Scientific Methodology And Enzyme Activity Reports Example

Objective: Investigate the effect of increasing temperature on enzyme activity.

Introduction: Enzymes are biological catalyst. All most all the functions in the cell are regulated by these enzymes. Except for RNAase, all most all the enzymes are proteins. Enzymes are very efficient and specific in the reaction they catalyze. All enzymes exhibit four common features: I. Enzymes do not make a reaction to occur; they can only fasten the reaction rate. II. Enzyme molecule is not permanently altered by the reaction, though they may be altered transiently. However at the end of the reaction they emerged unchanged and can be used over and over again. III. Enzyme can catalyze both forward and backward reaction; however thermodynamics favors one direction to another. III. Enzymes are highly specific for the reaction they catalyze. (Nelson et al.)
The three dimensional structure is very important for enzyme catalyst. A number of ionic and other non-covalent bonds contribute to the formation of the three dimensional structure. Within the three dimensional structure, enzyme catalysis happens at a site called the active site. The rate of catalysis depends on the three dimensional shape of the active site and the proximity of the enzyme active site to the substrate. (Nelson et al.)
A cells physical and chemical environment is very important for the enzyme activity. One of these physical environments is temperature. As the temperature of the reaction medium increases the kinetic energy of the system increases, and the rate of collision between the reacting molecules and thus the reaction rate increases. However, this does not continue linearly with linear increase in temperature. For a fixed enzyme concentration, rate of catalysis increases with gradual increase in temperature to maximum point, beyond which the rate of catalysis comes down with further rise in temperature. Temperature corresponding to the point of maximum rate in enzyme catalysis is called the optimum temperature. At this temperature the kinetic energy is optimum and the three dimensional shape of enzyme is intact. Beyond this optimum point, enzyme begins to denature with increase in temperature. The kinetic energy associated with heating, breaks the weak non-covalent bonds that maintains the three dimensional structure of the active site. Once the charges in the active site are disrupted or the shape of the active site is altered, the rate of catalysis comes down. (Ottaway)

Research question: What is the optimum temperature for enzyme activity?

Hypothesis: The rate of enzyme catalysis is highest at the optimum temperature.
Prediction: As a rule of thumb 100C rise of temperature doubles the reaction rate, till optimum temperature is reached. For most enzymes in an animal cell, the optimum temperature is close to body temperature which is 350C to 370C. Above and below this temperature the enzyme shows less than optimum activity. In contrast to animal and human enzymes, the enzymes in the thermophilic bacteria that live in hot spring have an optimum temperature close to 700C

The graph plotted between enzyme activities vs. temperature is expected to be as below:

Importance of this study: The optimum temperature at which the enzyme shows maximum activity depends on the type of enzymes. While enzymes from human work at an optimum temperature of 370C, the enzymes in animals living in the Arctic have a much lower optimum temperature. In PCR, reaction, the primer works best at an optimum annealing temperature, above and below which its efficiency is poor. Doing this lab project, will help to appreciate the characteristic feature of enzyme kinetics.
Methods: H2O2 is a toxic byproduct of metabolic reactions. It is removed from the body by an enzyme catalase. Catalase catalyses the following reaction: H2O2 2 H2O + O2.Though this reaction can occur by itself, catalase makes the reaction rate faster. In the following experiment, the rate of this reaction was measured, by measuring the concentration of the reaction product O2, using an O2 sensor. Since we intend to study the effect of varying temperature on the rate of a fixed enzyme concentration; temperature is the dependent variable and enzyme is the independent variable in this experiment. To begin with we took four test tubes and labeled each of them. To each, we added 5ml of 3% H2O2 and 3ml of water. Following this, each of the test tubes was placed in four different water bath set at four different temperatures namely: 0oC, 23oC, 37oC and 55oC for 10 minutes. At the end of 10 minutes, the first test tube was removed from the water batch set at 0oC (while rest of the test tube continue to remain in their respective water batch), and 10 drops of the enzyme solution was added to this. The stop watch was started immediately. The contents of this test tube was then transferred to a Nalgene bottle and mixed well. An O2 sensor was then positioned on the bottle as shown in Figure 1. Positioning the sensor will make the bottle air tight. (Biology Lab Manual)
Ref: www2.vernier.com
When the stop watch shows 30 second, the sensor was switched on immediately and the data was recorded for 180 seconds. At the end of 180 seconds, the graph of Time vs O2 concentration will be displayed by the monitor as shown in Figure 2. Using the linear regression formula: y=mx+ b, the reaction rate is calculated by the computer, for 1min, 2min, 3 min, 4 min ,5 min and 6min. Once the data collection was complete for 0oC, the same procedure was repeated one by one for the test-tube set at 23oC, 37oC and 55oC as well.
Image courtesy: (www2.vernier.com)

Results:

The data recorded in the lab are as follows:

Graph1: Reaction rate of enzyme vs temperature.

Discussion: Catalase is an enzyme, having a specific structure which helps it to bind to the substrate H2O2. At high temperature (such as 55oC), the structure of the protein is altered and hence its function is affected. At low temperature, though the structure is not much affected, the kinetic energy of the system is low and thus the reaction rate is not optimum. Catalase has the optimum activity at 37oC, since the kinetic energy at this temperature is optimum without compromising the structure of the active site. (Szaz) 

References:

Biology106 Lab Mannual: TCC Campbell Biology Concepts And Connection. 2nd ed. Tacoma Community College, 2014. Print.
Nelson, David L, Michael M Cox, and Albert L Lehninger. Lehninger Principles Of Biochemistry Absolute Ultimate. New York: W. H. Freeman, 2009. Print.
Ottaway, J. H. Regulation Of Enzyme Activity. Oxford: IRL Press, 1988. Print
Szasz, G. 'The Effect Of Temperature On Enzyme Activity And On The Affinity Of Enzymes To Their Substrates'. Clinical Chemistry and Laboratory Medicine 12.4 (1974): n. pag. Web.
www2.vernier.com/,. 'Enzyme Action: Testing Catalase Activity'. N.p., 2015. Web. 7 Feb. 2015.