REQUIRED PRACTICAL 1 - ENZYME CONTROLLED REACTION Flashcards
what is the aim of the first practical
the effect of a named variable, like pH for ex, on the rate of an enzyme controlled reaction
what factors effect the rate of reaction of an enzyme controlled reaction influenced by
- temp
- pH
- conc of substrate
- conc of enzyme
how can the effect of these variables be determined
through CHANGING a SINGLE variable and MEASURING its effect on the RATE OF REACTION
what is important when measuring the effect of a single variable on the rate of reaction and why is this important
- to keep all other variables CONSTANT
- so they dont influence the results
name all the equipment needed for this experiment
- powdered milk suspension
- trypsin solution (0.5%)
- distilled water
- HCL acid
- 5 cm3 syringes
- flat bottomed tubes
- water bath
- timer
explain the method
- 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
what is the formula for rate of reaction
RoR = 1/mean time
what are potential hazards
- broken glass
- HCL
- hot liquids
- enzymes
what is the risk for broken glass
cuts from a sharp object
what is the safety precaution for broken glass
- take care when handling glass objects
- keep away from the edge of the desk
what is the risk for HCL
may cause harm/irritation to eyes or in cuts
what is the safety precaution for HCL
- wear eye protection
- avoid contact with the skin (through gloves)
- tie up long hair
what is the risk for hot liquids
scalding/burning
what is the safety precaution for hot liquids
- handle with care
- use tongs to remove boiling tubes from water bath
- wear eye protection
- keep away from the edge of the desk
what is the risk for enzymes
allergies
what is the safety precaution for enzymes
- avoid contact with skin/eyes
- wear eye protection
name the 3 conclusions for this practical
- 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
how can temperature be controlled
- thermostatically controlled water bath
- monitor using a THEMOMETER at REGULAR INTERVALS and add hot/cold water if temp fluctuates
how can pH be controlled
- use of a buffer solution
- monitor using a pH meter at REGULAR INTERVALS
why was the test tubes, including the enzyme and substrate, left in the water bath for 5 mins
so the solution equilibrate/ reach temp of the water bath
explain why using a colorimeter to measure colour change is BETTER than comparison to colour standards
- objective/not subjective
- higher accuracy
explain a procedure that could be used to STOP each reaction
- 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
