Required practical 8- Dehydrogenase activity in chloroplasts

Outline the method to prepare chloroplast suspension:

• Place a small beaker in a larger beaker half filled with ice.

• In another beaker add some iosolation medium and crushed spinach leaves

• Then get a funnel add three layers of muslin and some drops of iololation medium and pour into the small beaker placed inside the ice beaker

Outline the method for testing the effect of dehydrogenase activity in chloroplasts:

• For tube y the purpose is to investigate the effect of ammonium hydroxide

• So, without ammonium hydroxide it takes shorter time to decolorise

• With ammonium hydroxide it takes longer time to decolarise, reducing the rate of dehydrogenase enzyme

• When removing the foil for tube A, which appears a lighter shade, shows that both light and chloroplasts are required for this reaction

The idea behind the practical

• Dehydrogenase enzyme catalysts the reaction NADP + H → NADPH during the light dependent reaction

• So to find the effect of ammonium hydroxide on the time taken for DCPIP to decolorise (from blue to colourless)

• When it becomes colourless the DCPIP is reduced

• So, DCPIP accepts the electrons instead of (NADP)

• So reduces the rate of dehydrogenase enzyme

Practical implications:

• When blending the spinach leaf, so not put the midrib or the stalk into the blender, as they are tough to blend, and they contain little amount of chloroplasts

• Muslin is used as filter paper, as it removes the large debris, but allows organelles such as chloroplast through

• Isolation medium: contains same water potential, buffer, and cold

  • So, less osmotic activity, no denaturing of enzymes and minimal enzyme activity

• End point is subjective, leading to inaccurate results being recorded, to overcome this make sure to use a colorimeter