Laboratorio

Effects of pH and temperature on Enzymes

Olivia Wilkins
Professor Barrett
Cells and Genes
Wednesday 5:30-8:30
Introduction:
Enzyme activity is constantly going on within our cells and contributes to the state of our well-being (Nadaraia et al., 2007). This is because they carry out, and catalyze, our biochemical reactions. Enzymes catalyze these reactions by lowering the activation energythat is required to form new bonds. Enzymes are a type of protein. Proteins consist of polypeptide chains that fold into secondary and then tertiary structures (Bugg 2001). The final three-dimensional structure of this specific protein has an important feature known as the active site. The active site is a crevice and makes up 10-20% of the entire structure. This feature is responsible for actingon the substrate (the molecule upon which the enzyme is acting on in a chemical reaction). It is made up of various acid residues that play a big role in recognizing substrates (Ghanem & Raushel 2007). Once the substrate is recognized, it binds to the active site which forms an enzyme-substrate complex. Then, this complex converts to an enzyme-product complex and the product is released. Oncethis process finishes the active enzyme is regenerated and will move on to catalyze another substrate (Bugg 2001).
Enzymes are known as incredibly efficient catalysts (Ghanem & Raushel 2007). However, there are a number of factors that can influence the activity of an enzyme, such as temperature and pH. For example, enzymes are only active in a certain range of pH. The point at which themaximum activity is reached can be recognized as optimal pH. If the pH is below or above this range, the enzyme functions poorly. This is because the structure of the protein changes, even to the point of denaturation. Temperature also affects enzyme activity. The rate of activity will increase as temperature increases until it reaches a certain point. Until reaching this point, the activity rateincreases with temperature because of energy. As temperature increases, molecules collide more often and more closely, which allows for a conversion from kinetic energy to chemical potential energy, which is what increases the activity rate (Nishiura 1999). However, an enzyme past its optimum temperature experiences the same effects caused by a low or high pH, and denature or lose its structure bychanging into its tertiary form (Scopes 2002). Both of these effects can be seen within this lab. In context, the optimal pH of a potato would be relatively basic because it is grown in soils (Bickelhaupt 2012). Similarly, the optimal temperature of a potato would be low because it is grown underground.
If the pH is high, then the enzyme activity will slow down because the protein will denature.If the temperature is high, then enzyme activity will increase because the energy of the molecules will increase.

Method:
The spectrophotometer was turned on and set to a wavelength of 420 nm. While it warmed up, 6 test tubes were obtained. 3 of these test tubes were left blank for the spectrophotometer and the other 3 were used for the experiment. The three blank tubes were labeled as 22B,40B, and 60B in correspondence with the experimental tubes which were labeled as 22, 40, and 60. 3mL of a pH 6 phosphate buffer was added to each tube. Next, 0.5mL of water and 0.5mL of potato juice (the enzyme) were added to the blank test tubes. 3 more test tubes were obtained, filled with 1 mL of potato juice, and labeled accordingly. Another 3 test tubes were obtained, filled with 1 mL ofcatechol, and labeled accordingly. Tube 22 and its blank 22b, a test tube of potato juice and a test tube of catechol were left on the lab bench for 10 minutes so that the buffer would reach room temperature. Tube 40 and its blank 40B, a test tube of potato juice and a test tube of catechol were placed in a hot water bath of 40°C for 10 minutes. Tube 60 and its blank 60B, a test tube of potato juice...