Introduction

Background Information

Carbohydrates such as the monosaccharide glucose play a fundamental role in the living organisms. This simple sugar is composed entirely of oxygen, hydrogen, and carbon. Disaccharides such as sucrose can be formed by linking two monosaccharides together with glycosidic bonds. Polysaccharides (macromolecules containing many monosaccharides) such as amylose and amylopectin are digested in the human gastrointestinal tract. Amylose consist of straight chains of glucose molecules while amylopectin consist of branched chains.

During digestion carbohydrates are first broken down in the mouth by a-amylase. After leaving the stomach the carbohydrates are further broken into dextrins, then di- , tri-, and oligosaccharides and finally into individual glucose molecules that are able to cross the intestinal epithelium into the blood stream.

Purpose

The purpose of this lab is to study the affects of hydrolysis of starch by the enzyme a-amylase. Iodine staining will be used in order to determine the amount of enzymatic activity.

Hypothesis

The amount of enzymatic activity will increase as a function of the concentration of a-amylase.

Procedure

An agar gel was prepared from 20ml of agar gel buffer and approximately 0.32g of powdered agar. Next 0.4ml of a mixed starch 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.

A saliva solution was prepared using one drop of the experimenters own saliva added to 1ml of agar gel buffer.

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

*These two samples were accidentally switched in comparison to the original key in the lab text.

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

For detection of enzymatic activity 5ml of diluted iodine was placed onto each petri dish and allowed to sit for 10-20 minutes after which time it was discarded and the dishes were rinsed with water. The diameter of the rings for the wells were then measured, recorded, and graphed.

Results

In petri dish A there was a progressive increase in enzymatic activity as the concentration of a-amylase increased. A standard curve was plotted to connect the ring diameters measurements of the well samples 2 - 6. (See Graph #1; Table #1).

In petri dish B there was no noticeable overall increase in enzymatic activity as the concentration of a-amylase increased. A standard curve was plotted to connect the ring diameters measurements of the well samples 2 - 6. (See Graph #2; Table #2).

Calculations

Conversion of Concentrations (Petri Dish A)

Saliva (Well #8)

Initial Concentration: 8.0mg/ 0.05ml

8.0mg/ 0.05ml = X mg/ 1ml
8.0mg * 1ml = X * 0.05ml
160mg = X

Converted Concentration: 160mg/ ml

Pancreatic Standard Extract (Well #9)

Initial Concentration: 80mg/ 0.05ml

80mg/ 0.05ml = X mg/ 1ml
80mg * 1ml = X * 0.05ml
1600mg = X

Converted Concentration: 1600mg/ ml

Pancreatic Extract from Experiment 1 (Well #7)

Initial Concentration: 1000mg/ 0.05ml

1000mg/ 0.05ml= X mg/ 1ml
1000mg * 1ml = X * 0.05ml
20000mg = X

Converted Concentration: 20000mg/ ml

Discussion

In analyzing the results of petri dish A, well samples 2 - 6 clearly support the initial hypothesis that enzymatic activity will increase as a function of increased a-amylase concentration. For well #1 the agar gel buffer served as a control and should have showed no enzymatic activity. However, a ring was indeed detected around the well perhaps indicating that there was some experimental error. Given that the activity of the different amylase concentrations in well samples 2 - 6 met with the original hypothesis it seems unlikely that there was something wrong with the agar gel itself. Contamination of the agar gel buffer either by means of tools used or by leaking from an adjacent well in the same petri dish may be the reason for the apparent enzymatic activity. In well #7 the Pancreatic enzyme from Experiment #1 showed unusually high enzymatic activity in comparison to the Pancreatic Standard enzyme in well #9. Reasons for this may include the fact that in preparing the enzyme the solution was hand shaken as opposed to be centrifuged. Use of this alternative technique may have inadvertently left fragments of the original enzymatic powder either undissolved or less dissolved than what would be considered ideal. This would in turn result in a solution that would be significantly more concentrated in certain parts and thus would manifest itself by showing a higher degree of enzymatic activity. The ring diameters for both the Pancreatic extract from Experiment #1 and the Pancreatic Standard Extract were plotted against the standard curve and then converted to a concentration in mg/ ml. (See Calculations) In well #8 the saliva showed signs of enzymatic activity. The ring diameter was plotted against the standard curve generated by well samples 2 - 6 and an a-amylase concentration of 8.0mg/ 0.05ml or 160mg/ ml was determined.

In analyzing petri dish B, well samples #2 - 6 did not support the initial hypothesis that enzymatic activity will increase as a function of increased a-amylase concentration. Reasons for this may include cross contamination of the wells by the tools used or experimental error in preparing the agar gel solution. For well # 1 enzymatic activity was once again detected. Reasons for this may include those mentioned in analyzing petri dish A. In well #7 the Pancreatic enzyme from Experiment #1 showed normal enzymatic activity in comparison to the Pancreatic Standard enzyme in well #9. For well # 8 the saliva showed signs of enzymatic activity just as in petri dish A. However, because of the abnormal standard curve generated by well samples 2 - 6 the concentration for both the Pancreatic extracts and the saliva solutions could not be determined from the graph.

In conclusion, this experiment showed that the enzyme a-amylase was effective at degrading starch in an agar gel. As demonstrated with the results from petri dish A the amount of enzymatic activity increases with the concentration of a-amylase. A comparison of class data and more experimentation following the procedure outlined is recommended for more conclusive results.