PRE LAB EXPT 4 (Day 1)

Enzymes

• compunds, usually globular proteins

• act as biological catalysts for biochemical reactions

• without them, life would not be possible since biochemical reactions would not occur as fast as they would

• classified according to the reactions they catalyze

Types of enzymes

• oxidoreductases

• transferases

• isomerases

• ligases

• lyases

Substrates

enzyme differ in terms of size, specificity, and reactivity to substances called…

Type of enzymes present within a cell

Dictates the tyoe of reactiond that occur within the cell

Factors that affect the activity of enzymes

• temperature

• pH

• enzyme concentration

• substrate concentration

Optimal conditions in cell and body

Must be maintained in order for enzymes to function properly and increase the rate of biochemical reactions

Objectives of day 1

• study the effect of enzyme concentration and pH on the enzyme's activity

• determine the enzyme's optimum pH

Reagents

• 0.2 M Phosphate buffer (pH 6.9)

• 1% starch solution

• Iodine solution (technical grade)

• pH buffer: 2, 4, 7, 10

Equipment

Temperature-controlled water bath

Materials to be borrowed from the stockroom

• thermometer

• (5) droppers

• spot plate

• distilled water in wash bottles

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

Procedures

• saliva collection

• effect of enzyme concentration

• effect of pH

Saliva collection

• collect 5 mL of your saliva into a beaker

• measure 20 mL distilled water in a graduated cylinder and add it to the beaker containing the collected saliva. mix thoroughly and this will be the stock solution

Effect of enzyme concentration

• label (5) test tubes 1-5

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

• transfer 3 mL of the saliva sample from the stock into test tube 5

• place all five tubes in a water bath at 37C for 5 minutes

• Obtain 5 clean droppers and label -- one for each solution in the respective test tubes

• remove the test tubes from the water bath

• gently shake each of the test tubes

• put back all 5 test tubes into the water bath. Record the time at which you added the saliva

• without removing the tubes from the water bath, transfer 4 drops of each reaction mixture (tubes 1-4) using the labeled droppers in Procedure B.5 for each to separate wells on a spot plate

• add one drop of iodine solution to each well. record your observations

• repeat steps 10 & 11 after the mixture have had 5 minutes of reaction time

• repeat steps 10 & 11 after the mixture have had 10 minutes of reaction time

Test tube 2 (effect of enzyme concentration)

add 3 drops of saliva from test tube 5

Test tube 3 (effect of enzyme concentration)

add 6 drops of saliva from test tube 5

Test tube 4

add 10 drops of saliva from test tube 5

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

• ensyme activity level: high (3)

Effect of pH

• obtain 4 clean and dry test tubes labeled: pH 2, pH 4, pH 7, pH 10

• place 2 mL of the stock saliva sample (prepared from proceure A) into each of the test tubes. add 2 mL of the respective buffer solutions

• obtain 4 additional test tubes, labeled as test tubes 1-4

• add 2 mL of 1% starch solution to these tubes

• place all 8 tubes in a water bath set at 37C for 5 minutes

• combine the content of each test tubes as follows:

  • test tube 1 + test tube pH 2

  • test tube 3 + test tube pH 7

  • test tube 4 + test tube pH 10

  • test tube 2 + test tube pH 4

• Gently shake the test tubes and return to the water bath

• wait for 15 minutes, transfer 3 drops of each reaction mixture (using clean droppers) top a spot plate. add one drop of the iodine reagent to each

• record your observations using the reference table in procedure B.11

Excess saliva collected and test tubes with starch (waste geenrated)

disposol container: sink

Samples in spot plate (waste generated)

disposal container: may be wiped with tissue then dispose