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NaCl effect on Lactase

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Jessica Hobbs

on 25 November 2016

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Transcript of NaCl effect on Lactase

NaCl effect on Lactase
To find out the natural salt content of the environment where lactase normally functions, to figure out the point in which the enzyme will begin to become denatured, and what the effects of high amounts of salt or low amounts of salt will have on the speed of the reaction.
Reasons for doing this experiment.
· H0: Salt concentration will not have any effect on the enzyme lactase.

· Ha: If the salt concentration is too high, then the enzyme will begin to be denatured, and if the salt concentration is not enough, then it will cause the reaction to be slowed down.
However when these elements
are put into a solution together salt has been shown to denature proteins. Since an enzyme is made up of both RNA and proteins, the salt concentration will cause the enzyme to break apart and it's active site, the site where the substrate binds to the enzyme to catalyze, to disappear or be misshapen. But enzyme activity needs a certain amount of salt to slow the reactions down. If there was no salt in the reaction, it would happen too fast, and if there was too much salt, the reaction would happen very slowly or not at all. There is an optimal salt concentration for enzyme activity. Blood for example has a salt concentration of .9%. This lab is meant to find the optimal salt concentration for lactase reactions.
This experiment was designed to test out the effects of salt on the enzyme lactase. We tested different concentrations of NaCl (20%, 10%, 5%, and 2.5%) and measured this under a spectrophotometer for the absorbance rate. We recorded our data every 20 seconds for 180 seconds per cuvette needing to be tested on.
Experimental Design
In order to complete our experiment, we first started a sterile dilution. We collected 8mL of 20% NaCl and placed it into Tube 1. We then removed 4mL from Tube 1 and placed this collection into Tube 2 and added 4mL of water into that same tube. We removed 4mL from Tube 2 to place into Tube 3. 4mL of water was also added into Tube 3. We again removed 4mL from Tube 3 and placed it into Tube 4 with an addition of 4mL of water. To make the volume the same as the other tubes for Tube 4, we removed 4mL from Tube 4 into a waste disposal. This completes the sterile dilution. For each tube we removed 1mL and placed them into their corresponding cuvettes (Tube 1 goes with Cuvette 1). Next we needed to make the enzyme lactase. We crushed a Lactaid pill until it became a powder. We mixed that powder with a 10mL phosphate buffer. After mixing, we filtered out the solution into a clean beaker. We then collected 2mL of that solution into another tube with 18mL of phosphate buffer. After developing lactase, we placed 1mL of lactase into each cuvette. In a 5th cuvette we added 1mL of lactase and 1mL of water to create the positive control. Before using the spectrophotometer, we blanked out the machine with a cuvette filled with only water. Right before it was each cuvettes’ turn to enter the spectrophotometer we added 1mL of ONPG to the cuvette and 2mL of lactase. We allowed each cuvette to run for 180 seconds and record data every 20 seconds. Once every cuvette has been tested, we cleaned our area analyzed our data.
Our data did not support a lot of our hypothesis. We assumed that the enzyme would be denatured by the amount of salt concentration, which was correct but we were incorrect about the speed of the reaction. There were many problems in our experiment unfortunately. Some of our beakers and substances were not labeled properly, we did not get to attempt the second trial run, and we did not do the experiment for the full 300 seconds.
Does the data support the hypothesis
In Figure 1 below we compared the different rates of absorbance to 5 different NaCL concentrations
As you can see the 0% concentration's reaction happened very quickly and flattened out before the trail was over. 10% and 20% concentration reactions both happened very slowly and flattened out at a low absorbency. Both the 2,5% and 5% concentrations had a steady slope between the
0% concentration and the 10/20% concentration. On 2.5% you can see a fluctuation and a large spike at the end. since we were unable to do a repeat trail we do not know if this was due to the concentration on due to human error. Based
on previous knowledge of the background of NaCl's affect
on enzyme activity we believe those two concentrations
are more optimal for enzyme activity than
the other concentrations
In Figure 2 we compared the five different concentrations at a certain time and graphed the delta absorbance
In this graph we can see that the higher concentrations seemed to have lower absorbency at 180 secs. we can infer that this is due to the higher salt concentration. the 0% concentration test had a high concentration at 180 seconds because the reaction had already reached it's maximum absorbency.
The 2.5% concentration's absorbency is higher than the 0% concentration because if you look at Figure 1 you can see a large spike towards the end of our trail time. From this graph we can infer that a low salt concentration, that is greater than 0%, may have the best
absorbency and that higher concentrations will not have as
much enzyme activity.
Future Research
There are many animals that have adapted to living in areas with high concentrations of salt. However the Dead Sea can't not sustain such abundant life, why is this true?

Our hypothesis is that the salt concentration in the dead sea is so much higher than that of most other oceans causing the protein in the enzyme to denature. This denaturing is so frequent that it is almost impossible for the environment to be able to preform the catalytic processes needed to sustain life.
Salt, or NaCl is an ionic compound between a positively charges sodium atom and a negatively charged chlorine atom. When placed in an aqueous solution NaCl completely dissociates, or breaks apart.
The enzyme lactase is an enzyme that breaks apart lactose, with is found in milk and other dairy products. ONPG, the substrate, was used in this experiment because it has a similar structure to lactose and can be catalyzed by lactase. In a aqueous solution lactase and ONPG with bind together to catalyze.

Diagram of a NaCl molecule
Interpretation of the Results
Figure 1:
Change in absorbance over time for NaCl concentrations gradually slow down at a similar rate. The 0% salt concentration absorbs the most but gradually slows its’ rate as time proceeds. 2.5% is next highest, then 5%, then 10%, then lastly 20%. This represents a degrading of lactase over time and with more concentrations of NaCl.

Figure 2:
The change in absorbance at 180 seconds has a rising peak from 0% to 2.5% concentrations of salt. This is the peak of lactase activity where it is most effective absorbing the salt. These are the only concentrations where the absorbance rates are increasing. After the concentration surpasses 2.5% the absorbance rate of salt takes a steep dip until the concentration has reached 4.5%. The dip symbolizes lactase’s lack of response to absorb all of the salt at the same rate as before. As the concentration of salt continues to increase the absorbance decreases at a slow gradual rate. The lactase activity is decreasing with more amounts of salt concentration meaning salt effected the enzyme lactase negatively.
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