PRACTICAL 1 -

PRE - LAB -

• the rate of reaction of an enzyme - controlled reaction is influenced by different factors -

  • temperature

  • PH

  • substrate concentration

  • enzyme concentration

• the effect of each of these can be determined by changing a single variable and measuring its effect on the reactions rate, but its important to keep all other variables constant so they dont influence the results

• initial rate of reaction is measures because the rate of an enzyme-controlled reaction is high, because enzymes act as biological catalysts, so the concentration of reactants change rapidly

• the initial rate of the reaction is the only point during the reaction where the concentration of the reactants and products is known

EQUIPMENT -

• powdered skimmed milk suspension (2%)

• trypsin solution (2%)

• 5 test tubes and a test tube holder

• stop watch

• two 5cm³ pipettes

• goggles

• colorimeter

• 2 cuvettes

• distilled water

METHOD -

• dilute stock solution of trypsin with distilled water to produce solutions with 0.2%, 0.4%, 0.6% and 0.8%

• make a control by adding 2cm³ of trypsin solution and 2cm³ of distilled water, use this to set the colourimeter absorbance to zero

• to another cuvette add 2cm³ of milk suspension and 2cm³ of the stock trypsin solution, mix and place in the colourimeter and measure absorbance at 15 second intervals for 5 minutes

• rinse the cuvette with distilled water and repeat with all the trypsin concentrations, but place the control back into the colourimeter between the different concentrations to set the absorbance back to zero

RISK ASSESMENT -

• BROKEN GLASS -

  • RISK - cuts from sharp objects

  • PRECAUTION - take care when handling glass objects, keep them away from edges of surfaces

  • EMERGENCY PROTOCOL - elevate cuts, apply pressure, dont remove glass from wounds and seek medical assistance

  • RISK LEVEL - low

• ENZYMES -

  • RISK - allergies / irritations

  • PRECAUTION - avoid contact with skin / eyes and wear gloves / eye protection

  • EMERGENCY PROTOCOL - seek assistance

  • RISK LEVEL - low

GRAPH / ANALYSIS -

• plot the absorbance readings (Arbitury units) against time (s)

• work out the mean rate of reaction for each concentration

CONCLUSION -

• milk contains a white protein called casein which when broken down causes the milk to turn colourless

• trypsin is a protease enzyme which hydrolyses the casein

• as the concentration of casein increases, the number of enzyme-substrate complexes forming also increases because the enzymes and substrates are more likely to collide

• this means the rate of reaction will also increase up to the optimum enzyme concentration

• the rate plateaus at the point where all the substrates occupy an active site, increasing the enzyme concentration wont increase the rate as the substrate concentration is limiting the rate