The Affect of pH anad Water on the Albumin Degradation

Abstract

Albumin is a protein found in egg white. Serum albumin is the most abundant protein in human blood and is the same type but in a different category as egg white albumin. Egg white albumin is disintegrated in the stomach by an enzyme called pepsin. The stomach environment is extremely acidic and so the enzymes in the stomach work best under acidic conditions. The enzyme pepsin, which is found in the stomach, has an optimal pH between 1.5 to 2; close to the pH of the stomach itself. To simulate the stomach, we added egg white albumin into a test tube and in three of those four pepsin was added. To see the effects of an enzyme in different environments, acetic acid, water, and sodium bicarbonate were added to corresponding test tubes. Using a spectrophotometer numbers of absorbance were recorded for the different mixtures, the higher the number the opaquest the solution was and therefore, the most chemical reaction occurred. The stomach tube with the highest absorbance number was the test tube with acetic acid and the lowest in the sodium bicarbonate stomach tube. This happened because pepsin works best under acidic pH conditions and not under basic pH conditions.

Introduction

Organisms are made of up of smaller units of life known as cells. Even though those cells are the smallest unit known, apart from atoms, there are also extremely complex even for their size. Cells go through many chemical reactions and processes with every second. However, these chemical processes would not be possible without the aid of biocatalysts. Biocatalysts can take different forms but enzymes are the most important ones. Enzymes speed up the rate at which chemical reactions occur by decreasing the activation energy needed to make a reaction occur. Without enzymes, the reactions that happen in our cells would be too slow and we would not be at this state of evolution we are now. (citation of lab manual).

During the 1820’s to 1840’s many enthusiastic scientists lead a series of experiments. These experiments ranged from studying different types of animal stomachs. However, the real ignition did not start until Johann Nepomuk Eberle identified an agent in an animal stomach that converted egg albumin into water soluble material. This discovery would lead many other scientists to start their own research and between 1825 to 1833 William Beaumont would become one of the first scientists to study the actual human stomach and expand this research until pepsin was eventually discovered (citation of the quarterly review of biology).

Pepsin is the most efficient enzyme in the stomach at its optimal conditions, generating 3 to 4 times the product that other enzymes can create. As long as the pH is in its optimal conditions the efficiency of pepsin will not be noticeably increased or decreased however, as soon as a pH of 5 is encountered there will be a noticeable decrease in product and the enzyme will become much slower. However, since the pH of the stomach tends to decline as new enzymes and hydrochloric acid is decreased, the enzyme tends to be always in its best environment to reduce the reaction time (citation of a dynamic stomach).

This experiment focused on how the enzyme pepsin is affected by pH and water by placing it in a tube stomach with egg white albumin. The use of egg white albumin will allow us to see if there is any change in the enzymatic activity since it can be degenerated by pepsin easily (citation of history). The albumin was paired with different mixtures in order to test the pepsin in different environments, which included acetic acid for an acidic pH, sodium bicarbonate for a basic pH, and water at a pH of 7 (citation of lab manual). A spectrophotometer, with the wavelength at 420nm, gave the absorbance number of the test tubes at different times w. When the absorbance numbers are greater than 0, or the reference point, and closer to 1 the solution in the tube is absorbing more light since it is foggier, and the closer it is to 0 the clearer it is so the less light is being absorbed. Based on everything mentioned above, it was hypothesized that the stomach tube with albumin, acetic acid, and pepsin will have the highest absorbance number since the enzymatic activity will be higher inside the tube. Furthermore, the stomach tube with albumin, sodium bicarbonate, and pepsin will have the lowest absorbance number since the basic pH will disturb the enzymatic activity of pepsin.

Materials and Methods

Materials

Five Spec test tubes along with a test tube holder given by lab instructor. A spectrophotometer that will have to be calibrated for the readings. 1ml pipets and beakers for separate solutions and a watch. Egg white albumin gathered from egg white, distilled water, the enzyme pepsin given by your lab instructor, sodium bicarbonate gathered from baking soda, and acetic acid derived from vinegar (lab manual).

Spectrophotometer Calibration

The spectrophotometer is a machine that measures degradation and amount of solution there is during a chemical reaction. This is done by beaming a light and recording how much light is absorbed during that reaction based on a reference point. The spectrophotometer, shown in Figure 1, uses a dimensionless scale, which means there is no unit to the number shown on the screen. As such, the spectrophotometer needs a reference point that is will be known as “zero” and that zero will be used to compare the test tube stomachs and notice any slight differences naked human eye cannot see. The calibration of the spectrophotometer for this experiment was by using a “blank” test tube that contained 3 ml of distilled water and 1 ml of albumin. The albumin is used since all the test tubes used in the experiment have albumin in the solution. The blank tube is then covered with parafilm and mixed thoroughly. Since the spectrophotometer uses light to notice any changes, there is different wavelength settings and for this experiment the wavelength has to be at 420nm. The MODE BUTTON on the spectrophotometer must be on absorbance, when this is done, the blank test tube is placed inside the spectrophotometer and the lid is closed as to prevent any outside light from interfering with the reading. The screen will be reading BLANK and when it reads zero the spectrophotometer is calibrated and the blank test tube can be taken out (lab manual).

Preparation of Test Tube Stomachs

To prevent any confusion the four leftover Spec test tubes were labeled 1, 2, 3, 4 according to the four solutions that will be used. All four test tubes contain different readings and solutions and Table 1 can show such measurements. Please note that it is better to do one by one since there will be a 0 minute reading needed. Test tube 1 contained a solution of 2ml of albumin and 2ml of distilled water. It was soon covered with parafilm mixed and placed on the test tube holder of the spectrophotometer for the 0 minute reading- this step was repeated for all test tubes as soon as the solution was mixed for more accurate readings. The absorbance number was then written down under the corresponding tube and time on a sperate table. Test tube 2 contained a solution of 2ml of albumin, 1ml of water, and 1ml of pepsin. Test tube 3 contained a solution of 2ml of albumin, 1ml of pepsin, and 1 ml of sodium bicarbonate. Finally, test tube 4 contained 2 ml of albumin, 1 ml of pepsin, and 1 ml of acetic acid (lab manual).

Test Tube Stomachs 15 Minute Reading Periods

To be able to note how pH and water affect the degradation of albumin by the enzyme pepsin, there needed to be more than one initial reading. The 15 minute periods correspond to after the initial 0 minute reading. There are two extra readings, a 15 minute reading and a 30 minute reading for all four test tubes. All the absorbance numbers from the spectrophotometer are placed on a data table, to have further data to compare, the tables of other groups were used. It is also important to notice that after a test tube was inside the spectrophotometer it was warm because of the light, this can also affect the reading and enzymatic activity of pepsin while it is inside the spectrophotometer (lab manual).