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Hill Reaction

The light-dependent stage of photosynthesis

Robert Hill was a British biochemist who, in a series of papers published between 1937 and 1940, showed that the first stage of photosynthesis involved the photolysis, (splitting), of water to yield oxygen.

When light energy is absorbed by chlorophyll electrons are raised to a high energy level and transferred to the electron carrier NADP. The electrons from chlorophyll are replaced by splitting water, yielding oxygen (O2), electrons (e-) and hydrogen ions (H+).

In the chloroplasts of plant cells NADP is reduced to NADPH when it combines with an electron from chlorophyll and a hydrogen ion from water.

The reaction can be demonstrated in isolated chloroplasts using DCPIP to replace NADP as the electron acceptor. DCPIP is reduced from blue to colourless by the addition of electrons.

It is much easier to get this to work than some textbooks and methods imply. It is good practice to keep everything cool, so use reagents from the fridge or keep them on ice. However, this is not critical for the practical to work. Make fresh suspension each day, though the chloroplasts will retain around 50% of their activity after 24 hours in the fridge.

Safety

DCPIP is irritant but at the concentration used here is a low hazard and does not need a warning label. caution

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Results

Varying the intensity of light

You can vary the intensity of light by

  • measuring the distance between the light source and the sample, (light intensity is proportional to the inverse square of the distance - I=1/d2)
  • controlling the light with a dimmer switch
  • placing neutral density filters between the light source and the sample.

    • You may want to consider how to make sure that the heat from the light source does not affect the result, but this is unlikely to be significant over the short period of time needed for the reaction.

 intensity of light

Varying the wavelength of light

To investigate the effect of different wavelengths of light place coloured filters between the light source and the reaction mixture. You must ensure that the intensity of light striking the reaction mixture is the same whatever colour of filter you use. We have selected a set of four filters, (blue, green, red and neutral), which each allow approximately 10% of the total light to be transmitted and will give good results.

 blue
green
red

Absorbance vs Time

Reaction mix exposed to a 50W halogen light at a distance of 25cm.

    absorbance vs time

Absorbance vs Time using different coloured filters

    These results were obtained using a set of filters that each transmitted the same total amount of light, (approx 10%). The reaction mix was exposed to a 50W halogen light at a distance of 25cm. Indandescent bulbs will not give a good result with the blue filter as they do not emit much blue light.
    different colours

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Methods
The reaction mixture contains a suspension of chloroplasts in buffer containing DCPIP . This should be kept in the dark until it is exposed to whatever lighting conditions are being investigated.

Read absorbance using red light, (635nm).

If you are not using a colourimeter you can get qualitative results by eye.

 Hill Reaction
Preparing a suspension of chloroplasts.

    You should do this in dim light and keep the chloroplast suspension in the dark.
    Fresh, young spinach works best in our experience, though other sources of chloroplasts could be tried. Water cress, cabbage and horse radish have all been reported to work.
  • Put 10g of fresh spinach into a pestle and mortar with a pinch of sand and grind to a paste
  • Add 20cm3 of Hill reaction buffer
  • Filter through 4 layers of butter muslin.

    • Keep the chloroplasts cold until you are ready to use them.

Reaction mixture

    The suggested reaction mixture for a 4cm3 cuvette is:
      DCPIP (0.1% solution) 0.1cm3
      Hill reaction buffer 2.8cm3
      Chloroplast suspension 0.1cm3
  • You can premix the DCPIP and buffer in sufficient quantity for a number of experiments and this will keep well.
  • The DCPIP/buffer should be warmed to the experimental temperature, (25°C works well).
  • Add the warmed DCPIP/buffer mix to the chloroplast suspension in a test tube and keep this in the dark.
  • The mixture can be poured into a cuvette to read absorbance, (red light), then transferred back to the test tube to be exposed to whatever lighting conditions you are investigating.
  • If you are not using a colourimeter you can get qualitative results by eye.

Hill reaction buffer - 0.15M phosphate buffer pH 6.5 with added glucose and KCl.

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Copyright 2012 Mystrica Ltd.