This experiment was to test how differentpH levels affect lactase activity. Enzymeshelp speed up reactions within the human body. Lactase helps break down lactose in the digestive system. Using an enzyme concentration of 1/1000 and 2.
5mMONPG, we observed the enzyme activity with 3 different pH levels. The result was that pH 4 had the highest averageactivity compared to pH 7 and pH 10. This shows that lactase increases its activity within acidic pH levelsand decreases within basic levels. IntroductionEnzymes arecatalysts; this means without permanently chemically changing itself, they canincrease the rate of a reaction of another substance (Freemanet al.
, 2017). Enzymes have an active site that allows themto bind substrates together, which helps break old bonds and form new ones toproduce a product (Freeman et al., 2017). All reactions need some amount of initialenergy input to be completed. Enzymeslower the activation energy (the amount of energy needed to reach the transitstate) which increases the speed of the reaction (Freeman et al.
, 2017). Every enzyme hasits optimal functional conditions. Certainfactors can affect the function of an enzyme. Things like the pH level, temperature level, interactions with differentmolecules, and changes to its primary structure can alter the function of anenzyme (Freeman et al., 2017). Temperature can affect an enzymes movementand folding pattern (Freeman et al.
, 2017). The pH can affect the charge on amino and carboxylgroups in side chains and the active site’s ability to react with the transferof protons and electrons (Freeman et al.,2017). When properly used, thesefactors can help regulate an enzyme’s activity. The human bodyuses enzymes to help with many functions.
Lactase is an enzyme located in the small intestine that helps breakdown a milk sugar called lactose in the human body (Gerbault, 2014). Since this enzyme is located within thedigestive system, it will encounter fluids within the human digestivetract. Gastric fluids are known to havea high acidic pH of 1.5-3.5 (O’Connor and O’Morain, 2014).
A common average pH level of the fluids inthe small intestine is 6-7 (Evans et al.,1988). This brought up the question, ifthe pH level in the small intestine was more acidic, would the lactase reactionrate increase? We conducted anexperiment to find out if different pH levels affect the reaction rate oflactase. We hypothesizedthat the pH level would affect the lactase reaction rate. Using different pH levels as the environmentalcondition, we can test to see if an acidic, basic, or neutral pH produces theoptimal lactase reaction rate. Since theenzyme is in the digestive system, lactase activity will increase as the pHgets lower. Which in turn, the lactaseactivity will decrease as the pH level gets higher.
Materials andMethodsWe blanked thespectrophotometer (or zeroed it out) at the wavelength 420 nm with 3 mL ofphosphate buffer in a cuvette. Next, wemade the enzyme concentration 1/1000 by crushing up a lactate pill with amortar and pestle. We then took thatpowder and put it into a beaker containing 10 mL of phosphate buffer init. After we stirred the solution andlet it sit for 2 minutes, we filtered the solution into a different beaker witha paper towel. The filtered solutionbecame our stock enzyme solution. Next, we filled 3large test tubes with 9 mL of phosphate buffer in each tube and then added 1 mLof the stock enzyme solution to the first test tube. We then performed a serial dilution with theremainder of the tubes and put the last 1 mL into the sink.
The third test tube contains the enzymeconcentration that we used for the rest of this experiment, which is1/1000. Then we filled 3cuvettes each with 1 mL of the 1/1000 enzyme solution. We added 1 mL of pH 7 to the first cuvette, 1mL 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 mLof 2.
5mM ONPG to it and quickly put the cuvette into the spectrophotometer. We recorded the absorbance in 30 secondintervals for 5 minutes. After 5minutes, we removed the pH 7 cuvette and repeated the process with the pH 4 andthe pH 10 cuvettes. We then repeatedthis experiment for 2 more trials for each pH level. Results The data for each 3trials of each pH level are demonstrated by Tables 1, 2, and 3. The absorbance from the spectrophotometerrepresents the lactase activity. Foreach pH level, the total absorbance at minute 5 lowered with each trial. During trial 2, the pH 10 absorbance was inthe negatives for over 3 minutes.
Figure1 is a visual representation of Table 1. PH 4 started with the lowest absorbance at time 0 with .017, but endedminute 5 with the highest at .584. PH 10started with the highest absorbance at .047, but ended with the lowest at only.
092. Figure 2 represents Table 2 andproduced 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 thesame absorbance at .143 and .140. At minute5, pH 7 had the highest at .311. Figure3 visually represents Table 3 and is closer to trial 1 rather than trial2.
PH 10 had the highest absorbance attime 0 with -.006. PH 4 at time 0 hadthe lowest absorbance at -.031, but ended minute 5 with the highest at.
243. For each trail, pH 10 absorbancestayed at a slow constant increase. Figure4 shows the average absorbance for each pH level at minute 3.
DiscussionOur results supported the alternate hypothesisof the lactase reaction rate being affected by different pH levels. The pH 4 solution had the highest averageabsorption rate compared to the pH 7 and pH 10 solutions (refer to Figure 4). This supports lactase having a quicker reactionin acidic solutions rather than basic. Havinga basic pH added to lactase will decrease the reaction time significantly.
The data in trial 2 (Figure 2, Table 2) didnot support our hypothesis. This couldhave been due to errors in the solutions or in the spectrophotometer. A “cloudy” cuvette can cause misreads in aspectrophotometer.
Not zeroing themachine out before starting the experiment can also lead to altered data. We did not keep the solutions coved tightlyduring the experiment, so the outside environment could have altered them. This could also be the reason that theoverall absorbance for each trail decreased. For future experiments, we could keep thesolutions in their original containers with seal on them, so they are notexposed to the environment.
The outsideof the cuvettes could be cleaned right before putting the ONPG into it to make sureit is clear. We could also have multiple trials running at the same time insteadof performing them one at a time. Thiscould cut down the experiment time and the amount of error possibility. To get significant results, there needs to bemore research done on how different pH levels affect lactase activity.
Different concentrations of lactase need tobe tested at different pH levels. Thistype of experiment would help distinguish if the neutral smallintestine/lactase relationship is supported or not.