This investigation was conducted to isolate chloroplasts for the purpose of determining the absorption spectrum of chlorophyll, the chromatography of pigments, and detecting the rate of photosynthetic reactions. Differential centrifugation was used to isolate chloroplasts to monitor their rate of photosynthesis using the electron acceptor DPIP. The Hill reaction method was used to measure electron transport.
Chloroplast Isolation and Detection of Photosynthetic Reactions
Photosynthesis is the process by which plants convert sunlight into sugar molecules, which they use for energy and growth. The equation for photosynthesis is 6CO2 + 6H2O + Energy →C6H12O6 + 6O2, which describes the conversion of six water molecules combined with six carbon dioxide molecules to produce one molecule of sugar and six molecules of oxygen (Photosynthesis, n.d.). There are two types of photosynthetic reactions: light dependent (formula: H2O + NADP++ Pi + ADP + Light →O2+ NADPH + H++ ATP) and light independent (formula: CO2 + NADPH + H++ ATP →glucose + NADP+ADP + Pi) (EDVOTEK, 2010). Light dependent reactions require light and occur in the thylakoid membrane while light independent reactions occur in stroma of the chloroplast and can occur in light or dark conditions (Photosynthesis, n.d.). Chlorophylls and carotenoids are the major pigments found in the thylakoids of the chloroplasts and can be isolated using organic solvents.
Chlorophylls are the primary photosynthetic pigments and absorb light within the blue and red spectrums (400-475nm and 600-700nm respectively), providing energy for the initiation of photosynthesis and producing NADPH and ATP for the biosynthesis of carbohydrates (Boyer, n.d.). There are two types of carotenoids: carotenes, which contain carbon and hydrogen, and xanthophyll, which contain hydrogen and oxygen in hydroxyl or epoxide functional groups (Boyer, n.d.). Carotenoids are responsible for the colors distinctions of plants and flowers, displaying colors such as yellow, red, pink, orange, green, and black due to high concentrations of carbon atoms (Boyer, n.d.). Below is a photograph of chloroplast cells and an illustration of the anatomy of a chloroplast cell.
The Hill reaction method was used to illuminate the electron reactions through the use of an accelerating agent to measure the sped of the transport of electrons from PSII to PSI (Green & Dunford, 1996; Walker, 2002). Although their exact function is not fully understood, carotenoids are believed to aid in the absorption of light and the transfer of excitement energy to chlorophyll to aid in photosynthesis (Boyer, n.d.). The pigment or color of the plant is determined by the color of the light not absorbed by the plant (Farabee, 2010). In order for photosynthesis to occur, the organism requires water, sunlight, and carbon dioxide although independent photosynthesis can occur without light if the energy carriers from the light process are present (Farabee, 2010). The purpose of this experiment is to isolate a biomolecule from its original source, extract the pigments, determine the pigment concentration, chromatography, and elution, and measure the visible absorption spectrum (Boyer, n.d.).
In conducting the experiment, chromatography was used to separate the pigments in the designated sample. The mixture of chloroplast pigments, containing chlorophyll, xanthophyll and its derivatives, and small amounts of β-carotene, are paced on a chromatography plate and developed in the solvent. A chromatography of a separate mixture of pigments containing chlorophyll a and b, xanthophyll, epoxide derivatives of xanthophyll, and β-carotene is also taken to aid in pigment identification. The samples are placed in the solvent and allowed to dry on the chromatographic plates for approximately 3 minutes (EDVOTEK, 2008).
The distance the sample travels from the origin divided by the distance the solvent travels from the origin is the Rf. Pigments travel at different rates because they have various degrees of solubility within the solvent, allowing them to be determined by their migration rates. The various structures of the pigment molecules allow for differentiating properties that determine their light absorption rates and polarity, which determines how they separate within the solvent. The Rf values were calculated for the samples to determine the light wave-length, light intensity, and photosynthetic rate of the sample plant (EDVOTEK, 2008).
The second part of the experiment uses DPIP to determine the reaction of photosynthesis in chloroplasts when DPIP is substituted for NADP+as the electron acceptor in chloroplast preparations. Blue when oxidized, DPIP becomes colorless as it is reduced and a spectrophotometer can be used to measure the increase in light transmittance as the DPIP loses its blue color over a period of time. These procedures will test the hypothesis that if living chloroplasts and light energy are necessary to photosynthesis, intact chloroplasts will produce electrons accepted by DPIP when exposed to light, which should result in a decrease in the color of the DPIP as it is reduced and the light transmittal will increase over time. It will also test the hypothesis that if living chloroplasts and light energy are required for photosynthesis, dark conditions or placement in an ionic detergent will decrease photosynthesis when chloroplasts are placed in this environment and DPIP and light transmittal will remain steady (EDVOTEK, 2008).
The mixture of chloroplast pigments, containing chlorophyll, xanthophyll and its derivatives, and small amounts of β-carotene, are collected by blending spinach leaves in a sucrose solution (EDVOTEK, 2008; Huber, 2011). The samples are placed a centrifuge on low speed (1000XG) for 1 minute, then transferred to another tube and placed in the centrifuge for another minute at 600XG (Huber, 2011). This should raise the chloroplasts to the surface, at which point he remaining supernatant may be discarded (Huber, 2011). The chloroplasts are boiled to destroy their ability to synthesize and then the mixture is cooled and this is used as the control (Huber, 2011). A properly calibrated spectrophotometer is used to take time 0 readings after the test and control samples are placed in front of the light source (Huber, 2011).
Readings are taken from tubes 1 and 2 at 5 minute intervals and tube 3 after 20 minutes (Huber, 2011). The continual reduction in levels of DPIP indicates a definitive photosynthetic reaction in tubes 1, 2, and 3 while the steady reading of 0 in the control tube indicates no photosynthetic reaction (Huber, 2011). The excitation energy absorbed is quickly transferred to the PSII and PSI levels as the photosynthetic reaction is stimulated by the exposure to the light source (Green & Durnford, 1996). When energy is absorbed, it can be dissipated as heat, emitted immediately as a longer wavelength (fluorescence), or a chemical reaction can occur, such as photosynthesis (Photosynthesis, n.d.). The isolation of the chloroplasts and stimulation through exposure to the light source confirmed this reaction through the lack of response from the control sample.
The use of the Hill reaction allows the photosynthetic process to be viewed and examined on a molecular level (Walker, 2002). With phosphate/chloride used as a buffer, the electron acceptor DPIP revealed the rate of photosynthesis of the chloroplasts extracted from the spinach leaves (Boyer, n.d.). the hypothesis that the greater the electron production, the more the DPIP would reduce provided a measuring tool by which to measure the photosynthetic process. It also proved that, conversely, the lack of photosynthetic process would allow DPIP and light levels to remain at a steady rate.
Boyer, R. F. (n.d.). Modern Experimental Biochemistry, Second Edition.
Benjamin/Cummings Publishing Co.
Farabee, M.J. (2010, May 18). Photosynthesis. Retrieved april 1, 2011 from
Green, B. R., and D. G. Durnford. "The Chlorophyll-Carotenoid Proteins of Oxygenic
Photosynthesis." Annual Review of Plant Physiology Plant Molecular Biology 47 (1996): 685-707.
Huber, M. C. (2011). Manual of laboratory protocols and assignments for Biol 210:
Scientific techniques. Retrieved April 1, 2011 from http://analytical.biochem.purdue.edu/~courses/undrgrad/322/wwwboard/handouts/exp5.pdf
“Photosynthesis." The Biology Web. Accessed April 1, 2011.
Walker, David Alan. "And Whose Bright Presence-An Appreciation of Robert Hill and
His REaction." Photosynthesis Research 73 (2002): 51-54.