1 - Factors affecting rate of reaction

Why is the initial rate of reaction measured?

There are no limiting factors & it is the only point when the concentration of the reactants & products are known

What are the four factors that affect the rate of an enzyme-controlled reaction?

• Temperature

• pH

• Enzyme concentration

• Substrate concentration

What equipment is required for this practical?

• Powdered skimmed milk suspension (2%)

• Trypsin solution (1%)

• 6 test tubes & holder

• Stop clock

• Two 5cm³ pipettes

• Colorimeter (before taking values, calibrate it with either trypsin or distilled water & set filter to complementary colour to solution; don’t use filter that is the same colour as the solution)

• 2 cuvettes (use the same cuvette, to ensure the wall thickness is the same & make sure it doesn’t have any scratches on it)

• Distilled water

What is the method for this practical?

1. Dilute a stock solution of trypsin with distilled water to produce solutions with concentrations of 0.2%, 0.4%, 0.6%, 0.8% & 1.0%

2. Make a control by adding 2cm³ of trypsin solution & 2cm³ of distilled water into a cuvette. Use this to set the colorimeter absorbance to zero

3. To another cuvette, add 2cm³ of milk suspension & 2cm³ of the stock trypsin solution. Mix & place the solution into the colorimeter & start the stop clock

4. Measure the absorbance immediately & then at 15 second intervals for 5 minutes

5. Rinse the cuvette with distilled water & repeat for each concentration

What are some control variables for measuring enzyme activity?

• Replace the trypsin solution with distilled water or boiled enzyme solution

• Temperature: use a thermostatically controlled water bath & check regularly with a thermometer (add cool water if it gets too hot or add warm water if it gets too cold)

• pH: use pH buffer to avoid changes & monitor regularly with a pH meter/probe

What should you include when describing control variables?

• Why does it need to be controlled?

• How to monitor it?

• What to do if it changes?

What is the equation to measure the rate of reaction?

Rate of reaction = 1 / mean time

• could multiply all values by 1,000 to avoid tricky decimals (make this clear in table & axes of graph)

• mean time:

  • point at which graph levels off / time it took (transmission by time graph)

  • if there are 3 lines on the graph with different variables, there’ll be 3 different mean times

What is the risk, safety precaution & in emergency for the hazard: broken glass

Risk:

• cuts from sharp object

Safety precaution:

• take care when handling glass objects

• keep away from edge of desk

In emergency:

• elevate cuts & apply pressure

• don’t remove glass from wound

• seek medical assistance

What is the risk, safety precaution & in emergency for the hazard: hot liquids

Risk:

• scalding & burns

Safety precaution:

• handle with care & use tongs to remove boiling tubes from water bath

• wear eye protection

In emergency:

• run burn under cold water

• seek medical assistance

What is the risk, safety precaution & in emergency for the hazard: enzymes

Risk:

• allergies

Safety precaution:

• avoid contact with skin/eyes (wear gloves)

• wear eye protection (e.g. goggles)

In emergency:

• seek assistance

What is the conclusion from this practical?

• Milk contains a white protein (casein), which when broken down, causes it to turn colourless → trypsin is a protease enzyme which hydrolyses the protein

• As trypsin concentration increases, the number of enzyme-substrate complexes increases, so rate of reaction increases

• Rate plateaus when all substrates occupy an active site → increasing enzyme concentration will have no effect, as substrate concentration is the limiting factor

How can a colorimeter determine the rate of reaction of trypsin & milk?

• As trypsin digests the proteins in milk, milk becomes more translucent & more light is transmitted through

  • a decrease in absorbance can be measured by the calorimeter

• As absorbance increases, transmission decreases (e.g. dark/opaque solution has greater absorbance, but lower transmission)

What is the effect of enzyme concentration on enzyme activity?

• As the concentration of enzyme increases, successful collisions to form enzyme-substrate complexes increase, so the rate of reaction increases to an optimum

• Beyond the optimum, the rate plateaus as substrate concentration becomes the limiting factor

What is the effect of substrate concentration on enzyme activity?

• Enzyme activity increases initially as substrate concentration increases (substrate concentration is the limiting factor) & higher concentration results in more successful collisions to form enzyme-substrate complexes

• Beyond a certain substrate concentration, enzyme activity plateaus, as all the enzyme active sites are saturated & enzyme concentration is limiting

How is the effect of substrate concentration on enzyme activity investigated?

1. Prepare a simple dilution of milk/substrate concentrations

2. Add each solution to 1cm³ of trypsin of a fixed concentration

3. Record the absorbance immediately & every 15 seconds for 5 minutes

How is the effect of pH on enzyme activity investigated?

1. Add fixed volumes of buffer solutions with a range of pH values to 1cm³ of trypsin & 2cm³ of milk (both of a fixed concentration)

2. Record the absorbance immediately & every 15 seconds for 5 minutes

What is the effect of pH on enzyme activity?

Enzyme activity is highest at the optimum pH:

• above or below the optimum pH, enzyme activity decreases as the unsuitable pH disrupts its tertiary structure & changes the shape of its active site, causing partial denaturation

• complete denaturation may occur at extreme pH values

How is the effect of temperature on enzyme (trypsin) activity measured?

1. Prepare thermostatically controlled water baths with a range of temperatures

2. Place 2cm³ of trypsin solution & 2cm³ of milk suspension in each water bath

3. Leave for 5 minutes to allow the solutions to equilibrate & reach the temperature of the water bath

4. Mix together & record the absorbance immediately & every 15 seconds for 5 minutes

What is the effect of temperature on enzyme activity?

Increasing temperature increases enzyme activity to an optimum:

• both substrate & enzyme molecules gain kinetic energy & move faster, so there are more successful collisions to form enzyme-substrate complexes

• beyond the optimum temperature, enzyme activity decreases as the high temperature disrupts the tertiary structure of enzymes & denatures them

What is the equation to calculate a dilution & what do the terms represent?

C₁ x V₁ = C₂ x V₂

• C₁ = concentration of the stock solution

• V₁ = volume you need to make the new dilution

• C₂ = concentration that you want

• V_{2 =} volume of the concentration you want