Required Practical 8 - Investigating the effect of light/CO2/temperature on dehydrogenase activity

role of dehydrogenase in photosynthesis

catalyses the reduction of NADP in the light dependent reaction

NADP gains electrons from photoionisation of chlorophyll

how rate of dehydrogenase activity in extracts of chloroplasts can be measured

Control 1 - set volume of DCPIP, water and chloroplasts in isolation medium covered in foil

Control 2 - set volume of DCPIP, water and isolation medium without chloroplasts

Standard - set volume of water and chloroplasts in isolation medium without DCPIP

Experiment - set volume of DCPIP, water and chloroplasts in isolation medium

Shine light on test tubes and time how long it takes DCPIP to turn from blue (oxidised) to colourless (reduced)

Compare to colour standard to identify end point

Rate of dehydrogenase activity = 1/ time taken

examples of variables that could be controlled

volume of chloroplast suspension

volume/ concentration of DCPIP

source of chloroplasts

Purpose of control 1

shows light is required for decolourisation of DCPIP

Shows that chloroplasts alone do not cause DCPIP to decolourise

explain why DCPIP in control stays blue

no light so no photoionisation of chlorophyll

so no electrons released to reduce DCPIP

purpose of purpose of control 2

shows chloroplasts are required for DCPIP to decolourise

shows that light alone does not cause DCPIP to decolourise

why DCPIP changes from blue to colourless

DCPIP is a redox indicator / DCPIP gets reduced by electrons

from photionisation of chlorophyll

limitation of this method

end point is subjective

use a colourimeter

measure light absorbance of sample at set time intervals

zero colorimeter using the colour standard