Grable, C. I.

Presley, J. E.

Templeton, George E.

English

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 18 Article 8 1964 Identification of Ninhydrin Positive Components in Ethanolic Extracts of Rice Panicles by Paper Chromatography C. I. Grable University of Arkansas, Fayetteville J. E. Presley University of Arkansas, Fayetteville George E. Templeton University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Plant Biology Commons, and the Plant Pathology Commons Recommended Citation Grable, C. I.; Presley, J. E.; and Templeton, George E. (1964) "Identification of Ninhydrin Positive Components in Ethanolic Extracts of Rice Panicles by Paper Chromatography," Journal of the Arkansas Academy of Science: Vol. 18 , Article 8. Available at: https://scholarworks.uark.edu/jaas/vol18/iss1/8 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. 31Arkansas Academy of ocicnce Frocecdmgs, v ol. io, 1904IDENTIFICATION OF NINHYDRIN POSITIVECOMPONENTS INETHANOLIC EXTRACTSOF RICE PANICLES BY PAPER CHROMATOGRAPHYC. I.Grable 1, J. E. Presley 2, and G. E. TempletonUniversity of ArkansasThe readily extractable amino acids and amides in ricepanicle tissue are being studied to determine their role in thenechanism of physiologic resistance to the kernel smut patho-gen, Tilletia barclayana (Bref.) Sacc. and Syd. Paper chromato-jraphy, because it is rapid and reliable, is ideally suited for ex-amining amino acid pools from tissue whose resistance has beenmodified by manipulation of the environment or for followingchanges in pool composition during growth, either preceding,during, or after infection by this localized parasite in a resistantor susceptible host.Since the origin of modern paper chromatography by Cons-len, Gordon and Martin (2), multitudinous modifications foreparation and identification of amino acids have been reported'1, 4, 5, 7, 8, 10). This report gives the method selected fromhese many modifications which has been found to be best suitedn our laboratory for extraction, desalting, separation and identi-ication of the 80 percent ethanol soluble, ninhydrin positiveomponents of rice panicles at anthesis by two dimensional des-ending paper chromatography.MATERIALS AND METHODS1. Extraction.The Bluebonnet 50 rice panicles at anthesis were dried toonstant weight at 70° C, ground to pass a 40 mesh seive in aWiley Mill,and extracted with 80 percent ethanol in an Omni-nixer at 8000 R.P.M.. A three gram sample of dried tissue was)lended three times for 15 minutes each in 50, 40 and 30 mljortions of ethanol in a stainless steel cup (nominal volume 50ml) at room temperature. The supernatant from each extractionwas decanted and filtered through Whatman No. 1 filter papernd the filtrates were combined.2. Desalting.(The combined filtrates were desalted with a cation ex-ange resin according to the procedure of Plaisted (6) in which>0 ml of filtrate was passed through a .9x3-5 cm columnesearch Assistant, Department of Plant Pathology.Farticipant-Undergraduate Research Participation Program in Plant Patho-logy-NSF 21692.31Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8Published by Arkansas Academy of Science, 196432Arkansas Academy of Science Proceedingsof acidic Dowex 50 W x 8(100-200) bedded in 80 percentethanol at a flow rate of 3 ml/min. After elution of the aminoacids from the column with ammonium hydroxide, the eluateswere taken to dryness in a Rinco rotary evaporator. The residuewas taken up in 10 percent isopropanol, made up to 10 ml in aglass stoppered volumetric flask, then stored at room temperatureuntil used for spotting papers for chromatography.3. Chromatography.The amino acid solutions were spotted on 1814 x 22^nch sheets of Whatman No. 1 filter paper and were separated>y two dimensional descending chromatography. The solventystems used were phenol: water (4:1) or methanol: ethanol:water: urea (45:45:10:0.5) v:v:v:w in the first direction[22]/2 inch length in the machine direction) and n-butanol:cetic acid: water (4:1:6) (upper layer) in the second direction.A spot, .5 to 1 cm in diameter, containing 100-200 lambda ofhe amino acid mixture was applied to each paper at the appro-mate corner, 3 inches from either edge of the paper with 5 or0 lambda, self-fillingmicropipettes. Ahair drver was used inter-mittently to hasten drying, plastic gloves were used to avoidontamination of the papers with perspiration and a glass platewhich supported the papers during spotting was cleaned withcetone after spotting each paper. The pipettes were cleaned inletergent solution, rinsed five times in de-ionized water andfried with acetone.The spotted papers were folded and developed in an insu-ated Chromatocab in a descending manner. (Fig. 1). Five to0 papers were developed at a time with 75 or 100 ml of theolvent per trough for one or two papers respectively. The tem-perature ranged from 23 to 28° C during the course of this workwith temperature variation during one development not exceed-ng 1° C. Time for development ranged from 15 to 21 hoursSpending upon solvent, number of papers, temperature, lengthof paper, etc. Development was begun without an equilibrationperiod and solvents were permitted to travel to within Vi inchof the bottom of the paper before being removed and dried.The folded edge was removed at the antisiphon rod line andhe paper was similarly refolded along the edge perpendicular tohe machine direction prior to development in the second sol-vent.!Thepapers were dried in a forced air oven at 30° C. Paperseveloped in phenol were dried for 8 hours before developing in:>e second solvent whereas papers developed in the other two)lvents were dried for 2 hours. After completion of dryingfter the second development, the papers were sprayed or dippedlninhydrin and heated for color development.32Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8https://scholarworks.uark.edu/jaas/vol18/iss1/833Ninhydrin Positive Components in Rice PaniclesANTI-SIPHON RODORIGINSOLVENT TROUGHANCHOR RODfigure 1. Method of marking and foldingpaper for two-dimen-onal descending paper chromatography.33Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8Published by Arkansas Academy of Science, 196434Arkansas Academy of Science ProceedingsThe phenol solvent was prepared immediately before use>y shaking together 4 parts liquified phenol (Merck 88%) andpart de-ionized water in a separatory funnel until completeolution was affected. One liter of this solvent was used repeat-dly in the bottom of the chamber together with a small con-ainer of .5 g sodium cyanide in 15 ml water to reduce break-lown of phenol during development in this solvent.Separate Chromatocabs were used for each solvent systemwhen practical, or cabinets were thoroughly cleaned with deter-gent and air dried between different solvent developments.I The methanol: ethanol: water: urea solvent was preparedy mixing 45 parts absolute methanol, 45 parts absolute etha-ol, 10 parts de-ionized water and 0.5 grams urea.The n-butanol: acetic acid: water solvent was prepared im-mediately before use by shaking together 4 parts n-butanol, 1>art glacial acetic acid and 6 parts de-ionized water. Two phaseswere allowed to form and 100 cc of the lower phase (water) wasplaced in the bottom of the Chromatocab. The upper phase(butanol) was used as the mobile phase for development of thehromatograms.After completion of the second development and drying,he papers were either sprayed with 0.2 percent ninhydrin in aolution of n-butanol: 2 Nacetic acid (19:1) using an all glassatomizer then heated at 80°C in the drying oven for 15 minutes.or dipped in 0.2 percent ninhydrin in 95 percent ethanol andseated at 65-70° C for 30 minutes in an atmosphere madenerobic with CO2 bubbling through ethanol. The center ofach spot was marked and the Rf value for each spot was cal-ulated in both solvent systems as follows: Rf equalsdistance travelled by spotdistance travelled by solvent front.Identification of the amino acids was accomplished by corn-ering Rf values of pure amino acids (Nutritional BiochemicalIo., Cleveland, Ohio) in the same solvent systems and by co-hromatography i.e. adding known amino acid solutions to thepot containing solutions from tissue extracts and chromato-raphing. In some cases color differences aided in identification.IThe chromatograms were preserved by fixing the developeddor withcopper and coating the paper with plastic. The papersere freed of traces of solvent by washing in equal parts of pet-leum ether: acetone (v:v) before treating with ninhydrin.fter the ninhydrin treatment and color development the driediromatogram was dipped in dilute copper nitrate (1 ml ofturated aqueous Cu (NO3) 2 plus 0.2 ml of 10% v/v HNO3luted to 100 ml with ethanol) and exposed momentarily to34Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8https://scholarworks.uark.edu/jaas/vol18/iss1/835Ninhydrin Positive Components in Rice Paniclesammonia vapor. The paper was then dried at room temperatureand sprayed with Krylon crystal clear spray coating No. 1303RESULTSTwenty two ninhydrin positive components were separat-ed by two-dimensional paper chromatography of the ethanolicextracts from Bluebonnet 50 rice panicles at anthesis. Eighteenof these were identified by color, co-chromatography and bycomparison of their Rf values in three solvent systems with Rfvalues of pure amino acids chromatographed in the same threesolvent systems. Four spots were not identified and were de-signated unknowns A, B, C, and D. A two-dimensional chro-matogram is shown in Figure 2 with phenol: water in the firstdirection (from right to left) and butanol: acetic acid: water inthe second direction (top to bottom). The number or letter ofeach spot coincides with the number or letter of the amino acidor unknown listed on the chromatogram. This solvent systemdoes not separate isoleucine from leucine, valine from methion-ne, alpha amino butyric acid from gamma amino butyric acidand the phenylalanine spot overlaps the isoleucine-leucine spot.Therefore the identification of valine, gamma amino butyricacid and phenylalaine, and the absence of methionine and alphaamino butyric acid on this chromatogram is based on the sep-aration of these in the methanol: ethanol: water:urea solvent.soleucine and leucine were not separated in any solvent used.The assumption that both are present is based on the fact that50th are ubiquitous in biological material. In addition to theompounds identified in extracts of Bluebonnet 50 rice, met-lionine sulfone, beta alanine, tryptophane and histidine weredentified and two unknowns were found in extracts from otherice varieties.The Rf values for 40 pure amino compounds in three sol-vent systems are given in Table Iand the number of each com-pound corresponds with the numbers used in the chromato-graphic maps inFigures 3 and 4, prepared from this data.DISCUSSION AND SUMMARYThe degree of separation and identification of the aminocids is deemed adequate for the physiological studies envisioned.he majority of the ninhydrin-positive spots were chromato-jraphically identified and those which were not, were in relative-y low concentration. These unknowns might be amino acids>reviously described (3) but not yet readily available from nor-mal commercial sources, undescribed amino compounds, or small>eptides. Unless these unknowns are found to be closely relatedo some disease reaction of rice it would not seem warranted to) through the laborious procedures necessary for unequivocalentification of these compounds. (11)35Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8Published by Arkansas Academy of Science, 196436Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8https://scholarworks.uark.edu/jaas/vol18/iss1/837Ninhydrin Positive Components in Rice PaniclesIThe absence of some of the common protein amino acidsch as cystine, cysteine, methionine, tryptophane is not inter-cted as an absence of these amino acids in rice tissue but a;k of their occurrence in detectable quantities using these pro-jures. In fact, the presence of methionine is strongly suggestedice no precautions were taken to prevent its oxidation in theseocedures. Likewise no precautions were taken to avoid break-iwn of glutamine or asparagine to their respective acids.LITERATURE CITED(BLOCK, R.J., E. L. DURRUM, and G. ZWEIG. 1958.A manual of paper chromatography and paper electro-phoresis. Second Edition. Academic Press. New York 710pp.ICONSDEN, R., A.H. GORDON, and A. J. P. MARTIN.1944. Qualitative analysis of proteins, a partition chroma-tographic method using paper. Biochemical Journal. 38:224-232.IGREENSTEIN, J. P. and M. WINITZ. 1961. Chemistryof the amino acids. Vol. 1. John Wiley and Sons, Inc. N.Y.pp. 25-36.IHEFTMAN, ERICH. 1961. Chromatograhpy. ReinholdPublishing Corporation. New York.(LAWRENCE, J.M., T. U.MILLER, and L. W. RAS-MUSSEN. 1960. Estimation of amino acids in plant tissueby paper chromatography. Technical Bulletin 32, Wash-ington Agriculture Experiment Stations.IPLAISTED, P. H. 1958. Clearing free amino acid solu-tions of plant extracts for paper chromatography. Contri-butions from Boyce Thompson Institute 19 (3):231-244.ISMITH, IVOR. 1960. Chromatographic and eletrophore-tic techniques. Volume 1, Second Edition. Interscience Pub-lishers, Inc. N. Y. 617 pp.ITHOMPSON, J. P., S. I.HONDA,G. E. HUNT, R. M.KRUPKA, C. J. MORRIS, L. E. POWELL, Jr., O. O.SILBERSTEIN, G. H. N. TOWERS, AND R. M.ZACHARIUS. 1959. Partition chromatography and itsuse in the plant sciences. The Botanical Review 25 :1-263.¦ THOMPSON, J. F., C. J. MORRIS and ROSE K. GER-ING. 1959. Purification of plant amino acids for paperchromatography. Anal. Chem. 31: 1028-1030.I. THOMPSON, J. F. and C. J. MORRIS. 1959. Deter-mination of amino acids from plants by paper chromato-graphy. Anal. Chem. 31: 1031-1037.I- Winitz,M. 1962. Identification of the elusive amino acid.InAmino Acid Pools. J. T. Holden Ed. Elscvier Publish-ing Co. N. Y.pp. 5-23.37Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8Published by Arkansas Academy of Science, 196438Arkansas Academy of Science ProceedingsTABLE 1Rf. values of pure amino acids in three solvent systemsAmino Rf values inSpotacid Phenol* n-butanol 2 MethanolNumber1 Cysteic acid •!1 .07Aspartic acid .19 .172*Cysteinc -25 .053Cystine .25 .064bbOrnithine .29 .09Djenkolic acid .32 .08.247*8*9*Glutamic acid .30 .23Lysine -38 .12 .12Serine .3 7 .18Dihydroxyphenylalanine .35 .271011* Asparagine 40 .13Glycine .40 .1812*Homocystine .40 .15K Homocysteine .40 .1715* Arginine .44 .14Threonine .49 .2316*17* Histidine .51 .1218* Glutamine .57 .16Alanine -57 .2819*Ethanolamine .55 .3120El*rBeta alanine -61 .30Tyrosine .65 .43Citrulline -61 .18ir Methionine sulfone .60 .22Hydroxyproline .64 .20Methionine sulfoxide .76 .18\r .24Sarcosine .7628 Alpha amino butyric acid .75Gamma amino butyric acid .75.37.38I29* Valine .77 .4830*31 Methionine -77 .4832* Tryptophanc .77 .55Phenylalanine -82 .6233*34* Leucine .79 .6435* Isoleucine .79 .64Diiodotyrosine .79 .7536Ethionine .80 .31(7-A7-B8*Ethionine .62Proline .83 .31 .56Pipecolic acid .89 .41 .7139*Beta amino butyric acid .95 .7940 .28IUnknown .25 .16Unknown .58 .18Unknown .39 .20Unknown .39 .24s Unknown .29 .13Unknown .3238Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8https://scholarworks.uark.edu/jaas/vol18/iss1/839Ninhydrin Positive Components in Rice Panicles(Table 1 continued)1—phenol :H«O (4:1)2 n-butanol :acetic acid :H^O (4:1:6)3—Methanol :ethanol : H»O :Urea (45 :45 :10 :0.5)?present in rice panicles39Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8Published by Arkansas Academy of Science, 196440Arkansas Academy of Science Proceedings40Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8https://scholarworks.uark.edu/jaas/vol18/iss1/8

Identification of Ninhydrin Positive Components in Ethanolic Extracts of Rice Panicles by Paper Chromatography

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 18 Article 81964Identification of Ninhydrin Positive Componentsin Ethanolic Extracts of Rice Panicles by PaperChromatographyC. I. GrableUniversity of Arkansas, FayettevilleJ. E. PresleyUniversity of Arkansas, FayettevilleGeorge E. TempletonUniversity of Arkansas, FayettevilleFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Plant Biology Commons, and the Plant Pathology CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academyof Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu, ccmiddle@uark.edu.Recommended CitationGrable, C. I.; Presley, J. E.; and Templeton, George E. (1964) "Identification of Ninhydrin Positive Components in Ethanolic Extractsof Rice Panicles by Paper Chromatography," Journal of the Arkansas Academy of Science: Vol. 18 , Article 8.Available at: http://scholarworks.uark.edu/jaas/vol18/iss1/831Arkansas Academy of ocicnce Frocecdmgs, v ol. io, 1904IDENTIFICATION OF NINHYDRIN POSITIVECOMPONENTS INETHANOLIC EXTRACTSOF RICE PANICLES BY PAPER CHROMATOGRAPHYC. I.Grable 1, J. E. Presley 2, and G. E. TempletonUniversity of ArkansasThe readily extractable amino acids and amides in ricepanicle tissue are being studied to determine their role in thenechanism of physiologic resistance to the kernel smut patho-gen, Tilletia barclayana (Bref.) Sacc. and Syd. Paper chromato-jraphy, because it is rapid and reliable, is ideally suited for ex-amining amino acid pools from tissue whose resistance has beenmodified by manipulation of the environment or for followingchanges in pool composition during growth, either preceding,during, or after infection by this localized parasite in a resistantor susceptible host.Since the origin of modern paper chromatography by Cons-len, Gordon and Martin (2), multitudinous modifications foreparation and identification of amino acids have been reported'1, 4, 5, 7, 8, 10). This report gives the method selected fromhese many modifications which has been found to be best suitedn our laboratory for extraction, desalting, separation and identi-ication of the 80 percent ethanol soluble, ninhydrin positiveomponents of rice panicles at anthesis by two dimensional des-ending paper chromatography.MATERIALS AND METHODS1. Extraction.The Bluebonnet 50 rice panicles at anthesis were dried toonstant weight at 70° C, ground to pass a 40 mesh seive in aWiley Mill,and extracted with 80 percent ethanol in an Omni-nixer at 8000 R.P.M.. A three gram sample of dried tissue was)lended three times for 15 minutes each in 50, 40 and 30 mljortions of ethanol in a stainless steel cup (nominal volume 50ml) at room temperature. The supernatant from each extractionwas decanted and filtered through Whatman No. 1 filter papernd the filtrates were combined.2. Desalting.(The combined filtrates were desalted with a cation ex-ange resin according to the procedure of Plaisted (6) in which>0 ml of filtrate was passed through a .9x3-5 cm columnesearch Assistant, Department of Plant Pathology.Farticipant-Undergraduate Research Participation Program in Plant Patho-logy-NSF 21692.31Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8Published by Arkansas Academy of Science, 196432Arkansas Academy of Science Proceedingsof acidic Dowex 50 W x 8(100-200) bedded in 80 percentethanol at a flow rate of 3 ml/min. After elution of the aminoacids from the column with ammonium hydroxide, the eluateswere taken to dryness in a Rinco rotary evaporator. The residuewas taken up in 10 percent isopropanol, made up to 10 ml in aglass stoppered volumetric flask, then stored at room temperatureuntil used for spotting papers for chromatography.3. Chromatography.The amino acid solutions were spotted on 1814 x 22^nch sheets of Whatman No. 1 filter paper and were separated>y two dimensional descending chromatography. The solventystems used were phenol: water (4:1) or methanol: ethanol:water: urea (45:45:10:0.5) v:v:v:w in the first direction[22]/2 inch length in the machine direction) and n-butanol:cetic acid: water (4:1:6) (upper layer) in the second direction.A spot, .5 to 1 cm in diameter, containing 100-200 lambda ofhe amino acid mixture was applied to each paper at the appro-mate corner, 3 inches from either edge of the paper with 5 or0 lambda, self-fillingmicropipettes. Ahair drver was used inter-mittently to hasten drying, plastic gloves were used to avoidontamination of the papers with perspiration and a glass platewhich supported the papers during spotting was cleaned withcetone after spotting each paper. The pipettes were cleaned inletergent solution, rinsed five times in de-ionized water andfried with acetone.The spotted papers were folded and developed in an insu-ated Chromatocab in a descending manner. (Fig. 1). Five to0 papers were developed at a time with 75 or 100 ml of theolvent per trough for one or two papers respectively. The tem-perature ranged from 23 to 28° C during the course of this workwith temperature variation during one development not exceed-ng 1° C. Time for development ranged from 15 to 21 hoursSpending upon solvent, number of papers, temperature, lengthof paper, etc. Development was begun without an equilibrationperiod and solvents were permitted to travel to within Vi inchof the bottom of the paper before being removed and dried.The folded edge was removed at the antisiphon rod line andhe paper was similarly refolded along the edge perpendicular tohe machine direction prior to development in the second sol-vent.!The papers were dried in a forced air oven at 30° C. Paperseveloped in phenol were dried for 8 hours before developing in:>e second solvent whereas papers developed in the other two)lvents were dried for 2 hours. After completion of dryingfter the second development, the papers were sprayed or dippedlninhydrin and heated for color development. 32Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8http://scholarworks.uark.edu/jaas/vol18/iss1/833Ninhydrin Positive Components in Rice PaniclesANTI-SIPHON RODORIGINSOLVENT TROUGHANCHOR RODfigure 1. Method of marking and foldingpaper for two-dimen-onal descending paper chromatography.33Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8Published by Arkansas Academy of Science, 196434Arkansas Academy of Science ProceedingsThe phenol solvent was prepared immediately before use>y shaking together 4 parts liquified phenol (Merck 88%) andpart de-ionized water in a separatory funnel until completeolution was affected. One liter of this solvent was used repeat-dly in the bottom of the chamber together with a small con-ainer of .5 g sodium cyanide in 15 ml water to reduce break-lown of phenol during development in this solvent.Separate Chromatocabs were used for each solvent systemwhen practical, or cabinets were thoroughly cleaned with deter-gent and air dried between different solvent developments.I The methanol: ethanol: water: urea solvent was preparedy mixing 45 parts absolute methanol, 45 parts absolute etha-ol, 10 parts de-ionized water and 0.5 grams urea.The n-butanol: acetic acid: water solvent was prepared im-mediately before use by shaking together 4 parts n-butanol, 1>art glacial acetic acid and 6 parts de-ionized water. Two phaseswere allowed to form and 100 cc of the lower phase (water) wasplaced in the bottom of the Chromatocab. The upper phase(butanol) was used as the mobile phase for development of thehromatograms.After completion of the second development and drying,he papers were either sprayed with 0.2 percent ninhydrin in aolution of n-butanol: 2 Nacetic acid (19:1) using an all glassatomizer then heated at 80°C in the drying oven for 15 minutes.or dipped in 0.2 percent ninhydrin in 95 percent ethanol andseated at 65-70° C for 30 minutes in an atmosphere madenerobic with CO2 bubbling through ethanol. The center ofach spot was marked and the Rf value for each spot was cal-ulated in both solvent systems as follows: Rf equalsdistance travelled by spotdistance travelled by solvent front.Identification of the amino acids was accomplished by corn-ering Rf values of pure amino acids (Nutritional BiochemicalIo., Cleveland, Ohio) in the same solvent systems and by co-hromatography i.e. adding known amino acid solutions to thepot containing solutions from tissue extracts and chromato-raphing. In some cases color differences aided in identification.IThe chromatograms were preserved by fixing the developeddor withcopper and coating the paper with plastic. The papersere freed of traces of solvent by washing in equal parts of pet-leum ether: acetone (v:v) before treating with ninhydrin.fter the ninhydrin treatment and color development the driediromatogram was dipped in dilute copper nitrate (1 ml ofturated aqueous Cu (NO3) 2 plus 0.2 ml of 10% v/v HNO3luted to 100 ml with ethanol) and exposed momentarily to34Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8http://scholarworks.uark.edu/jaas/vol18/iss1/835Ninhydrin Positive Components in Rice Paniclesammonia vapor. The paper was then dried at room temperatureand sprayed with Krylon crystal clear spray coating No. 1303RESULTSTwenty two ninhydrin positive components were separat-ed by two-dimensional paper chromatography of the ethanolicextracts from Bluebonnet 50 rice panicles at anthesis. Eighteenof these were identified by color, co-chromatography and bycomparison of their Rf values in three solvent systems with Rfvalues of pure amino acids chromatographed in the same threesolvent systems. Four spots were not identified and were de-signated unknowns A, B, C, and D. A two-dimensional chro-matogram is shown in Figure 2 with phenol: water in the firstdirection (from right to left) and butanol: acetic acid: water inthe second direction (top to bottom). The number or letter ofeach spot coincides with the number or letter of the amino acidor unknown listed on the chromatogram. This solvent systemdoes not separate isoleucine from leucine, valine from methion-ne, alpha amino butyric acid from gamma amino butyric acidand the phenylalanine spot overlaps the isoleucine-leucine spot.Therefore the identification of valine, gamma amino butyricacid and phenylalaine, and the absence of methionine and alphaamino butyric acid on this chromatogram is based on the sep-aration of these in the methanol: ethanol: water:urea solvent.soleucine and leucine were not separated in any solvent used.The assumption that both are present is based on the fact that50th are ubiquitous in biological material. In addition to theompounds identified in extracts of Bluebonnet 50 rice, met-lionine sulfone, beta alanine, tryptophane and histidine weredentified and two unknowns were found in extracts from otherice varieties.The Rf values for 40 pure amino compounds in three sol-vent systems are given in Table Iand the number of each com-pound corresponds with the numbers used in the chromato-graphic maps inFigures 3 and 4, prepared from this data.DISCUSSION AND SUMMARYThe degree of separation and identification of the aminocids is deemed adequate for the physiological studies envisioned.he majority of the ninhydrin-positive spots were chromato-jraphically identified and those which were not, were in relative-y low concentration. These unknowns might be amino acids>reviously described (3) but not yet readily available from nor-mal commercial sources, undescribed amino compounds, or small>eptides. Unless these unknowns are found to be closely relatedo some disease reaction of rice it would not seem warranted to) through the laborious procedures necessary for unequivocalentification of these compounds. (11)35Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8Published by Arkansas Academy of Science, 196436Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8http://scholarworks.uark.edu/jaas/vol18/iss1/837Ninhydrin Positive Components in Rice PaniclesIThe absence of some of the common protein amino acidsch as cystine, cysteine, methionine, tryptophane is not inter-cted as an absence of these amino acids in rice tissue but a;k of their occurrence in detectable quantities using these pro-jures. In fact, the presence of methionine is strongly suggestedice no precautions were taken to prevent its oxidation in theseocedures. Likewise no precautions were taken to avoid break-iwn of glutamine or asparagine to their respective acids.LITERATURE CITED(BLOCK, R. J., E. L. DURRUM, and G. ZWEIG. 1958.A manual of paper chromatography and paper electro-phoresis. Second Edition. Academic Press. New York 710pp.ICONSDEN, R., A.H. GORDON, and A. J. P. MARTIN.1944. Qualitative analysis of proteins, a partition chroma-tographic method using paper. Biochemical Journal. 38:224-232.IGREENSTEIN, J. P. and M. WINITZ. 1961. Chemistryof the amino acids. Vol. 1. John Wiley and Sons, Inc. N.Y.pp. 25-36.IHEFTMAN, ERICH. 1961. Chromatograhpy. ReinholdPublishing Corporation. New York.(LAWRENCE, J. M., T. U.MILLER, and L. W. RAS-MUSSEN. 1960. Estimation of amino acids in plant tissueby paper chromatography. Technical Bulletin 32, Wash-ington Agriculture Experiment Stations.IPLAISTED, P. H. 1958. Clearing free amino acid solu-tions of plant extracts for paper chromatography. Contri-butions from Boyce Thompson Institute 19 (3):231-244.ISMITH, IVOR. 1960. Chromatographic and eletrophore-tic techniques. Volume 1, Second Edition. Interscience Pub-lishers, Inc. N. Y. 617 pp.ITHOMPSON, J. P., S. I.HONDA,G. E. HUNT, R. M.KRUPKA, C. J. MORRIS, L. E. POWELL, Jr., O. O.SILBERSTEIN, G. H. N. TOWERS, AND R. M.ZACHARIUS. 1959. Partition chromatography and itsuse in the plant sciences. The Botanical Review 25 :1-263.¦ THOMPSON, J. F., C. J. MORRIS and ROSE K. GER-ING. 1959. Purification of plant amino acids for paperchromatography. Anal. Chem. 31: 1028-1030.I. THOMPSON, J. F. and C. J. MORRIS. 1959. Deter-mination of amino acids from plants by paper chromato-graphy. Anal. Chem. 31: 1031-1037.I- Winitz,M. 1962. Identification of the elusive amino acid.InAmino Acid Pools. J. T. Holden Ed. Elscvier Publish-ing Co. N. Y.pp. 5-23.37Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8Published by Arkansas Academy of Science, 196438Arkansas Academy of Science ProceedingsTABLE 1Rf. values of pure amino acids in three solvent systemsAmino Rf values inSpotacid Phenol* n-butanol 2 MethanolNumber1 Cysteic acid •!1 .07Aspartic acid .19 .172*Cysteinc-25 .053Cystine .25 .064bbOrnithine .29 .09Djenkolic acid .32 .08.247*8*9*Glutamic acid .30 .23Lysine-38 .12 .12Serine .3 7 .18Dihydroxyphenylalanine .35 .271011* Asparagine 40 .13Glycine .40 .1812*Homocystine .40 .15K Homocysteine .40 .1715* Arginine .44 .14Threonine .49 .2316*17* Histidine .51 .1218* Glutamine .57 .16Alanine -57 .2819*Ethanolamine .55 .3120El*r Beta alanine -61 .30Tyrosine .65 .43Citrulline -61 .18ir Methionine sulfone .60 .22Hydroxyproline .64 .20Methionine sulfoxide .76 .18\r .24Sarcosine .7628 Alpha amino butyric acid .75Gamma amino butyric acid .75.37.38 I29* Valine .77 .4830*31 Methionine -77 .4832* Tryptophanc .77 .55Phenylalanine -82 .6233*34* Leucine .79 .6435* Isoleucine .79 .64Diiodotyrosine .79 .7536Ethionine .80 .31(7-A7-B8*Ethionine .62Proline .83 .31 .56Pipecolic acid .89 .41 .7139*Beta amino butyric acid .95 .7940 .28I Unknown .25 .16Unknown .58 .18Unknown .39 .20Unknown .39 .24s Unknown .29 .13Unknown .3238Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8http://scholarworks.uark.edu/jaas/vol18/iss1/839Ninhydrin Positive Components in Rice Panicles(Table 1 continued)1—phenol :H«O (4:1)2 n-butanol :acetic acid :H^O (4:1:6)3—Methanol :ethanol : H»O :Urea (45 :45 :10 :0.5)?present in rice panicles39Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8Published by Arkansas Academy of Science, 196440Arkansas Academy of Science Proceedings40Journal of the Arkansas Academy of Science, Vol. 18 [1964], Art. 8http://scholarworks.uark.edu/jaas/vol18/iss1/8

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Identification of Ninhydrin Positive Components in Ethanolic Extracts of Rice Panicles by Paper Chromatography

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