phosphate buffer in each tube and then added 1 mL
of the stock enzyme solution to the first test tube. We then performed a serial dilution with the
remainder of the tubes and put the last 1 mL into the sink. The third test tube contains the enzyme
concentration that we used for the rest of this experiment, which is
Then we filled 3
cuvettes each with 1 mL of the 1/1000 enzyme solution. We added 1 mL of pH 7 to the first cuvette, 1
mL of pH 4 to the second cuvette, and 1 mL of pH 10 to the third cuvette. Next, we took the pH 7 cuvette and added 1 mL
of 2.5mM ONPG to it and quickly put the cuvette into the spectrophotometer. We recorded the absorbance in 30 second
intervals for 5 minutes. After 5
minutes, we removed the pH 7 cuvette and repeated the process with the pH 4 and
the pH 10 cuvettes. We then repeated
this experiment for 2 more trials for each pH level.
The data for each 3
trials of each pH level are demonstrated by Tables 1, 2, and 3. The absorbance from the spectrophotometer
represents the lactase activity. For
each pH level, the total absorbance at minute 5 lowered with each trial. During trial 2, the pH 10 absorbance was in
the negatives for over 3 minutes. Figure
1 is a visual representation of Table 1.
PH 4 started with the lowest absorbance at time 0 with .017, but ended
minute 5 with the highest at .584. PH 10
started with the highest absorbance at .047, but ended with the lowest at only
.092. Figure 2 represents Table 2 and
produced different results than trial 1.
PH 4 at time 0 had the highest absorbance at .013. At 150 seconds, pH 4 and pH 7 had roughly the
same absorbance at .143 and .140. At minute
5, pH 7 had the highest at .311. Figure
3 visually represents Table 3 and is closer to trial 1 rather than trial
2. PH 10 had the highest absorbance at
time 0 with -.006. PH 4 at time 0 had
the lowest absorbance at -.031, but ended minute 5 with the highest at
.243. For each trail, pH 10 absorbance
stayed at a slow constant increase. Figure
4 shows the average absorbance for each pH level at minute 3.
Our results supported the alternate hypothesis
of the lactase reaction rate being affected by different pH levels. The pH 4 solution had the highest average
absorption rate compared to the pH 7 and pH 10 solutions (refer to Figure 4). This supports lactase having a quicker reaction
in acidic solutions rather than basic. Having
a basic pH added to lactase will decrease the reaction time significantly.
The data in trial 2 (Figure 2, Table 2) did
not support our hypothesis. This could
have been due to errors in the solutions or in the spectrophotometer. A “cloudy” cuvette can cause misreads in a
spectrophotometer. Not zeroing the
machine out before starting the experiment can also lead to altered data. We did not keep the solutions coved tightly
during the experiment, so the outside environment could have altered them. This could also be the reason that the
overall absorbance for each trail decreased.
experiments, we could keep the solutions in their original containers with seal
on them, so they are not exposed to the environment. The outside of the cuvettes could be cleaned
right before putting the ONPG into it to make sure it is clear. We could also
have multiple trials running at the same time instead of performing them one at
a time. This could cut down the
experiment time and the amount of error possibility. To get significant results, there needs to be
more research done on how different pH levels affect lactase activity. Different concentrations of lactase need to
be tested at different pH levels. This
type of experiment would help distinguish if the neutral small
intestine/lactase relationship is supported or not.