thylakoid membrane.

The sources of these protons are the splitting of water, which occurs on the lumenal side
of the thylakoid membrane, and the transport of protons from the stromal side across the membrane into the
lumen by the electron transport chain components plastoquinone and cytochrome b/f complex. In the second
step, this gradient of protons is released when the protons diffuse through the membrane-spanning ATP-
synthase molecule, which couples proton movement to the synthesis of ATP from ADP and Pi.
As is routine in studies using mitochondria and chloroplasts, we will supply an artificial electron acceptor to
monitor "partial reactions" of the electron transport. These are chemicals that accept electrons at positions of
the electron transport chain where exogenous compounds normally do not act in vivo. Frequently these
electron acceptors are dyes specifically selected so their reduction can be monitored by spectrophotometry.
The dye reagent we are using in this experiment is 2,6-dichlorophenol indophenol (DCPIP). It is blue when
oxidized and colorless when reduced. DCPIP accepts electrons between the electron chain components
plastoquinone and cytochrome. The electrons are ultimately derived from water.
In 1937 Robert Hill showed that this partial reaction of the electron transport chain using DCPIP could be used
to investigate the rate of oxygen evolution (from the splitting of water molecules in PSII) and thus the rate of
photosynthesis in thylakoids of isolated chloroplasts. The reaction is now known as the Hill reaction and is still
used today to determine photosynthetic rates in chloroplast preparations.

A. In vivo reaction

B. In vitro reaction
Figure 4. A. In vivo reduction reaction of NADP to NADPH in chloroplasts B. In vitro reduction reaction of
DCPIP in photosystem II