Bruce A. Bohrn
Department of Botany Universitv of British Columbia Vancouver
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The Incorporation of Glucose Into Chelidonic Acid A n experiment in plant biochemistry
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is added to the feeding vial drop by drop to "wash" all recent study completed in the author's labeled compound into the plant. After several such laboratory' involving the biosynthesis of chelidonic washings, the plant is transferred to a beaker of water acid, 7-pyrone-2,6-dicarboxylic acid (I), offers an for the remainder of the metabolism period. excellent laboratory exercise which could be performed in an advxnccd ~rndrrgraduateor u graduate l ~ ~ l ~ o r a ~ o r y The feedmg experiment is set up in the following manner. Using a razor blade, the plant is cut approxivoursv in p13m biochemistry. The cxaminution of thl. mately one-half inch above the ground. The cut end incorporation of a precursor into a naturally occurring is immediately placed in a beaker of water. With a molecule involves many techniques helpful in biosyndrop of water adhering to the cut end, the plant is carethetic research and would afford a student an excellent fully transferred to the feeding solution contained in a opportunity to integrate some of the techniques with small vial. For purposes of support, the diameter of the which he may already be acquainted. It would be vial neck should be only slightly greater than that of the possible for a student to carry the study fromacquisition stem of the plant. The vial should be firmly anchored of plant material, through feeding and isolation, to by means of a clamp or small test tube rack. location of label in chelidonic acid by means of a Illumination for the experiments in our laboratories chemical degradation. is provided by a bank of cool-white fluorescent lamps The plant used for the study was Cmvallariamajalis located about 6 in. from the plants. A small electric L. commonly known as lily-of-the-valley. It was obfan at a distance of about 5 ft supplies circulating air to tained from a commercial florist and is easily available assist transpiration. No difEculty with temperature during the Easter season. The plants are usually kept was observed with the fluorescent lamps even without in a cool, dimly lit room to hold them back before sales. the fan. Serious overheating of the plants occurs when Several days in a green house or on a sunny window sill incandescent lamps are used unless special precautions is sufficient to bring about unrolling of the leaves and are taken. An overall metabolism period of 24 hr is production of flowers. The administration of labeled used for our work, but any metabolism period from 20 precursor is done with a plant in bloom. to 30 hr would be satisfactory. Glucose and ribose were incorporated into chelidonic After the metabolism period, the plant is removed acid with reasonable efficiency. The low cost of glucose from the water, cut into pieces, and dropped into boil-U.L.-14Cand its availability in license-exempt packages ing 95% ethanol. In the event that absorption is inmakes its use for a laboratory exercise practical. For complete, the cut end of the plant should be rinsed example, New England Nuclear Carp. (Boston) lists 50 carefully with water to remove any mechanically of this compound for $30. This would allow ten adhering labeled precursor. The unabsorbed precursor pairs of students to administer 5 r c of precursor a t a and the rinses should be pooled and saved for analysis. cost of $3 per feeding. (Even if absorption of precursor appears complete, it is The glucose is dissolved in water and stored in a a good practice to assay the residual liquors for radiosmall plastic bottle in a freezer prior to its use. Genactivity. This, of course, is necessary in order to detererally, suflicient water is used so that the amount of mine the exact amount of label which was taken up by precursor to be offered to the plant is contained in 0.5the plant, but it is also a mark of good radiochemistry 1.0 ml of solution. By keeping the volume small, technique.) fairly rapid uptake of the compound can be expected. Extraction with boiling ethanol is continued until the I n our experience, uptake has been essentially complete plant material has been freed of pigments. About four in approximately 4 to 6 hr. Absorption of precursor changes of solvent are necessary for complete extracsolution can also be accelerated somewhat by maintaintion. The combined extracts are diluted to a known ing the plants on low water for a day or two before volume with ethanol and aliquots taken for determinafeeding. As the solution is taken up by the plant, water tion of total ethanol soluble radioactivity. The solvent is now removed on a steam bath or in a This work was supported by National Science Foundation rotaly film evaporator and the residue extracted with Grant GB-4538, and was done at the University of Rhode Island. boilmg water. After filtration through a bed of Celite B o n ~B. , A., Amh. Bioehern. Biophys., 115, 181-6 (1966). filter-aid, the aqueous extract is adjusted to pH 6.0 and 'DEAN, F. M., "Naturdly Occurring Oxygen Ring Compasscd through a column of Amberlite CG400 resin pounds," Bottemorths, London, 1963, p. 107. in the OH- form. Neutral and basic components are WILDE,C., Ann. 127, 167 (1963). Volume 43, Number 10, October 1966
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eluted with distilled water; the acidic components are removed with a mixture of ethanol, water, and concentrated hydrochloric acid (4: 5: 1). The eluate, which is yellow, is extracted with ether using a continuous liquid-liquid extraction system. (If this apparatus is not available, repeated extraction using a separatory funnel can be done. The hazard of radioactive contamination is increased by this method.) The ether extract is evaporated to dryness, the rcsidue taken up with a small volume of 80y0 aqueous ethanol and applied to a sheet of Whatman No. 3hfhl paper, and the paper run in a solvent consisting of ethanol, concentrated ammonium hydroxide, and water (20 :1:4). Chelidonic acid runs as a compact band with R, = 0.55 which can be detected by its quenching of ultraviolet light (2537 A). Rechromatography under the same conditions yields pure chelidonic acid. The acid is eluted from the paper with 80y0 ethanol and the extract diluted to a known volume. Aliquots are taken for ultraviolet analysis (E,,, = 8830 a t 277 mp) and radioactivity measurements. Standardization of the ultraviolet method was done with chelidonic acid obtained from the Aldrich Chemical Co. (hfilwaukee). From the information obtained above, it is possible to measure the efficiency of incorporation of precursor by determining the "dilution value" which is defined as the ratio of the specific activity of the precursor to that of the product. I n addition to the e5ciency of incorporation of a precursor, it is vital to learn where in the product the labeled carbon atoms are to be found. Often, only when the fate of the labeled carbons of the precursor is known, can a possible mechanism of incorporation be postulated. In the case of chelidonic acid, several degradation procedures have been ment i ~ n e d . ~The method chosen for our work and the one described here is that of Wilde.% In this procedure, chelidonic acid is oxidatively cleaved with bromine to yield pentabromoacetone and oxalic acid. Two equivalents of oxalic acid derive from carbons 1, 2 and 6, 7 (numbering pattern shown below) and the pentabromoacetone derives from carbons 3, 4, and 5.
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CBr,CCHBr, f 2HOOC-COOH C-3, 4, 5
C-1.2 and C.6, 7
After aliquoting for the analyses described above, a carefully weighed quantity of chelidonic acid in the
range 50 to 100 mg is added to the volumetric flask. After making to volume with ethanol, aliquots are taken for radioactivity measurements. The solution is then evaporated to dryness. To the dried acid is added 2 or 3 ml of water and several drops of bromine. The mixture is heated gently to about 50°C on a steam bath and the flask swirled from time to time. As the hromine is vaporized, more is added. I n about 45 min, the chelidonic acid will have dissolved and a heavy oil (occasionally a crystalline solid) can be seen in the flask. Bromine is added once again and the reaction continued for 20 to 30 min. After the mixture has been cleared of bromine, it is cooled at 1°C for several hours. The solid in the flask, which is a mixture of the two products, is collected on a small suction filter. After drying on the filter for a short time, the solid is washed with several 1-ml portions of chloroform. The washings are combined and evaporated to dryness to yield crude pentabromoacetone. The product is recrystallized from a small volume of acetic acid to which a few drops of water have been added. The crude oxalic acid remaining on the filter is recrystallized from 1 or 2 ml of water. The products obtained in this way are sufficiently pure for radioactivity measurements. The overall procedure is a lengthy one, but the work involved can be distributed over several laboratory periods. Often only a small amount of preparation is required for an otherwise time consuming process, e.g., banding the chromatogram prior to overnight irrigation. (Note: Although the solvent will run off the paper in an overnight irrigation, chelidonic acid will remain on the paper and can be located by running a spot of authentic acid on each sheet.) The feeding process itself can be set up in less than half an hour and can be timed so that the extraction procedure can he done during one workmg period. The final analytical procedures and the degradation can be done in one session. The radioactivity measurements will, of course, depend upon the equipment available. X'leasurements in our laboratory have been done by two methods: counting the C02 from comhusted samples using a Dynacon Electrometer; and direct counting using a liquid scintillation counter. (Both were Nuclear Chicago devices.) Liquid scintillation is much faster and would be the method of choice. As an example of the incorporations observed typical data are given. An original quantity of glucose weighing 0.59 mg and possessing an activity of 10 pc was administered. Of this 0.35 pc was not taken up by the plant; total ethanol soluble activity was4.78 pc. Incorporation into chelidonic acid was 2.25 X 10-8 pc which amounts to 0.047%.
The word gas wss first used in the seventeenth century. Explosions, strange noises, snd lurid flames had been seen in m i n ~ eaves, , etc. The alchemists, whose earthen vessels often exploded with terrific violence, commenced their experiments e t h prayer, and placed on their crucibles the sign of the onxs-hence the name cruciblefrom crux (gen. crucis), a cross. . .
. . .Taken from the 1873edilim ofSteele's "Fourteen Weeks in Chemistry" published by A . S. Barnes and Co.
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