Enzymes Lab Report Inroduction In this lab we explore an enzymes activity and how it can be affected by changes to its environment. An enzyme is a protein and is a catalyst to chemical reactions. It helps accelerate reactions by lowering the activation energy, which is needed for reactions in cells to progress at a higher rate. Activation energy is the minimum amount of energy needed for a chemical reaction to occur, yielding products from a given set of reactants. (Unit 7: Enzymes lab)
Products are results of the an enzyme cleaving to a specific substrate, by means of an induced fit. The induced fit is located at the active site of the enzyme or region of the enzyme where the substrate is bound. The substrate is the reactant within the reaction that fits with the enzyme like a key into a lock. Once the substrate enters the enzyme’s active site the enzyme can flexibly change shape to more snugly bind, via the induced fit, to form an enzyme-substrate complex.
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The substrate is then metabolized or broken down, resulting in a product, which can be utilized to energize cells. Once the product is released from the active site the enzyme returns to it’s original form. The main objective of this lab was to take the enzyme lactase and observe how well it acts as a catalyst to the substrates lactose and maltose while in varying environments. Lactase’s effectiveness was studied in an environment with differing temperatures, pHs, and while placed with a cofactor, an enzyme catalyst assistant.
The environment’s pH can range from 1-14, 7 being neutral, 7-1 being more and more acidic towards 1, and 8-14 being more and more basic towards 14. The product that was measuring to determine the enzyme’s performance was glucose, a monomer or small molecule, of the polymers, consisting of bound monomers, lactose and maltose. We hypothesized that lower temperatures would cause the enzyme to slow down and at higher temperatures it would unravel or go through denaturation, causing it to cease activity.
Since lactase is formed in the small intestine and the body temperature (~ 40 degrees C) is it’s optimal temperature and the normal pH of the small intestine is it’s optimal pH (pH=8) we also hypothesized that moving in either direction away from the norm would hinder the production of glucose. Thirdly, since lactase is specific the the substrate lactose (Lactose Intolerance and Health, Evidence Report/Technology Assessment Number 192) we hypothesized that lactose would have a higher metabolic rate than maltose.
Finally, since EDTA is most commonly added cofactor to laboratory solutions to bind and remove metal ions from the solution in order to slow undesired enzymatic reactions (Unit 7: Enzymes lab) we assumed that the breakdown of lactose into glucose by lactase was a desirable reaction and that EDTA would, not inhibit, but enhance the reaction. Methods Temperature experiment Microfuge tubes were labelled with 0, 40, 60, or 100. A plastic pipette was used to fill each tube up to the 0. 5 line with lactase solution. The volume of the lactase solution was 500mL. Each tube was place in a water bath or beaker and let to sit for 5 minutes.
An alternate plastic pipette was used and mild was added to the tube until the mixture of milk and lactase reached the 1. 0 line. After 10 minutes a glucose strip was placed into the tube for one second and then removed to sit on the bench top for 30 seconds. At the end of 1 minute the coloration on the strip was compared to the chart provided and the amount of glucose was determined in mg/dL. (Unit 7:Enzymes lab) pH experiment Three microfuge tubes were labelled acidic, neutral, and basic. A plastic pipette was used to fill the tube up to the 0. 5 line with milk.
Next, a clean plastic pipette was used to add 1 drop of 6M HCl to the acidic tube, and the solution was tested for pH with pH paper to verify that the solution had a pH of 2. Next, a clean plastic pipette was used and 1 drop of distilled water was added to the neutral tube. Then, it was tested with pH paper to verify that the solution had a pH of 7. Following that, a clean plastic pipette was used to add 1 drop of concentrated NaOH tho the basic tube, and was then tested with pH paper to verify that the pH was 12. Once the pH had been attained for each tube, lactase was added to bring the olution up to the 1. 0 line of the tube. All tubes were then placed in a 40 degrees C water bath and incubated for 10 minutes. After 10 minutes, a glucose strip was placed in each tube for one second and removed to compare the coloration of the strip to the chart provided and the amount of glucose was determined in mg/dL. (Unit 7: Enzymes lab) Specificity experiment One microfuge tube was labelled “Lactose” and another tube was labelled “Maltose”. Then, a clean plastic pipette was used to add milk up to the 0. 5 line of the lactose and also a clean pipette was used to do the same in the maltose tube.
Next, a clean plastic pipette was used to ad lactase to each tube until the level of mixture in each tube came up to the 1. 0 line. Both tubes were then placed in a 40 degrees C bath and incubated for 10 minutes. After 10 minutes a glucose strip was placed in each tube for one second, removed, and allowed to sit on the bench top for one minute. At the end of one minute the strip coloration was compared to the chart provided and the amount of glucose was determined in mg/dL. (Unit 7: Enzymes lab) Cofactor experiment One microfuge tube was labelled “Control” and another one “EDTA”. . 5 M EDTA was added to the EDTA tube until the solution reached the line between 1. 0 and 1. 5 lines on the tube. Distilled water was added to the control tube until the water reached the line between 1. 0 and 1. 5 on the tube. Then, three drops of milk were added to each tube and allowed to sit for 1 minute. Next, three drops of lactase were added to each tube and placed in the 40 degrees C water bath. They were left there for 10 minutes. After 10 minutes, a glucose strip was placed in each tube for one second, removed, and allowed to sit on the bench top for one minute.
At the end of one minute the strip coloration was compared to the chart provided and the amount of glucose was determined in mg/dL. (Unit 7: Enzymes lab) Results Temperature experiment | |0 C | | |Glucose | | |(mg/dL) | |100 |250 | | | | Control-Glucose (mg/dL) |EDTA-Glucose (mg/dL) | |Mean= |250. 5 |94 | |Sample size= |98 |98 | |t= 50. 1 df= 193 p < 0. 0001 reject the null hypothesis Discussion Although lactase is a very effective catalyst to lactose metabolism, we see that it is very sensitive. As we expose it to change from it’s normal environment temperature and pH it became almost immobilized rapidly, as it’s environment went farther from it’s optimal temperature and pH. In fact, we also discovered that EDTA plays a significant role in lowering the activation level of lactose.
It is now possible to see from our results that lactase is an enzyme that functions very well at optimal levels whether in the body or outside the small intestine. The finding that lactase is specific to the substrate lactose shows us that lactose intolerance is very widespread, affecting millions of individual, a is probably due to the lack of the enzyme lactase. Lactose intolerance is a condition that prevents an individual from metabolizing lactose. (Unit 7: Enzymes lab).
However, due to our findings in this lab that the environment is very influential on lactase, there may be a basis to say that lactose intolerance may be induced by a imbalance in the biological system. Studies have shown that the biological system is also affected by other enzymes too. Food is a very active vehicle that many people associate as being a catalyst for the production of vital energy and some are associating it with healthy enzymes. However, different forms of food are seen to affect us in varying ways.
Some produce an instant feel of increased energy and others seem to put us into a slumber. These varying results is not a figment of imagination. (Dr. Paul Kouchakoff, The Institute of Clinical Chemistry in Lausanne, Switzerland) For instance, raw food has been found to be more fitting to our biological system than cooked foods because the high temperatures of cooked food cause the enzymes in the food to be denatured, which in turn forces the body to produce the enzymes need to break down the food into energy.
On the other hand, raw food or slightly cooked food preserves the enzymes used to catalyze the molecular breakdown without the body needing to do the enzymatic work. (Biological Adaptations:Diet is Species Specific) If we want to know how the biological system of an individual responds to raw and cooked foods I propose that an experiment is done and that we do a Temperature experiment, similar to the experiment in this lab, on the glucose levels of subjects after eating raw foods and cooked foods.
Literature Cited anonymous. Lactose Intolerance and Health. Evidence Report/Technology Assessment Number 192. Minnesota Evidence-based Practice, Minnesota, MN. Kouchakoff, P. 1930. Influence of Food Cooking on the Blood Formula of Man. pages 1-4. Baker, M. Fossil Implies Our Early Kin Lived in Trees, Study Says. National Geographic News. November 21, 2002. page 1.