REQUIRED PRACTICAL 1 - ENZYME CONTROLLED REACTION

Question 1

what is the aim of the first practical

Answer 1

the effect of a named variable, like pH for ex, on the rate of an enzyme controlled reaction

Question 2

what factors effect the rate of reaction of an enzyme controlled reaction influenced by

Answer 2

• temp
• pH
• conc of substrate
• conc of enzyme

Question 3

how can the effect of these variables be determined

Answer 3

through CHANGING a SINGLE variable and MEASURING its effect on the RATE OF REACTION

Question 4

what is important when measuring the effect of a single variable on the rate of reaction and why is this important

Answer 4

• to keep all other variables CONSTANT
• so they dont influence the results

Question 5

name all the equipment needed for this experiment

Answer 5

• powdered milk suspension
• trypsin solution (0.5%)
• distilled water
• HCL acid
• 5 cm3 syringes
• flat bottomed tubes
• water bath
• timer

Question 6

explain the method

Answer 6

• variable = temp
• make 2 control samples
• add 5 cm3 of MILK SUSPENTION in 2 flat bottomed tubes
• add 5 cm3 of DISTILLED WATER to one tube : indicating the ABSENCE of enzymes activity
• add 5cm3 of HCL to the other tube : control indicates the colour of a COMPLELTELY HYDROLYSED SAMPLE
• take 3 test tubes and measure 5cm3 of milk into each
• place them in a water bath at 10 degrees C for 5 mins for equilibration
• add 5cm3 of trypsin to each test tube SIMULTANEOUSLY and start the timer immediately
• record how long it takes for the milk samples to completely HYDROLYSE and become colourless
• repeat steps 2-3 for temps 20, 30, 40 and 50 degrees C
• find the MEAN TIME for the milk to be hydrolysed at EACH TEMP and use this to work out the rate of reaction

Question 7

what is the formula for rate of reaction

Answer 7

RoR = 1/mean time

Question 8

what are potential hazards

Answer 8

• broken glass
• HCL
• hot liquids
• enzymes

Question 9

what is the risk for broken glass

Answer 9

cuts from a sharp object

Question 10

what is the safety precaution for broken glass

Answer 10

• take care when handling glass objects
• keep away from the edge of the desk

Question 11

what is the risk for HCL

Answer 11

may cause harm/irritation to eyes or in cuts

Question 12

what is the safety precaution for HCL

Answer 12

• wear eye protection
• avoid contact with the skin (through gloves)
• tie up long hair

Question 13

what is the risk for hot liquids

Answer 13

scalding/burning

Question 14

what is the safety precaution for hot liquids

Answer 14

• handle with care
• use tongs to remove boiling tubes from water bath
• wear eye protection
• keep away from the edge of the desk

Question 15

what is the risk for enzymes

Answer 15

allergies

Question 16

what is the safety precaution for enzymes

Answer 16

• avoid contact with skin/eyes
• wear eye protection

Question 17

name the 3 conclusions for this practical

Answer 17

• milk contains a PROTEIN called CASEIN and when that is broken down, it causes the milk to turn COLOURLESS
• trypsin is a PROTEASE enzyme which HYDROLYSES the CAESIN protein
• as the temp increases from 10 degrees C, kinetic energy INCREASES so MORE E-S COMPLEXES FORM
• meaning the rate of reaction INCREASES up to the OPTIMUM TEMP
• at temps BEYOND the OPTIMUM, the BONDS in the enzymes TERTIARY STRUCTURE BREAK - changing the shape of the AS
• meaning the SUBSTRATE and ENZYME are NO longer complementary and E-S complexes will NO longer form

Question 18

how can temperature be controlled

Answer 18

• thermostatically controlled water bath
• monitor using a THEMOMETER at REGULAR INTERVALS and add hot/cold water if temp fluctuates

Question 19

how can pH be controlled

Answer 19

• use of a buffer solution
• monitor using a pH meter at REGULAR INTERVALS

Question 20

why was the test tubes, including the enzyme and substrate, left in the water bath for 5 mins

Answer 20

so the solution equilibrate/ reach temp of the water bath

Question 21

explain why using a colorimeter to measure colour change is BETTER than comparison to colour standards

Answer 21

• objective/not subjective
• higher accuracy

Question 22

explain a procedure that could be used to STOP each reaction

Answer 22

• boil, add strong acid, add strong alkali to DENATURE the enzyme
• put in ice, this will LOWER KINETIC ENERGY so NO E-S complexes form
• add a high conc of inhibitor, this would cause NO E-S complexes to form