Plant extracts and weedkillers

One of the key experiments that demonstrates a number of fundamental principles in photosynthesis is the demonstration of electron transfer in isolated chloroplasts. The original experiment was carried out by Robert (sometimes Robin) Hill, after whom the reaction is often named. In fact at the University of Sheffield, we have an Institute named after him: The Robert Hill Institute.

In the Hill reaction, biologically active, but isolated chloroplasts are used in conjunction with an artificial electron acceptor, to illustrate that electron transfer takes place in intact photosynthetic systems. In a nutshell, the Hill reaction results from photon stimulated electron transfer from water molecules to an artificial electron acceptor (importantly, from a Biological perspective, against the chemical potential gradient). This discovery, made over 80 years ago, demonstrates that oxygen production is a separate process from carbon dioxide fixation into sugars

The basis of the experiment is to isolate a suspension of chloroplasts and use them to drive the reaction in which the electron acceptor 2,6 Dichlorophenol indophenol (DCPIP) (shown below)

is reduced. The enzyme that catalyses the supply of electrons is NAD/P/H dehydrogenase The reaction is easy to observe in principle, since the oxidised form of DCPIP is blue while the reduced form is colourless.  However, there are some practical issues with this experiment that need to be considered.

Extraction The leaves (spinach is common) used should be as fresh as possible, and this applies to all biochemical preparations, since cells and tissues are constantly "turning over" proteins and metabolites and it wont come as a surprise to find that old, dehydrated, poorly maintained tissue samples are less likely to yield reproducible (text book) results. The buffer used to extract the material into is designed to maintain an optimum pH and osmolarity, and it is traditional to keep samples cold on ice, to minimise denaturation or enzymatic loss of key components. Biochemists assume that keeping tissues and fractions on ice is of the default option (until proven otherwise). 

Reaction mixture The DCPIP concentration should be sufficient to be a clear blue colour and to facilitate a reaction rate that is measured in a few minutes (rather than seconds, or hours). The instructions provided by the A level curriculum documents read as follows:

DCPIP solution (per 250 cm3 ) 
Dissolve 0.01 g DCPIP and 0.93 g KCI in phosphate buffer solution and make up to 250 cm3 with phosphate buffer solution. Keep cool until required

If the Mr for DCPIP is 268: what is the concentration provided and what is the concentration in your final reaction tube?

Ammonium hydroxide (AH) is used to mimic the effect of a number of weed killers which act by subverting the normal flow of electrons. The addition of AH leads to a reduction in dehydrogenase activity. The concentration of AH added is around 1M and so it could act as a denaturant, it could significantly change the pH of the reaction, both of which could result in a loss of activity. It could be a competitive inhibitor of the dehydrogenase? Which is most likely? Are there any other explanations? DCPIP has a higher affinity for electrons than ferredoxin and the photosynthetic electron transport chain can reduce DCPIP as a substitute for NADP⁺

These experiments (carried out without a spectrophotometer), rely on the quality of the chloroplast preparation and the optimum concentration of DCPIP. If the reaction is too fast or too slow, you should investigate where the problem lies. The difficulty in observing colour changes by eye, illustrates why soectrophotometers became the gold standard. How would you optimise the Hill Reaction for school laboratories?