:How~T-.TDo-It- I:. The Hil Reaction of Photosynthesis in Isolated Chioroplasts A Quantitative Approach Rob L. Dean Downloaded from http://online.ucpress.edu/abt/article-pdf/58/5/303/47660/4450156.pdf by guest on 02 October 2021 The assay to determine the rate of + 2H20 - 4H 4e + 02 Stock Solutions photoreduction of 2,6-dichloropheno- lindophenol (DCPIP) described here Variants of this assay are widely 1. Chloroplast isolation buffer has been used in a new course entitled used in student laboratories but in the (modified from Hopkins 1990): "Scientific Methods in Biology." The published and unpublished laboratory 50 mM tricine, 400 mM sorbitol, purpose of this laboratory course is to manuals that I have encountered, the 10 mM NaCl, 2.5 mM MgCl2- acquaint students with experimental considerable potential for the quanti- 6H20, 1.25 mM MnCl2, 0.3 mM design and teach them how to evalu- tative analysis of the data that it pro- Na2EDTA. Adjust pH to 7.8 with ate and communicate their results. vides is not fully exploited. By refer- NaOH. One goal is to enhance the scientific ence to a standard curve of absorbance 2. Chloroplast reaction buffer (5X literacy of our students by integrating at 600 nm against DCPIP concentra- concentrated, modified from basic chemistry and arithmetic, used tion, one can determine the change in Hopkins 1990): routinely in research laboratories, into molarity and hence the number of 250 mM tricine, 500 mM sorbitol, their practical experience. moles of DCPIP reduced in a given 100 mM KCl, 25 mM MgCl2. The principles on which the assay is time. Since 1 mole of DCPIP contains 6H20, pH adjusted to 8.0 with based were worked out by Robin Hill 6.0247 x 1023 (Avogadro's number) NaOH routinely but pH 6.0-8.0 in the 1930s. Hill used artificial elec- molecules of DCPIP, the number of will work. tron acceptors, including DCPIP, to molecules of DCPIP that have been 3. 90% aqueous acetone. trap electrons passed to the electron reduced can be determined. Conse- 4. 0.2 mM 2,6-dichlorophenolindo- transport chain from photosystem II quently, from the stoichiometry of phenol (DCPIP) (BDH Chemicals when isolated chloroplasts are ex- Equations 1 and 2, the number of Ltd.). posed to light. As the blue, oxidized electrons transferred to reduce the 5. 2 mercaptoethanol (BDH Labora- form of DCPIP becomes reduced it DCPIP, the amount of water oxidized tory Supplies). becomes colorless, so the progress of to produce these electrons, and the the reaction can be monitored by the amount of oxygen evolved in the pro- change in absorbance at 600 nm of the cess can be easily estimated. Such es- Procedure DCPIP solution (Equation 1). timates, based on reactions that occur Quantities given are those I use for within the chloroplasts, refocus the one lab period with nine groups of DCPIP2H+ +2e students' attention on the biological four students. (blue) process under investigation. Chloroplast Isolation DCPIP - H2 Weigh 25 g of barley coleoptiles or (colorless) spinach leaves from which the petioles Equipment and major veins have been removed. The electrons involved in the reduc- Rinse leaves in ice water and pat dry. tion of DCPIP ultimately come from Spectrophotometers and suitable Cut the leaves into smaller fragments the oxidation of water by the oxygen- cuvettes or tubes with scissors and place them in a evolving complex of photosystem II Lamps with 100-watt frosted bulbs blender cup that has been stored in the (Equation 2). Domestic blender freezer. Add 100 ml of cold chloroplast 10-ml volumetric pipets isolation buffer to which 60 p1 of Variable volume micropipets (10- 2-mercaptoethanol has been freshly 100 j1) with tips added. Blend leaves in buffer with x Rob L is Cell 3 Dean, Ph.D., the Biology Parafilm Course Coordinatorin the Zoology De- 5 second bursts at full speed. Filter partment, Biology & Geology Building, Li-Cor-L1-189 quantum/radiometer/ the blended mixture (called a brei) Universityof Western Ontario, London, photometer with a quantum sen- through four layers of cheesecloth into ON N6A5B7, Canada. sor a 250-ml beaker on ice. Transfer 10 ml Clinical centrifuge aliquots of the filtrate into six 15-ml PHOTOSYNTHESIS303 centrifuge tubes. [I prefer graduated Pyrex tubes because they facilitatethis Table 1. Contents of the reaction tubes for the sample experiment. and a later volume adjustment]. Cen- x 10-4 5 x Chloroplast trifuge the filtrate for five minutes at 2 M Reaction Distilled 1300 x g (this is setting #7 on a Tube DCPIP(ml) Buffer(ml) H20 (ml) Prep(,ul) standard clinical centrifuge containing 1 Exp (5 cm) 1.0 1.0 3.0 swinging buckets with a 15-cm radius 2 Exp (10 cm) 1.0 1.0 3.0 from the axis of rotation). Discard the 3 Exp (15 cm) 1.0 1.0 3.0 supernatants and add chloroplast iso- 4 Exp (20 cm) 1.0 1.0 3.0 A volume lation buffer containing 0.6 pl/ml of 5 Exp (25 cm) 1.0 1.0 3.0 containing 25mg 2-mercaptoethanolto each pellet up to 6 Exp (30 cm) 1.0 1.0 3.0 of chlorophyll the 0.4 ml mark. Resuspend the pellets 7 Exp (40 cm) 1.0 1.0 3.0 (to be calculated) with a paintbrush and pool them in a 8 Exp (50 cm) 1.0 1.0 3.0 single tube. This is the chloroplast 9 Control (dark) 1.0 1.0 3.0 preparation. It is far from pure as it 10 Blank - 1.0 4.0 contains nuclei and other cellularcom- ponents, but since the chloroplastsare the only photoactive constituents, fur- tion mixture, that volume is given by: (in this case, 31 ,u) to the blank tube ther purificationdoes not improve the 25 (#10). Mix thoroughly and use the assay. 259= 13.2 pJ of chloroplastsuspension. blank to set the spectrophotometerto Downloaded from http://online.ucpress.edu/abt/article-pdf/58/5/303/47660/4450156.pdf by guest on 02 October 2021 an absorbanceof 0. Estimationof the Total A Sample Experiment The remainderof the experiment is ChlorophyllConcentration in the performed in a darkened room. The Chloroplast Suspension This experiment was designed to chloroplast preparation has shown measure the effect of photon fluence good stabilityover the three-hourlab- To make valid comparisonsbetween rate on the rate of photoreduction of oratoryperiod. However, to avoid bi- results obtained using differentchloro- DCPIPby isolated chloroplasts. Chlo- asing the data if there is any loss of plast preparations, it is necessary to roplasts were isolated and chlorophyll activitywith time, the students should standardize the amount of chloro- concentrationwas determined as de- run the reactions at random or, most plasts added. In photosynthesis re- scribed before. The chloroplasts were conservatively, from the furthest dis- search, this is accomplished by isolated from six-day-old barley co- tance from the lamp to the closest. estimating the total chlorophyll con- leoptiles grown under continuous Workingquickly, add 31 p1 of chloro- centration in the chloroplast prepara- light one foot from a 100-wattbulb. plast preparationto Tube #1, cap the tion and adding a volume of chloro- Tape a piece of masking tape to the tube with parafilm, and invert three plast preparation containing a bench and rule it at 5 cm increments times to mix the contents. Read the standard amount of chlorophyll to from 0 to 50 cm. Place a lamp with a absorbance at 600 nm (= A600 at 0 each reactionmixture. The following is 100-watt frosted bulb over the 0 cm minutes), place the tube in the 100-ml based on the method of Arnon (1949). ruling. Reaction tubes are placed in a beaker astride the 5 cm mark on the It is not the most accurate method to 100-ml beaker containing water at tape and turn on the lamp. As the estimate chlorophyll concentration, room temperatureto shield the reac- lamp is turned on, note the time. After but it is rapid and reproducible. tion tube from the heat emitted by the 55 seconds have elapsed, quickly dry Add 50 ,l of the chloroplastsuspen- lamp. The reactiontubes in the 100-ml the tube and take the one minute A600 sion to 5.0 ml of 90% acetone. Cover beakers can be placed astride any of reading. Immediately return the tube the tube with parafilmand shake thor- the ruled lines to change the photon to the 100-mlbeaker. Repeat this pro- oughly to mix the contents. Centrifuge fluence rate for each reaction. The cedure at 55 seconds into the 2nd, 3rd at 1300 x g for three minutes to pellet photon fluence rate at each distance and 4th minutes. precipitatedprotein and other particu- from the lamp can be recordeddirectly Repeat the above procedure for the late matter.Read the absorbanceof the in ,moles of photons per meter remainingtubes at their respectivedis- supematant at 652 nm (=A652)using squared per second using a quantum tances from the lamp. Do not add the 5.0 ml 90% acetone and 50 p1 of chlo- sensor attached to a Li-Cor-L1-189 chloroplast preparation to any tube roplast isolation buffer as your blank. quantum/radiometer/photometer[Li- until you are ready to start A600 read- The chlorophyll concentration in the Cor, Inc., Lincoln, Nebraska]. ings. The control is treated identically chloroplastpreparation is derivedfrom: Prepare spectrophotometer tubes except that it is wrapped in aluminum according to Table 1. The following foil between A600readings. 100 table is preparedfor each reactionmix- A652x 3 = x mg chlorophylllml ture (Tubes 1-9, Table 1). * chloroplast suspension Rate Calculations e.g.