BABS1201 Group Enzymes Project Group Protocol Including Equipment and Reagents List

Lab Day: Lab Time: Wednesday 10am – 1pm

Are you in Lab G20 (furthest from BSB Student Office) or Lab G21 (closest to BSB Student Office)?: Demonstrator Name: Daniel Winters Names of Group Members: Johnny Nguyen, Therese Pham, Linda Tang

Name of Enzyme You are Investigating: Amylase Brief Background: Amylase is a digestive enzyme, produced mainly by the salivary glands and the pancreas, to break down starch in food into smaller carbohydrate molecules and disaccharides such as maltose. It can be found in humans and some other mammals. Some plants and bacteria may also produce amylase. After being broken down into smaller carbohydrate molecules, it can be converted into a monosaccharide such as glucose, which fuels processes for organism function. There are two variations of this molecule but the human body has alpha amylase. Food that contains large amounts of starch will have a slightly sweet taste when chewed because of this breaking down of starch into sugar by amylase. Alpha Amylase's official name is 1,4-­‐a-­‐D-­‐Glucan glucanohydrolase; EC 3.2.1.1. It breaks down starch into maltose via hydrolysis. Amylase is folded into a tertiary shape and has an area called the “active site” where the substrate, carbohydrate, is broken down. Its structure consists of a single chain of amino acids, which forms three distinct regions called domains, where each domain has a specific biological function assisting in the breakdown of carbohydrates. Several amylases require the presence of a cofactor to function. A cofactor is a chemical species necessary for the enzyme to function but does not necessarily undergo chemical reaction. Amylase requires calcium and chloride ions for activity in animals and plants. The amylase from the bacteria Bacillus subtilis contains zinc ions. Hypotheses: Substrate Concentration In the more concentrated starch solution, there will be an increase in enzyme-­‐substrate reaction until all the enzyme active sites have been used, whereby the reaction rate will slow. As a result, there will be a relatively quick change in colour that gradually slows down. When amylase is added to the less concentrated starch solution, there will be a quick enzyme-­‐substrate reaction resulting in a rapid colour change Enzyme Concentration A higher concentration of amylase added to the same concentration of starch solution will result in an increasing rate of reaction. The more amylase added to the solution, the faster the reaction until the point where all the substrate is used up and amylase is in excess. Therefore, the colour change will be rapid then slow to a standard rate.

Equipment and Reagents: • • • • • • •

Test tubes Test tube rack Timers Pipettor Amylase (0.025mg) Iodine solution (0.002M) Starch solution (0.75mg)

Method:

Concentration of substrate 1. Collect 7 test tubes and label them from 1 through to 6. Label a 7th test tube with “control”. 2. Add 1mL of starch solution into the first test tube using a pipettor and increase in increments of 0.5mL along the test tubes. Continue so that the last test tube has 3.5mL. Place 3mL in the control. 3. Add one drop of iodine solution to each test tube. 4. Note the colour of each solution. 5. Add 3mL of the enzyme amylase into every solution except for the control. 6. Start the timer and add another drop of iodine to every test tube after 30 seconds and slightly shake the test tube, observing the colour and comparing it to the control. 7. Continue doing this until the reaction is complete (shown by a discoloration of the iodine in the test tubes). 8. Record the time that is taken for the iodine to change to clear.

9. Complete this trial 3 times. Concentration of enzyme 1. Collect 8 test tubes and label them 1 through to 6, with the 7th labelled with “positive control” and the 8th with “negative control”. 2. Add 1mL of amylase into the first test tube with a pipettor increase in increments of 1mL along the test tubes ending with 6mL in test tube 6. Place 4mL in each control test tube. 3. Place a drop of iodine solution into each test tube. 4. Note the colour of each solution, including the controls. 5. Add 2mL of starch to each test tube (except for the controls) and note the colour of the solutions. 6. For the positive control, you will add 2mL of another enzyme known to break down starch. Your negative control should only have 4mL of amylase and one drop of iodine. Use this as something to compare the colour of your other test tubes to. 7. Add another drop of iodine to all test tubes in 30-­‐second intervals (except for the negative control). 8. Start the timer and note down the time that it takes each test tube to go clear, or for the colour to remain constant (i.e. the same shade of blue)

Diagrams of Procedures to be Carried Out and Apparatus to be Employed: Concentration of Substrate

Concentration of Enzyme

Risk Assessment: Type of Hazard

How hazard may arise Burns may result from touching the water bath surface, water or lifting the lid which may result in trapped steam burning the wrist Risk Control Strategies Emergency

Procedure Hazard equipment Water bath Physical injury – Burns

Check temperature of water bath

In the event of being burnt, place Do not touch external surfaces under cold Where there is a lid, lift it carefully to running water for 15 minutes, alert slowly release trapped steam demonstrator PPCE – lab coats, thermal protection gloves Tie hair back, secure loose clothing Temporarily switch off water bath when retrieving or placing sample within it Leave scalpels on bench area Cut away from oneself Always place in a safe position and orientation to avoid possible accidental injury to others i.e. pointing downwards and away from others. PPCE – lab coats, covered footwear Do not use damaged glassware Inspect all glassware for chips or cracks Immediately switch off equipment if any part is caught

Physical injury

Physical hazard

Loose clothing, body parts may get caught in water baths with ‘moving tray’ mechanism Scalpel removed from bench area i.e. walking around with scalpel Position of blade left pointing towards a person

Scalpel

Physical injury – Cuts

In the event of being cut, immediately wash wound liberally with soap and water and cover with a dry dressing Immediately wash wound liberally with soap and water and cover with a dry dressing Eye-­‐ irrigate eye immediately

Glassware e.g. beakers, test tubes

Physical injury – Cuts

Glass breakage, cuts from chipped glassware

Iodine solution

Chemical hazard

Physical injury – tingling, redness or inflammation when in contact with the skin

Inhalation, ingestion, Avoid having face too close to the skin/eye contact iodine to prevent inhalation and ingestion

Skin-­‐ wash with Keep head a safe distance away from soap immediately chemical and use a squash pipette to Breathing-­‐ transfer the liquid respiratory Use lowest possible concentration of support iodine Swallow – PPCE – lab coats, gloves Medical attention immediately

Results: Part 1 Trial 1 Test tube 1 2 3 4 5 6 2 1 2 3 4 5 6 3 1 2 3 4 5 6

Volume of starch (mL) 1.0 1.5 2.0 2.5 3.0 3.5 1.0 1.5 2.0 2.5 3.0 3.5 1.0 1.5 2.0 2.5 3.0 3.5 Time for iodine to change colour (min:sec) 0:02 4:20 1:08 11:46 10:58 6:27 0:28 1:29 2:52 4:35 4:43 5:20 2:26 2:36 2:45 2:55 3:06 3:10

Part 2 Trial 1 Test tube 1 2 3 4 5 6 2 1 2 3 4 5 6 3 1 2 3 4 5 6

Volume of amylase (mL) Time for iodine to change colour (min:sec) 1.0 2.0 3.0 4.0 5.0 6.0 1.0 2.0 3.0 4.0 5.0 6.0 1.0 2.0 3.0 4.0 5.0 6.0 -­‐ -­‐ 8:12 1:24 15:25 1:21 3:53 3:08 2:17 1:05 0:50 0:49 2.40 2:06 1:53 1:42 1:30 1:05

References http://www.eng.umd.edu/~nsw/ench485/lab5.htm http://www.123helpme.com/view.asp?id=121479