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A method of Hill reaction activity assessment allows to study the photochemical processes on the level of thylakoid membrane of chloroplasts. The research is focused on the changes in pigment-protein complexes during the absorption and transfer of radiation energy and the electron flow from photosystem II (PSII) to photosystem I (PSI). Activity of Hill reaction could be used for the assessment of both the effect of various compounds (heavy metals, herbicides) and environmental factors (temperature, irradiation etc.) on plants.

Principle of the method

Intact chloroplasts isolated from photosynthetic tissues are able in specific conditions to sustain for some time period their photosynthetical activity. When stimulated by light all primary photochmical reactions are running, including photolysis of water (Hill reaction). But electrons excused from this process are caught by artificial electron acceptor, e.g. 2,6-dichloro-indophenol (DCIP). This compound is reduced by the electrons and its optical features are changed. This change is measurable spectrophotometrically.

Procedure

Activity of Hill reaction was assessed in pea plant (Pisum sativum L.) as a change in photosynthetic activity of isolated chloroplast suspension directly treated by fluoranthene (FLT) or acquired from leaves of plants cultivated in the presence of FLT. For isolation of chloroplasts fully developed and photosynthetically active leaves are choosed, after 18 days of cultivation the 3^rd and 4^th leaf, after 25 days the 5^th and 6^th leaf. The method for isolation was optimalised for the highest possible yield of intact chloroplasts, especially chemical ingrediences and their concentrations in isolation medium and resuspendation medium and also the ratio of leaf biomass weight and the volume of isolation medium needed.

Optimalisation of the chloroplasts isolation method

The whole process must be done at the temperature of 0 - 4°C. All solutions and media needed must be prepared from the previous day. The adjustment of pH value (pH = 7) should be done before the isolation process.
Exact procedure (see the graphic version in PDF):

• Ø      homogenization of leaves in a cooled mortar with a small addition of sea sand.
• Ø      addition of cooled isolation medium
• Ø      centrifugation at 1370 RPM for 5 min
• Ø      repeated centrifugation of supernatant at 3240 RPM for 10 min
• Ø      - resuspendation of sediment in cooled resuspendation medium by gentle shaking

Assessment of Hill reaction activity

• Ø      placing of reaction mixture (electron acceptor and chloroplast suspension) in a Petri dish
• Ø      irradiation by flood light reflector (400 µmol m^-2 s^-1) with water cooled filter for 4 min at 20 ± 1°C
• Ø      assessment of absorbance of the reaction mixture spectrophotometrically at 630 nm
• Ø      conversion of the absorbance value to the percentage of reduction of the electron acceptor.

Exact amounts and ratios of chemicals in the solutions, appropriate volume of chloroplast suspension, length of exposure period and the method of conversion of absorbance value to the percentage of reduction of artificial electron acceptor are dependent on the plant species, type of electron acceptor and used chemicals.

More infomation is available in a scientific paper published in Chemosphere called "Evaluation of fluoranthene phytotoxicity in pea plants by Hill reaction and chlorophyll fluorescence" (Kummerová, M., Krulová, J., Zezulka, Š., Tříska, J. (2006), Chemosphere 65(3): 489-496).

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