required practical 8

investigate the effect of a named variable on the rate of dehydrogenase activity in extracts of chloroplasts

an enzyme found in plant chloroplasts which is essential for the light dependent reaction, as it catalyses the acceptance of electrons by NADP

using a dye like DCPIP which accepts the electrons instead - this means it is reduced and changes colour from blue to colourless

the time taken for the solution to change from green to blue-green (DCPIP doesn’t go colourless because chloroplasts are present)

whether ammonium hydroxide is present or not

• label 5 test tubes A,B,C,X and Y and stand them in a large beaker in ice • place a lamp 10cm from the beaker and turn it on • set up test tube A,B and C (standards): A = chloroplast suspension, DCPIP, buffer, wrapped in foil B = DCPIP, buffer, distilled water C = chloroplast suspension, buffer, distilled water • set up tube X with the chloroplast suspension and buffer, then add the DCPIP, quickly mix the contents and start a timer • record how long it takes for the contents of tube X to go green (use tube C as a reference) • repeat the process with tube X 4 times • set up tube Y with chloroplast suspension, buffer and ammonium hydroxide, then add the DCPIP, quickly mix the contents and start a timer • record how long it takes for the contents of tube Y to go green (use tube C as a reference) • repeat the process with tube Y 4 times • calculate the mean time taken for the colour change in each test tube

• the colour change takes longer in tube Y becasue ammonium hydroxide is present • the ammonium hydroxide is alkaline so can denature dehydrogenase, or pick up electrons instead of NADP/DCPIP

for comparison - acts as control to show that reaction doesn’t occur without light

for comparison - acts as control to show that reaction doesn’t occur without chloroplasts

acts as standard to use as reference for when colour change is complete