Experiment #3 - Proteases and Factors that Influence Enzyme Activity

Introduction

Background Information

Proteins are an essential part of any living organism having a role in structure, communication, defense, cell regulation and enzymatic reactions. Proteins are made up of amino acids containing an amino group (NH2), a carboxylic group (COOH), a H atom, and a R group all attached to the alpha carbon. There are approximately 20 naturally occurring amino acids, each with a different R group. Amino acids are linked together by peptide bonds to form di-, tri-, and polypeptides. A protein usually contains between 50 - 100 amino acid residues linked together. These peptide chains are then folded into a unique and highly ordered structure based off the sequence of amino acid residues ultimately producing a diverse array of three dimensional proteins.

One factor that influences the enzymatic activity of a protein is its shape. Unfolding the protein is called denaturation and inhibits its biochemical activity. Denaturation usually occurs at temperatures above 50°C although there is a temperature optimum below that point at which the enzyme has its fastest catalytic rate. Denaturation can also occur when the protein is in an environment that is either too acidic or too alkaline.

During digestion proteins are first acted upon by pepsin which upon activation in the gastrointestinal tract cleaves some of the peptide bonds of the targeted proteins. The action of this enzyme is terminated in the small intestine due to a change in pH from acidic to neutral. Other proteolytic enzymes such as trypsin, chymotrypsin, carboxypeptidase and elastase are all secreted from the pancreas into the small intestine where they break down proteins into amino acids allowing them to cross the intestinal wall into the bloodstream.

Purpose

The purpose of this lab is to study the affects of pH and temperature on the activity of proteolytic enzymes from the stomach and pancreas.

Hypothesis

The enzyme will show no activity if exposed to a temperature >50°C or if exposed to either a strong acid or a strong base.

Procedure

An agar gel was prepared from 20ml of agar gel buffer and approximately 0.32g of powdered agar. Next 0.8ml of a casein solution was added.. The solution was then placed in a boiling water bath for 2 - 3 minutes after which time it was taken out and allowed to cool before pouring it into petri dishes. Gels were allowed for set for 15 minutes after which time wells were cut and labeled using the large end of a Pasteur pipette. On agar plate B 5ml of 5% acetic acid was added.

For sample application, 0.1ml of sample was added to the prenumbered wells in accordance with the key below:

Lids were placed on to the petri dishes and the plates were allowed to sit for 12 - 24 hours.

For analysis 5ml of 5% acetic acid was placed on to agar plate A and allowed to sit for 15 minutes in order to stop further enzymatic action. After the elapsed the time both plates were rinsed and observed for the presence of transparent rings around the wells The diameter of the rings were then measured, recorded, and graphed.

Results

In both agar plates A and B there was no visible sign of enzymatic activity as would be evidenced by the presence of transparent rings around the well samples. A standard curve could not be plotted.

Discussion

In analyzing the results of both agar plates A and B the apparent lack of activity may have been the result of a time prolonged degradation of the experimental enzymes used. Given the fact that no other member of the class was able to obtain results, the lack of enzymatic activity is likely to be more attributed to defective enzymes more so that experimental error in gel preparation of well contamination.

In theory well sample 3 number one should have yielded no enzymatic activity as it contain no proteolytic enzymes, thus serving as a control for the other well samples. Well samples number 2 - 5 should have yielded increasing levels of enzymatic activity as evidenced by consistently larger ring diameters. Once recorded these values would be used to draw a standard curve. Well sample 6 should have yielded no enzymatic activity because the proteolytic enzyme would have been temperature denatured from the process of boiling. Well sample 7 should have yielded normal enzymatic activity and mostly served a s a control for well sample 6. Well sample 9 may or may not have had an enzymatic effect. Although saliva does contain the enzyme a-amylase this enzyme is specific to starch and carbohydrate substrates and thus should not have any effect upon the protein casein. However, saliva also contains a number of other proteins some of which may have had a small (if any) enzymatic effect upon the casein substrate.

In conclusion, this experiment should have showed that both temperature and pH have an adverse activity of proteolytic enzymes. However, because of defective enzymes the experiment was unable to validate this hypothesis. A repeat of the experiment using fresh proteolytic enzymes is recommended in order to obtain desired results.