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. 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.
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...