Lab #1 Test for effect of enzyme concentration on catalysis.

Introduction Starch is a polysaccharide found in most plant parts that is made up mostly of amylase and amylopectin. These two molecules comprise twenty and seventy- nine percent of this polymer respectively while the other one percent is composed of substances such as phosphates or fatty acids. Amylase has a structure which is a helical unbranched chain held together by 1, 4 glycosidic bonds. As opposed to amylopectins highly branched molecular chain with its 1, 4 and 1, 6 glycosidic bonds. During digestion starch is broken down first in the mouth by salivary amylase which as the name suggests is secreted by the salivary glands. This amylase is a hydrolytic enzyme i.e. it works on its substrate (the starch) by catalyzing the breaking of the bonds that link the molecules together and releasing water in the process. Maltose is the result/product of this condensation reaction. Salivary amylase is activated or released via the act of chewing. Like all enzymes amylase works best at certain pH which is 6.5-7.5. In this laboratory exercise students are attempting to see how enzyme concentration at varying states of dilution affected catalysis while the substrate used is fixed. In this experiment chromophoric group (in this case iodine solution) is used to indicate whether or not catalysis has occurred.

Material Water Buffer solution (pH 6.8) 2 % starch solution Amylase solution (saliva may be used as a substitute) Time 12 test tubes

Method 1. Four test tubes are labeled A-D. A serial dilution of amylase solution was

generated as instructed. 2. Forty drops of buffer solution was added to test tube A. 3. Eight test tubes were labeled 1-8 and 2 drops of iodine solution was added to each. 4. 0.5ml 2% starch solution was added to tube A. 5. One drop of the amylase plus starch from tube A is added to iodine tube #1. 6. After one minute a drop of the mixture from tube A was added to iodine tube # 2. 7. The one minute intervals were repeated until iodine tube # 5 was reached. 8. Observations were recorded. 9. The contents of test tube # 1-8 were discarded; the test tubes are then rinsed and dried. 10. Steps 2-9 were repeated with test tubes B-D.

Results Time (Minute) 0 Red-brown solution 1 Red-brown solution 2 Red-brown solution 3 Red-brown solution 4 Red-brown solution 5 Red-brown solution 6 Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Red-brown solution Test tube A Test tube B Test tube C Test tube D

Red-brown solution

Red-brown solution Red-brown solution

Red-brown solution Red-brown solution

Red-brown solution Red-brown solution

Red-brown solution

Discussion/Interpretation of results In this laboratory exercise students expected a chromophoric change through the usage of iodine solution to indicate for any presence or absence of starch. A blue-black colour would be expected if starch was still present and catalysis had not taken place. A colour change of a light yellow-brown would have been a sign that the product maltose is present. However there appeared to be no change in the red brown colour if the iodine when added in any of the test tubes #1-8. This can be accounted as a result of experimental error in way of the measuring of the reagents or some kind of contamination during the exercise. Also the quality and concentration of the salivary amylase may be questioned. Another explanation lies in the production of dextrin as a result of the hydrolysis of the starch. Dextrin is soluble and leaves a red colour in solution with iodine indicator.

Conclusion There was no starch present so catalysis had taken place.

Reference: Toole Glen, Toole Susan; Understanding Biology, For Advanced Level Fourth Edition. Stanley Thornes (Publishers) Ltd. 1999; United Kingdom. Weil J.H; General Biochemistry; 2006; New Age International Publishers; India. Jayaraman J; Laboratory Manual in Biochemistry; 2007; New Age International Publishers; India.