PRE-LAB EXPT 4 (Day 2)

Objectives of day 2

• study the effect of temperature and the presence of inhibitors on the enzyme's activity

• determine the enzyme's optimum temperature

Materials to bring by students

ice

Reagents

• 0.2 M Phosphate buffer (pH 6.9)

• 1% starch solution

• iodine solution (technical grade)

• 1% NaCI

• 95% ethanol AgNO3

Equipment

temperature-controlled water bath

Materials to be borrowed from the stockroom

• thermometer

• distilled water in wash bottles

• spot plate

Safety

• wear PPE and lab goggles at all times

• wash hands thoroughly with soap and water before leaving the laboratory

• dispose gloves properly after performing the experiment

Saliva collection

• collect 2 mL of your saliva into a beaker

• mesaure 25 mL phosphate buffer (pH 6.9) in a graduated cylinder and add it to the beaker containing the collected salova. mix thoroughly and this will be the stock solution

Effect of temperature

• label six test tubes 1-6

• to test tubes 1-3, place 2 mL each of the stock saliva solution prepared in procedure A

• to test tubes 4-6, place 2 mL of 1% starch solution

• incubate the following for 10 minutes:

  • test tubes 1 and 4 in an ice bath

  • test tubes 2 and 5 at 37C

  • test tubes 3 and 6 at 100C

• After incubation, combine the following tubes together:

  • test tube 1 + test tube 4

  • test tube 2 + test tube 5

  • test tube 3 + test tibe 6

• gently shake the test tubes and return to the respective temperature setups

• incubate for another 10 minutes

• transfer 4 drops of each mixture onto the spot plate using clean droppers

• add 1 drop of iodine solution to each well

• record your observations using the reference table

Dark blue-black (iodine test for starch)

• amount of starch remaining: all

• enzyme activity level: none (0)

Blue (iodine test for starch)

• amount of starch remaining: most

• enzyme activity level: low (1)

Light brown (iodine test for starch)

• amount of starch remaining: some

• enzyme activity level: moderate (2)

Gold (iodine test for starch)

• amount of starch remaining: none

• enzyme activity level: high (3)

Effect of inhibitors

• label 3 clean test tubes 1-3

• add the following to corresponding test tubes

• obtain 3 additional test tubes, label as test tubes 4 to 6

• transfer 2 mL of 1% starch to test tubes 4 to 6

• place all 6 test tubes in a water bath set at 37C for 5 minutes

• after 5 minutes, combine the contents of each test tube

  • test tube 1 + test tube 4

  • test tube 2 + test tube 5

  • test tube 3 + test tube 6

• gently shake the mixture then return to the water bath and incubate for another 10 minutes

• transfer 4 drops of each of the contents of the test tubes to a spot plate using 3 separate clean droppers

• add one drop of the iodine reagent to each. record your observations using the reference table in procedure B.10

Test tube 1 (effect of inhibitors)

add 2 mL of saliva sample + 10 drops 1% NaCI

Test tube 2 (effect of inhibitors)

2 mL of saliva sample + 10 drops 95% ethanol

Test tube 3

add 2 mL of saliva sample + 10 drops AgNO3

Excess saliva collected, test tubes with starch in Procedure B, mixture in test tube 1 + test tube 4, test tube 2 + test tube 5 (waste generated)

disposal container: sink

Mixture in test tube 3 + test tube 6 (waste generated)

disposal container: metal-containing waste

Samples in spot plate (waste generated)

disposal container: may be wiped with tissue then dispose