Lab Report: Analysis Of Enzyme Activity Under Different Conditions

Abstract

An enzyme is a protein that serves as a biological catalyst during a chemical reaction (Gonzalez, 2018). A catalyst is any substance that increases the derivative of an established chemical reaction forming, this is done by lowering the activation energy of the reaction level. In this experiment we used a spectrophotometer to measure the activity of an enzyme (pepsin) under different conditions. In this experiment, chemicals in solution-based labs absorb and reflect light in different rates. The amount of absorbed light determined the proportional to the amount of chemical present in a current solution. Our team collated on creating an experimental design to find out the numerical value of the absorbance values of a chemical reaction, letting us measure how much product has been formed (or destroyed) and will be able to calculate the rate of reaction (Gonzalez, 2018). For this experiment, we used a spectrophotometer to measure the absorbance and transmittance of our solutions.

The amount of enzyme was kept constant for this experiment along with the albumin degradation. We believe the first tube without pepsin is the control, and the basic or acidic pH will either slow or speed up the enzymes at the site. Introduction Living organisms produce enzymes to speed up the chemical reactions in their cells’ performance (Bohager, 2006). Enzymes break down molecules which are called substrates. In the chemical process, each enzyme has only one substrate in which it breaks down. The location enzymes are produced in the cells of the human body. The four categories that do influence enzyme activity rates are temperature, pH, enzyme concentration and the presence of inhibitors.

In every cell, catalase is produced (Bohager, 2009). The very purpose is to break down the chemical compound, hydrogen peroxide (H2O2). Hydrogen peroxide is known as an active waste product of cellular activity meaning it is poisonous to its cells. Catalase speeds up the decomposition of hydrogen peroxide into harmless water and oxygen gas (2 H2O2- catalase 2 H2O+O2). Having excessive heat will denature enzymes. Denaturation is loss within its structure, for instance. When this occurs during the ionic process, hydrogen bonds tend to break. Explaining in a little further detail, salty environments (excess Na+ and Cl-), acidic environments (too much H+), and alkaline environments (too little H+ ) break ionic and hydrogen bonds for instance (Bohager, 2009). At the finish stage of this process, this will interfere with their electric charges. Heat causes movement within molecules. This impact disturbs their relatively weak bonds. The pH of a solution will also disturb the charge of acidic and basic amino acid side chains on a protein. By this, this will cause, the interactions that lead to, and what we them as tertiary and quaternary protein structure.

At the denatured stage, most proteins will not reform their original form or stage (Bohager, 2009). Enzymes, conduct oneself, faster near prime temperature (Brooker, 2008). The ideal temperature for the human enzyme is 40 degrees C, while the optimum temperature for enzyme from hot springs prokaryotes is 70 degrees C, for example. The purpose of this lab was to determine and analyze several conditions that affect enzymatic rates and to measure the activity of an enzyme by spectrophotometry. (Gonzalez, 2018)

Materials/Methods

In this lab, we use a spectrophotometer which has a tube holder, wavelength knob, scale, on/off switch, sensitivity scale switch, absorbance/transmittance knob, and a mode button (which factors in the absorbance, transmittance, and concentration). This instrument measures color changes that result in accumulation from accumulation or degradation of chemicals during reactions. We also used a clean test tube, so that we can measure the absorbance of the transparent liquid, which is egg white. Albumin has been used in the past in medicine to treat heavy metal intoxication which is the protein in egg whites. Some of the tests we did, we contributed pepsin into the mixture, which is a digestive enzyme in the stomach, which breaks down proteins into polypeptides. Sodium bicarbonate is a basic pH and was also part of the tests that we did, including acetic acid that is an acidic pH, we did this to see the difference in the enzymes reactions. We used vinegar which was part of the acetic acid. We use distilled water for our control to test against it. We also used a timer to time the absorbances of the product. We began to take a clean test tube and added one milliliter of albumin and three milliliter of distilled water in in tube B. We covered the test tube with parafilm and mixed the contents, we then put the test tube in the spectrophotometer and set the wavelength to 420 nanometers. We tested our control first so that we can compare the tubes with added acetic acid and basic acid.

For the next three test tubes we added pepsin, one with sodium bicarbonate and another with acetic acid or vinegar. We timed the tubes up to thirty minutes to see the changes and to see if our hypothesis was right. We go on to record our findings in the tables below so that we can compare with other teams.

Conclusion

After we added the water to tube A, albumin (egg whites) to tubes B, C, D, pepsin to all four tubes, acetic acid (acidic pH) to tube C and sodium bicarbonate (basic pH) to tube D, they were all placed in a spectrophotometer for time intervals of 0, 15, 30 and 45 minutes.

As predicted in the hypothesis, the presence of pepsin broke down the egg whites, and when exposed to high or low pH, enzymatic activity slowed due to the enzyme’s bonds being broken down. The absorbance values seen in the spectrophotometer support this, showing lower values in tubes C and D. This is due to the fact that improper environments can break down the enzyme’s chemical bonds (Gonzalez 2018). Tube A did not have any activity detected from the pepsin enzyme, because the water acts as a control and is not a substrate. The average absorbance at 0 minutes peaked in tube B, which indicates higher levels of enzyme activity. The absorbance in tubes C and D were a bit lower, as some of the enzyme was being destroyed. At 15 minutes, tube B peaked again.

The absorbance in tubes C and D dropped even lower, as with time the pH levels broke down the pepsin even further. Fifteen more minutes passed, and the enzyme activity began to die down a bit, as the concentration of enzyme to substrate began to drop. Comparing the results from team 2-4, we were not able to give a full explanation with details of the effects concerning the experiment. The teams only comparable answer was “0” mins because that was the only results all teams shared. With the lack of time, all the teams listed above, needed to finish the experiment correctly.

The results were only comparable to us with the “0” mins, everything further than that was invalid to our research. Our results of this experiment demonstrate the functions of enzymes when they come into contact with a substrate. The tubes with an acidic or alkaline environment tested and proved that enzymes can denature when they’re in the wrong environment, such as one that is too hot or has the wrong pH. These results prove that enzymes, while useful at breaking down substances such as albumin, can be denatured easily.