.‘1. ... . - l.,$ Q0... . . 33 ‘..O a l.‘ ’0. k u. I "five ... .u ..x. . .H .. .. Y fl? . .. S ,H . nfioa .I n a a L . . . a «\u s v y 0 I \. . oglu_~~ 1‘.“ K 5“ 2:) .... a . r. . . t .n . n .. .. I‘ V .u .“ W“ 5 fl“ 0 .K, 1.. . .vw b I V 1': v .P 2: ‘.‘ b ’ ‘s; . . :L‘ga: '2 ». .323: v v ~v h: b 3. . .' * O y Kc . “I’m ”8 J ... . H rub» Hid» “on. I _ S. a Q.“ s . .Q q I A Q ..h . ._ h ,9. ...3 . !.\.. fisk um . ...G .99.\ .3 . - ..~ 1. s s . v o. 01 d ‘ do 1"“; o: o . I k r” K 5.1% . .u. - \ lg .13 .. a... .. . > .0 O g1.-\ mll‘ . ”I“. O ...- .l. .— o a x. h o? . u ..., a .. o . ... ..«H 3 I.” “I. c“ ..aa ..\ I . rt. .- a ’Q A c .R .. .....w. ...... H .. m 0.. o b o- o O I d .4. o -4 5 ..-... z. u _ ..u A. ...... A g .- 1...“ a . I‘.‘ I“ J. . . o .. 2? . T _:_‘_:_:__:_E:______:__:__:__ MICHIGAN STATE UNIVERSITY EAST LANSING, MICHIGAN THESIS L [B R A R Y Michigan State A) University TEE RELATICRCHIP OF FOLIC ACID COfiPOUHDS T0 OPE-ChRBOH EETAEOLISE IN THE TOBICCO PLANT By Gail Denise Brown A THESIS Submitted to the College of Science and Arts, Kichigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1958 ACKNOWLEDGMENT The author would like to express her appreciation to Dr. R.U. Byerrun for his interest, guidance, and cooperation which have helped a great deal in the progress of the present study. Acknowledgment is also due to the flichigan State University Department of Chemistry and the National Institutes of Health for funds provided in support of this work. eeeeeeeeee eeee es ii VITA The writer was born in Detroit, Michigan in 1934 and received her secondary education at Busch High School in Center Line, Michigan. In 1955 she received a Bachelor of Science degree from Michigan College of Mining and Tech- nology with a major in medical technology. Since that time she has worked in the clinical chem- istry department of E.W. Sparrow Hospital in Lansing, Michigan and spent one year as a Graduate Teaching Assistant at Michigan State University. During summer and tall terms of 1957 she was a Special Graduate Research Assistant under the auspices of the National Institutes of Health. iii THE RELATIONSHIP OF FOLIC ACID COMPOUNDS TO ONE-CARBON METABOLISM IN THE TOBACCO PLANT BY Gail Denise Brown AN ABSTRACT Submitted to the College of Science and Arts, Michigan State University of Agriculture and Applied Science in partial fulfillment fo the requirements for the degree of MASTER OF SCIENCE Department of Chemistry Year 1958 **§ (- APPPOVOC Z: . 1/ v/L’)?j-cxlz_~iucc1w~ iv ABSTRACT The present study was undertaken in an attempt to elucidate the role of "folic acid” compounds in one-carbon metabolism of higher plants. 01‘ was administered to tobacco plants Formaldehyde- both hydroponically and directly through the stem. The plants were allowed to grow for a period of one or four days. Fluorescent materials which possessed radioactivity and resembled 'folic acid" compounds in ultraviolet absorption spectra and paper chromatographic characteristics were isolated from the plants. In a further study a mixture of tetrahydropteroyl- glutamic acid and formaldehyde-C14 was administered to isolated leaves of a tobacco plant by vacuum infiltration. After allowing the leaves to metabolize in the presence of light for 10.5 hours a single fluorescent material which possessed radioactivity was recovered from the leaves. It was found to be identical in ultraviolet absorption spectrum and paper chromatographic characteristics with a fluorescent material which was a component of the administered mixture. TABLE OF CONTESTS IE‘ITRODUCTION ......O'.........OOOOOOOOOC......0.... EXPEBIKENTATIQN AND RESULTS eeeeeeeeeeeeeeeeeeeeeee Preparation of Formaldehyde-Cl4 Solution ....... Uptake Studies ............OOOOOOOOOOOOOOOO..... Isolation and Identification of "Folic Acid" Compounds eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 9 ISOlation 0.0.0.0..........QOOOOOOOOOOOOOOOOC 9 Paper Chromatography of Reference Compounds . lO Chromatography and Counting of Plant Extracts 16 Ultraviolet Absorption Spectra .............. 18 Leaf Infiltration Experiment‘ ......OOOCOOOOOOOC 20 l 5 Preparation of Tobacco Plants .................. 2 8 DISCUSSION ......COOOOOOCOOO...OOOCOOOOOOOOOOCOOOOO 22 SURTAZFAIRY COO-......OOOOOOOOOO......OOOIOOOOICOOOOOIOO 29 REFEREFICES .0 O. O O O O O O O O O O O O O O O 0 0 O O O O. O O O O O O O O O O. .0 0 30 APPENDIX .....IOOOIOOOOOOOOOOOOO......OOOOOOOOOI... 33 INTRODUCT ION INTRODUCTION The nature of the "active one-carbon unit" which is utilised in many biosynthetic pathways in plants and animals has been under intensive investigation in the last ten years. Workers have concluded that the compound or com- pounds in question are derivatives of the “folic acid" group of bacterial growth factors. These derivatives are found in many tissues, and are especially abundant in green leafy plants, liver, and yeast. ”Active Cl” has been implicated as a cofactor in the biosynthesis of purines (1,2), in serine-glycine intercon- version (3). 1n the metabolism of glutamic acid, glycine, and methionine (4,5,6) and in synthesis of the thymine methyl group (7,8). The exact chemical identity of the active cofactor is still unknown. Research has been complicated by the fact that a multiplicity of related compounds can function as growth factors for Lactgbacillus casei, Streptococcus ca 1 , and Leuconogtog gitrovorum (Pedigcgccug gerevisiag). All of the 'folic acid"compounds contain the pteroylglutamic acid nucleus. Differences lie in the state of oxidation of the pyrasine ring, the number of glutamic acid residues present, and the one-carbon constituent at the N5 or N10 position. Further complication arises as the compounds ean he letabolically interconverted through a series of ATP and DPN dependent reactions (3,8,15,17) and can also be altered 1 in isolation procedures (9,10,11). ‘ Host recent investigations have demonstrated that der- ivatives of 5,6,7,8-tetrahydropteroylglutamic acid (tetra- hydrofolic acid, FH4) are carriers of the one-carbon unit. these are the 5-formyl derivative (citrovorum factor, leuco- vorin, rsrai)(12), the lO-formyl derivative (floFH‘) and the 5- or lO-hydroxymethyl compounds (hSFH4 and hloFH4 respect- ively)(3,6,9,14-16). The formyl derivatives which are at the oxidation level of formate are required for the biosyn- thesis of the purine ureide carbon, whereas the hydroxymethyl derivatives which are at the oxidation level of formaldehyde seem to be active in serine-glycine interconversion and the formation of methionine from homocysteine. The above metabolic pathways have been elucidated largely through work with animals and microorganisms. The role of "folic acid” derivatives in plant metabolism has not been extensively studied. Since many.1eafy plants contain appreciable amounts of these compounds, it can be postulated that reactions similar to those in animals can take place. Studies in this laboratory concerned with transmethylation in higher plants have shown that formate-Cl‘ can be incorpor- ated into the methyl group of methionine and the methoxy group of lignin (18) and into the N-methyl group of nicotine in tobacco plants (19,20). Formaldehyde-C14 has been shown to be incorporated to a greater extent into the N-methyl group of nicotine than formate (21). Similar results have been observed in animal tissue, that is, greater incorporation of formaldehyde than formate in certain reactions (3,6,22). this seems consistent with the concept that formate must undergo reduction to be utilised at the hydroxymethyl oxidation level and subsequently in the formation of methyl groups. Zakrsewski and Nichol, using formats-014, demonstrated its invorporation into the S-formyl group of citrovorum factor by cells of E. {gagglig (23). Since formaldehyde is apparently a more direct precursor of methyl groups in tobacco plants than formate, it seemed of interest to attempt to demonstrate its incorporation into "folic acid" compound(s) in the intact plant. The formaldehyde would presumably enter the compound or compounds as an hydroxymethyl group if meta- bolic pathways in higher plants are analogous to those in animals. _ Formaldehyde-C14 was administered to growing tobacco plants and an attempt was made to isolate compounds of the 'folic acid" type which had incorporated the radioactivity of the formaldehyde. Because of difficulties encountered .in isolation and identification of these compounds, no absol- ute proof of the incorporation of formaldehyde-C14 can be put forth at this time. However, results of paper chromat- ographic separation seem to indicate that the radioactivity was taken up by fluorescent compounds with Rf values in the same range as known "folic acid" derivatives. Ultraviolet absorption spectra of the plant compounds also reveal certain similarities to ”folic acid” derivatives. EXPERIMENTAIION AND RESULTS EXPERIMENTATION AND RESULTS Ezeparatiog g; tobaccg Rléflifi It has been demonstrated previously in this laboratory that tobacco plants can absorb organic compounds from a nutrient solution through their root systems (19,21,24la tobacco plants used in this study were of a high nicotine strain, fliggfiiggg zugtigg L., var. humilis. They were chosen for their availability and to extend one-carbon studies which have been done on this particular species. Seeds were planted in flats containing vermiculite (commercial heat expanded mics) and transplanted when seed- lings were two to three weeks old. Plants were watered daily with tap water and twice weekly with a nutrient sol- ution containing 1.0 gm. lgSog°7H20, 1.0 gm. KzflPdg, and 5.8 gm. Ca(N03)204EQO in four liters of tap water. Plants were removed from the flats and vermiculite was washed from the roots with tap water. For hydroponic admin- istration of formaldehyde-C14 the plants were placed in 125 I1. Erlenmeyer flasks containing 50 ml. of a 1:3 dilution ‘ of nutrient solution with the following composition. Water 1 1. Magnesium sulfate 250 mg. Calcium nitrate 1 gm. Ammonium sulfate 250 mg. Potassium chloride 250 mg. Potassium dihydrogen Ferric chloride 2 mg. phosphate 35° .3. One ml. of an aqueous solution containing 0.5 mg. of aureo- mycin was added to each flask to reduce the microbial popu- lation. Five ml. of a solution of formaldehyde-C14 containing approximately 2 mg. of formaldehyde was added to each flask. A cotton plug was placed around the plant stem at the neck of the flask to prevent loss of formaldehyde by volatilisation. Plants were allowed to grow for periods varying from one to four days in a special fume hood to remove any radioactive carbon dioxide which may have formed. During the growing period water was added to the flasks as needed to maintain the 50 ml. volume of solution. The light source consisted of two 36 inch 30 watt fluor- escent tubes and one 100 watt incandescent bulb which were lighted for 12 hours during each 24 hour period. Light intensity was approximately 200-250 foot-candles at the tap of the plants. Administration of formaldehyde-014 directly through the stem of the plant and by vacuum infiltration of leaves will be described later. Egepaggtigg g; {ogmaldggggg-Ql‘ golution Formaldehyde-01‘ solution was prepared by adding radio- active formaldehyde stock to a solution of formaldehyde con- taining approximately 2 mg. in 5 ml. Formaldehyde-C14 was obtained from Volk Radiochemical Company, Chicago. This solution was standardized in triplicate as to radioactivity and formaldehyde content by precipitation with dimedon (5,S-dimethylcyclohexanedione-l,3) according to the method of Ice and Reid (25). A 10 ml. aliquot of formaldehyde solution was added to a solution containing 50 ml. of acetate buffer (pH 4.6) and 10 ml. of 0.8 per cent dimedon in 95 per cent ethanol. the mixture was allowed to stand at room temperature overnight. The fluffy white dimedon derivative was removed by filtration through a tared sintered glass crucible and washed with water slightly acidified with acetic acid. The precipitate was dried at 75?c. for one hour and weighed. multiplication or the weight of the derivative by a factor of 0.1027 gave the weight of formaldehyde in the sample. a sample of the precipitate from each determination was transferred to a tared aluminum planchet and plated for radioactivity measurement. Counting was done on a Nuclear- Chicago scaler, model 192x, with a fracerlab 80-16 internal gas-flow Geiger-Muller tube. Efficiency of the instrument was 40 per cent. Counts were corrected to ”infinite thinness" by use of a self-absorption curve. Three formaldehyde solutions were prepared and standard- ised in this way. Five ml. of solution contained 1.99 lg. of formaldehyde with radioactivity of 1.47x 105 cpm., 1.88 rig. of formaldehyde with radioactivity of 3.00 X 105 cpm. and 6 2.12 mg. of formaldehyde with radioactivity of 1.05 X 10 cpm. Entakg ltugieg Ringler (21) had shown previously that formaldehyde is taken up from nutrient solution by tobacco plants under the conditions which were employed in this experiment, and further, that formaldehyde in the concentration used was not toxic to the plants. He also demonstrated that the uptake of formal- dehyde was true absorption rather than adsorption on the plant roots.‘ Eighty-nine per cent of a 2 mg. dose was taken up by the plants in two days in his experiments. To confirm the uptake results of Ringlar two groups of 1‘ solution. two plants each were given 5 ml. of formaldehyde-C One group was allowed to grow for 24 hours and the other for four days. The remaining nutrient solution was filtered and the flasks washed with distilled water to bring the total volume up to about 30 ml. Three m1. of dimedon solution was added and the pH was adjusted to 4.6 with acetic acid. The mixture was then allowed to stand overnight and the precipitate treated as before. The average uptake of formaldehyde by the two plants was 88 per cent in 24 hours and 96 per cent in four days. The fact that a sample of formaldehyde was quantitatively recovered from a mixture of formaldehyde and nutrient solution indicates that the nutrient solution did not interfere with the analytical procedure. Isolatigg and identification 2;,1ggligwggid" compounds Iaalgtion.l In 1950 Keresztesy and Silverman first reported the isolation and partial purification of a growth factor for L.‘citrovorug from liver extract (26). The iso- lation was effected by making a water extract of the liver, adjusting the pH to 5.5, and adsorbing the active compound on charcoal. The charcoal was eluted with an ethanol-water- sodium hydroxide mixture and the eluate was extracted with water-saturated butanol. The butanol fraction then was washed with water, the aqueous extract acidified and the growth promoting activity precipitated as the silver salt. The factor was further purified by precipitating it as the barium salt and by chromatography of it on an aluminum oxide column. Modifications of this procedure have been used by other investigators to isolate citrovorum factor (27, 28), polyglutamyl-folic acids (27,29), RIO-formylfolic acid (30), land some unidentified compounds with activity for L. gi§§_- 12m and. do £929.21». (31% ' Sauberlich in 1951 (28) reported the isolation of citro- vorum factor from fresh spinach by allowing a water extract of the plant to autolyse for 15 hours, precipitating the proteins with ethanol, and adsorbing the compound on charcoal._ This was followed by elution, extraction with butanol, and psecip- itation of the activity as the barium salt. 10 The procedure used for isolation of ”folic acid” comp- ounds in this study was a modification of the methods of Silverman etial. (30) and Sauberlich. Plants which had been fed formaldehyde-01‘ were removed from the nutrient solution and rinsed with distilled water. Roots were removed and dis- carded; the leaves and stems of the plants (usually two plants) were cut into small pieces with scissors. The plant material was then placed in e Waring blendor containing 100 m1..of an 8 per cent solution of NagHPO4°12H20. The air in the Waring blendor had been displaced with nitrogen to minimise air oxida- tion of reduced "folic acid" compounds. The plant material then was homogenized for 15 seconds, the solidmaterial re- moved by filtration, and the filtrate transferred to an Erlen- meyer flask. Nitrogen was bubbled through the mixture for five minutes. Ten m1. of toluene was added to reduce microbial growth and the mixture was incubated overnight at 37°C. It was assumed that the incubation would allow autolysis of any polyglutamyl compounds to take place. Fifty m1. of 95 per cent ethanol was added to precipitate proteins and the mixture heated to 83-85°C. for five minutes while stirring with a stream of nitrogen. The proteins were removed by filtration. About 2 gm. of Norite A was added to the filtrate to absorb the aromatic organic compounds. The solution was 11 adjusted to pH 6-7 with hydrochloric acid and heated to 35-4000. for 30 minutes. Charcoal was removed by filtration and the filtrate treated again with charcoal. The charcoal cake was eluted two times with a mixture of ethanol-ammonium hydroxide-water (4:133) for 30 minutes at 35-4000. The total volume of eluate was about 200 ml. The tan colored eluate was evaporated to dryness under reduced pressure at room temperature and the resulting tan solid material stored at 6°C. Paper ghromatographz 2; reference compounds. Paper chromto- graphic characteristics of “folic acid" compounds were deter- mined in the following manner. A 45 X 15 cm. glass cylinder was used as a chromatographic chamber for ascending develop- ment of papers. Approximately 50 ml. of solvent was placed in the bottom of the cylinder and allowed to saturate the air in the cylinder by standing at least 24 hours. Sheets of Whatman No. 1 filter paper were cut into 16 3 l4 inch pieces and samples of plant material and reference com- pounds were placed two inches apart one-and-one-half inches from the narrower end of the paper. Samples were applied with a micro pipet in spots about one cm. in diameter and dried between applications by a stream of warm air. The paper was stapled together to form a cylinder in such a manner that the edges of the paper were not in contact with each other. The self-supporting cylinder was placed in 12 the chromatographic chamber and allowed to develop at room temperature for about four hours in the case of the aqueous solvent or 18-20 hours for the organic solvent. Reference compounds used in chromatography were pteroyl- glutamic acid, leucovorin, acid-treated leucovorin, tetrahydro- pteroylglutamic acid, and tetrahydropteroylglutamic acid treated with formaldehyde-014. Pteroylglutamic acid was obtained from General Biochemicals, Incorporated, Chagrin Falls, Ohio. Leucovorin was the gift of Dr. Harry P. Broquist, Lederle Laboratories Division, Amer- ican Cyanamid Company, Pearl River, New York. Acid transformation products of leucovorin were prepared by making a solution of leucovorin in 0.1 M hydrochloric acid and allowing the mixture to stand overnight at room temperature. Treatment of leucovorin with mineral acid has been reported ‘to result in the formation of transformation products which are Ns-Nloeimidazolinium derivatives(32,33). ' Tetrahydropteroylglutamic acid was prepared by hydrogen- ation of pteroylglutamic acid in glacial acetic acid over platinum. The reduction was carried out in a flask attached to a Warburg manometer in a constant temperature water bath at 25°C. The procedure was a modification of that of O'Dell, et a1. (35) which was also used by Blakley (16). Pteroyl- glutamic acid takes up two moles of hydrogen to form the tetra- hydro derivative under theae conditions. Twenty-five mg. of pteroylglutamic acid was placed in the side arm of a 125 ml. Warburg flask; 5 m1. of glacial acetic acid and 13 mg. of platinum oxide were placed in the bottom of the flask. Hydrogen gas was introduced into the system to reduce the catalyst and saturate the acetic acid until the gas pressure as recorded on the manometer remained con~ stant. The pteroylglutamic acid was tipped into the mixture and the reaction was allowed to progress until 80 per cent of the theoretical amount of hydrogen had been taken up. The catalyst and unchanged pteroylglutamic acid were removed by filtration and the filtrate was poured into 50 ml. of ethyl ether.. the tan precipitate which resulted was re- covered by centrifugation and washed three times with 30 m1. volumes of ethyl ether. The product was dried in a desiccator under reduced pressure over potassium hydroxide and stored in the dark. ‘ Tetrahydropteroylglutamic acid prepared as described above was added to an equimolar solution of formaldehyde-014, assuming that the tetrahydro compound was pure. Blakley (16) reported the equimolar condensation of tetrahydropteroylglu- tamic acid with radioactive formaldehyde in zigggp The results of his studies indicated that the compound formed was the S-hydroxymethyl derivative. Since the “folic acid“ group of compounds have character- 13t1¢ absorption 0f ultraviolet light or distinct fluorescence, completed chromatogrsms were examined under an ultraviolet lamp 14 and the fluorescent or absorbent zones were marked with a pencil. ' Following are the Rf values obtained using the refer- ence compounds mentioned above. gompound - Pteroylglutamic acid Leucovorin Acid-treated leucovorin Tetrahydropteroyl- glutamic acid Tetrahydropteroyl- glutamic acid and formaldehyde mixture Solvent EBKW‘ KQHPO4’* EBAW KZHPO4 Shaw K2HPO4 EBAW KZHPO4 EBAW K2HPO4 UV Character A1 Fit ’WW'IJW" yellow F blue F F F F blue F F , F green-blue F blue F ' blue F F yellow F blue F ' blue F P F green-blue F blue F blue F F yellow F blue F blue F F *Ethanol-n—butanol-ammomia-water (50315310325) (29) ’*0.1 I dipotassium hydrogen phosphate (34) tAbsorbant 1r Fluorescent 3.13192 27 50 2o 31 21 32 44 71 1 16 20 0 24 56 73 0 15 21 35 O 21 29 44 74 l 14 22 35 1 21 32 46 76 The R: values reported above are the average of two or more chromatographic runs. A tabulation of R: values of these and other “folic acid" compounds in similar solvents which have been reported in the literature by other workers is shown’in the appendix. In chromatography of the tetrahydro compound and form— aldehyde mixture an area of high radioactivity correSponding to formaldehyde-*014 (Rf 0.60 in EBAK) was detected by use of a chromatographic strip counter (Nuclear-Chicago windowless rate meter coupled with an Esterline-Angus recorder, efficiency approximately 4 per cent). Counting with this instrument indicated that there was a considerable excess of formaldehyde and hence the tetrahydropteroylglutamate preparation was not pure. The detection of four or five fluorescent spots on the chromatograph of the preparation also indicates impurity or reoxidation to the more stable pteroylglutamic acid. Greenberg (l), Blakley (16) and Kisliuk (36) also reported the presence of a number of products upon hydrogenation of pteroylglutamic acid. Tetrahydropteroylglutamic acid and the tetrahydropteroyl- glutamic acid and formaldehyde mixture seem to have identical Rf values by the chromatographic methods employed._ It will be shown later by ultraviolet absorption spectra that at least one of the fluorescent materials of the same Rf value in the two preparations is not the same. Chromatography and counting g; plant extracts. The plant preparations were chromatographed under the same conditions as the reference compounds. A portion of the dry plant extract ‘was resuspended in 200to 500 p1 of distilled water and spotted on the papers. Fluorescent material was detectable on com— pleted chromatograms but resolution was poor in both solvents. In the first experiment two plants were used and were allowed to metabolize the radioactive formaldehyde for 24 hours. The radioactivity of the hydroponically administered formalde- hyde was 1.47 X 105 cpn. per plant. Chromatography of the plant extract in EBAW revealed the presence of fluorescing material in a streak extending approximately 6 cm. No radio- activity in this area could be detected on the chromatographic strip counter. ' 'In order to increase the possible radioactivity of the _Cl4 fluorescent compound(s), a new formaldehyde solution was prepared and standardized. This solution contained 3.00 X 105 cpm. in 5 ml. The new formaldehyde solution was administered to two groups of two plants each. One group was allowed to grow in the presence of formaldehyde for 24 hours and the other was allowed to grow for four days. Chromatographic results were .similar to those of the first experiment. Again no radioactiv- ity was detected in the fluorescing material. In a third experiment two plants were given formaldehyde containing 1.05 X 106 cpm. in 5 ml. and were allowed to grow for four days. The plants were worked up in the usual 1? manner. The plant extract was applied to the paper in a one inch strip at the origin, rather than in a single Spot, to increase the concentration of material on the paper. A number of fluorescent areas were noted on the completed chromatograms which gave reproducible peaks of radioactivity when measured on the chromatographic strip counter. These spots were cut out of the chromatograms and eluted with water directly into tared aluminum planchets for counting with the internal gas-flow Geiger-Muller tube. Elation was performed by stapling a rectangular filter paper wick to one end of the paper containing the material and a pointed wick to the other end. The rectangular wick was dipped into a water reservoir and the water allowed to elute the paper by capillarity. The pointed end of the paper was placed in the aluminum planchet. When approximately 0.3 ml. had been eluted the paper was removed and the liquid in the planchet was evaporated at 30-40°C. under a stream of air. Weight of eluted material in each case was less than one mg. Counts were corrected for background. Since the weight of material was very small no correction for self-absorption was necessary. The following data were obtained in this experiment. 18 2 Solvent fl! Qharacter fir g _Q Con.(mgo EBAE fluorescent 27 29 fluorescent 37 60 fluorescent ' 53 115 absorbent 82 8 K23P04 fluorescent 50 (streaked) 5 fluorescent 80 " 12 In order to get formaldehyde into the plants in a shorter period of time a fourth experiment nas performed. Th3 roots of two plants were removed by cutting diagonally across the stem one-half inch above the roots. The cut end of the stem was placed in a mixture of 20 ml. of diluted nutrient solution and 5 ml. of formaldehyde solution contain- ' ing 1.05 x 105 cpm. After 22 hours the plants both had taken up approximately 22 ml. of the mixture. The plants were worked up in the usual manner and the extract was separated by paper chromatography as was done in the third experiment. .Elution and counting with the internal gas-flow tube gave the following results. Solvent g1 Character 3f §_;_ EBAW fluorescent 3O 29 fluorescent 37 37 fluorescent 45 81 - absorbent $1 11 K2HPO4 fluorescent 50 (streaked) - fluorescent 80 " 8 Ultraviolet absorption spectra. Absorption Spectra of refer- ence compounds and materials isolated from the plants were determined using a Beckman DK-Z recording spectrophotometer. 19 These are shown in the appendix. Since the compounds in question were ultraviolet absorbing or fluorescent the wave length range of 220 to 340 up was chosen. Absorption spectra of leucovorin, pteroylglutamic acid, and tetrahydropteroylglutamic acid were run on diluted stock solutions. For materials which had been eluted into planchets and dried for counting it was found convenient to wash the planchets with approximately two to three ml. of distilled water and determine the spectra of the resulting solutions. In some cases when the material had not been counted (for example, the products of acid-treated leucovorin) the spots from the chromatograms were cut out, placed in two to three ml. of distilled water and allowed to stand at least one hour with occasional agitation to form a solution for the determination of spectral absorption. Because of the limited amount of material eluted from the chromatograms the ultraviolet absorption of the water solutions were determined first. Then the solutions were divided into two equal portions and one volume of approximately 0.2 M sodium hydroxide or hydrochloric acid was added and the ultraviolet absorption spectra were determined again. This method gave spectra in acid or base at one-half the concen- tration of those in water. 20 Leaf infiltration eXperiments In order to determine how the plants metabolize pre- formed "folic acid" compounds a fifth experiment was under— taken. This experiment also served to increase the concen- tration of these compounds in the plants. Since nothing was known about the absorption of such materials through the roots of the plants another method of administration seemed desir- able. Therefore the vacuum infiltration procedure of Kursanov (37) was employed. Three isolated leaves of a tobacco plant which had been weighed previously were immersed in a mixture of 4.0 mg. of tetrahydropteroylglutamic acid preparation and 10 ml. of form~ aldehyde solution containing 4.24 mg. of formaldehyde and 2.10 x 106 cpm. Air was removed from the leaves by reducing the pressure to 25 mm. of mercury. As air was readmitted to the system the air spaces of the leaves were filled with the surrounding liqu1d. The leaves were removed from the liquid, blotted dry, and weighed again. Average absorption of the liquid mixture was 0.3 gm. per leaf. The total absorption was 0.90 gm. or approximately 1.56 X 105 cpm. The leaves were allowed to metabolize in the presence of light for 10.5 hours. They then were worked up in the manner described previously and chromatographed in EBAW and KQHPO4.‘ The following results were obtained. 21 §olvant E! gharacter gr 3 go? 92m,(mg. V EBAW green-blue fluorescent 21 --- blue fluorescent 31 --- K2HP04 fluorescent l (faint)--- fluorescent 75 34 The fluorescent material at Rf 0.75 in KZHPO4 was eluted into an aluminum planchet and counted with the internal gas-flow tube. Since chromatography of the tetrahydropteroyl- glutamic acid and formaldehyde-C14 mixture revealed a fluor- escent area at R, 0.76 in KQHP04, this material was also eluted and counted. Radioactivity was 17 cpn./mg. above back- ground. In order to further identify the material isolated from the plant tne ultraviolet absorption spectra of all materials in the reference compounds which had R: values near 0.75 in KQHPO4 were determined. These were 1) Rf 0.76 material from the tetrahydropteroylglutamic acid and formaldehyde mixture, 2) R: 0.74 material from the tetrahydropteroylglutamic acid preparation, 3) R: 0.56 material from acid-treated leucovorin, and 4) Rf 0.73 material from acid treated leucovorin. Results of these determinations are shown in the appendix. DISCUSSION DISCUSSION The identity of the radioactive fluorescent materials isolated from the plants after administration of formalde- hyde-C14 is difficult to establish from the data presented in this study. Examination of the paper chromatographic data on “folic acid“ compounds that have been presented in the literature reveals certain disparities in the behavior of individual compounds. This is especially evident in the case of the reduced compounds. Zakrzewski and Nichol (38) have shown that the mobility of certain pteroylglutamic acid der- ivatives changes with the pH of the develOping solvent, and also that their mobility increases with decreasing concentra- tion of salts in the solvent. This may account for some of the apparent discrepancies, since the solvent was not the same in all cases. The Bf values reported for dihydro and tetra- hydropteroylglutanic acids seem to indicate some disagreement as to the chromatographic identity of these compounds, however. Since the conditions of isolation and chromatography under which the present study was conducted were not rigor- ously anaerobic, it seems reasonable to expect the more oxygen- sensitive "folic acid“ compounds to be oxidized to more stable forms during these procedures. lay, et a1. (41) have shown that the N10 substituted derivatives are more oxygen-labile than those that are N5 substituted. The work of Cosulich, et a1. (32) indicates that the NS-Nlo-imidazolinium derivative 23 is especially stable to oxygen because of the possibility of resonance of the quaternary nitrogen. This reasoning cannot be strictly applied to the 85-N10-methylene (or hydroxymethyl, as it is sometimes named) bridge compounds since no quaternary nitrogen is involved. Cosulich and Smith (42) synthesised RIO-methyl pteroic and filo-methyl pteroylglutamic acids and showed that Nlo-methyl pteroic acid is stable to aerobic alkaline oxidation. a comparison and correlation of the ultraviolet absorp- tion spectra and Br values of the compounds isolated from the plants and those of reference compounds in the light of the above considerations reveals similarities which are as follows. Experiment two. No definite Rf data was obtained, but the absorption spectra in acid, alkali, and distilled water (Appendix II, Figure 6), show an absorption maximum at approx- imately 26? up with little change in acid or alkali. None of the reference compounds which were investigated show this type of behavior in ultraviolet light. may, et a1. (41) reported the absorption Spectrum of RIO-formylpteroylglutamic acid in 0.1 I sodium hydroxide solution in which a characteristic absorption maximum at 260 mp was observed. Since it is not known whether the material that was isolated from the plants possessed any radioactivity or whether the compound was chromatographically pure, it seems inadvisable to attempt to draw any conclusions as to its 24 identity. Experiment three. It is unfortunate that absorption spectra in acid and alkali were not determined on all of the fluor- escent materials isolated from the plants in this experiment. It is difficult to make any comparisons without knowledge of their behavior in these solvents. The fact that four fluor- escent materials were separated by EBAW solvent whereas only two were obtained in KZHPO‘ may be rationalized when one considers that EBAW is a basic solvent containing 10 per cent .ammonium hydroxide. May, at al.($l) showed that interconver- sions of leucovorin and its derivatives may be brought about under the influence of alkali. The Ns-Nlo-imidazolinium tetra- hydro compound can be converted to filo-formyltetrahydroptaroyl- glutamic acid or leucovorin under appropriate conditions of high pH. Further aerobic oxidation of the RIO-formyltetra- hydro compound gives Nlo-formylpteroylglutamic acid. The RIO-formyl group may also be hydrolyzed by dilute alkali. If an analogy can be drawn in the case of the N5-N10- methylene compounds it may be postulated that such transform- ations could have occurred in EBAW solvent during chromatography. The absorption spectra of the material which exhibited an Rf of 0.80 in KZHPO4 (Appendix II, Figure 11) shows a distinct absorption maximum at 305 mpuin 0.1 M sodium hydroxide. A compound which was separated from acid treated leucovorin (Appendix 115 Figure 14) shows an absorption maximum at this 25 wave length, although the curve is not as sharp, and it also has maxima at 287 and 234 ups. Cosulich, et al. (32) have reported the absorption spectra in 0.1 M hydrochloric acid of N5-N10- imidasolinium tetrahydropteroylglutamic acid, anhydroleucovorin A, and anhydroleucovorin B, all of which are Rs-Nlo-methenyl derivatives of tetrahydropteroylglutamic acid. They maybe interconverted at various pH values from 1,3 to 4.0. All three compounds exhibit absorption minima near 300 my in 0.1 a hydrochloric acid. inhydroleucovorin B shows a maximum at 304 up. The spectrum of 5-formyl-lo- methyltetrahydropteroylglutamic acid also shows absorption at 310 mp(32). It is tempting to postulate that the compound isolated from the plants is an NS-Nlo-methylene bridge com- pound, which would be identical to neither the NS-Nlooimidaso- linium or the methyl derivatives, but resemble them in ultra- violet absorption. The B: of the NS-Hlo-methylene bridge compound as reported by Osborn is 0.25 in 0.1 M phosphate buffer, pH 8.0. The absorption spectra of the compounds isolated from acid treated leucovorin at B 0.56 and 0.76 in K2HP0‘ (Appendix II, Figures f 14 and 15) appear to be very similar, although their R values f are quite different. This variation in at of apparently simi- lar compounds may lend some support to thehypothesis that the compound isolated from the plants may be an HS-Nlo-methylene derivative, even though its Rf is 0.80. 26 Experiment four. The results of experiment four are for the most part similar to those of experiment three. It may be noted however that the radioactivity of the fluorescent com- pounds isolated fron the plants is lower than that in experi- ment three. This is not without explanation, since the plants in experiment four were only allowed to metabolize the form- aldehyde-C14 for one day whereas those in experiment three were grown for four days in the presence of formaldehyde-01‘. Experimant five. Thefluorescent material isolated from the leaves which had been infiltrated with a formaldehyde-C14 and tetrahydropteroylglutamic acid mixture (Rf 0.75 in K2HP04) is not the same as that isolated from the formaldehyde-C14 fed plants, although the R value is very nearly the same. The absorption Spectra in :cid, alkali, and distilled water (Appen- dix II, Figure 17) are distinctly different than those of experiments three and four. It is also different than the fluorescent material (Rf 0.74 in KZHPO‘) which was a component of the tetrahydropteroylglutamic acid preparation (Appendix II, Figure 13). Comparison of the spectra of this plant material and that of the fluorescent material (R 0.76 in KZHPO4) which was r a component of the formaldehyde—C14 and tetrahydropteroylglu- tamic acid mixture reveals very close similarity. A comparison may also be made with the component of acid- treated leucovorin (Rf 0.73 in KZHPO4.Appandix II, Figure 15). 27 The absorption spectrum in 0.1 M hydrochloric acid and distilled water of both of the compounds appear similar, although that in 0.1! sodium hydroxide is quite different. If the material from acid-treated leucovorin is assumed to be an N5-Nlo-imidazo- liniun derivative with a quaternary nitrogen, its behavior in alkali would be expected to be different than that of an NS- N10. methylene compound, but perhaps it would be similar in acid. It is interesting to note that this was essentially the only fluorescent material recovered from the leaves that had been fed the mixture, and that the radioactivity of the plant material was greater than that of the administered component. Radioactivity of the fluorescent materials has been re- corded as cpm./mg. The material which was eluted into planchets for counting and weighed probably contained a certain amount of KZHP°4 from the solvent, so that the eeight observed was not a true weight of the fluorescent materials. The small amount of material isolated precluded any elaborate purifi- cation procedures. The higher radioactivity of the plant material therefore may be more apparent than real. Further study of this problem should include 1) an unequi- vocal synthesis of N5-H10-methylene pteroylglutamic acid der- ivatives to be used as reference compounds for comparison filth plant materials, 2) better control of the tetrahydropteroyl- glutamic acid synthesis and purification of the product, 3) determination of the ultraviolet absorption spectra:hi acid 28 and alkali of those plant materials which can be separated by EBAW, 4) larger scale separation of fluorescent plant materials so that they may be further purified and identified, and 5) hydrolysis of the Ns-and/or N10 substituent and exam- ination of that carbon for radioactivity. SWEAR! SWEAR! 1e 3. SUMMARY Formaldehyde-C14 was administered to tobacco plants both hydroponically and directly through the stem. An attempt was made to isolate and identify ”folic acid” compounds which may have incorporated the radioactivity of the formaldehyde. Fluorescent materials recovered from the plants resembled 'folic acid" compounds in paper chromatographic characteristics and ultraviolet absorption spectra, but could not be positively identified. A mixture of tetrahydropteroylglutamic acid and form- aldehyde-C14 was administered to isolated leaves of a tobacco plant by vacuum infiltration. A.single fluorescent material was recovered from the leaves which was shown by paper chromatography and ultraviolet abporption spectra to be identical sith a component of the administered mixture. ' A discussion of the possible identity of the fluorescent materials derived from the plants and suggestions for further study were presented. REFERENCES 5. 6. 7. 8.A 9e. 10. 11. 12. 13. 14. 15. 16. 17! 3O EFEfiENCES Greenberg, G.R., Fed. Proc. $1, 745 (1954). €uc§anan, J.M. and Schulman, H.P., J. Biol. Chem. 202, 241 19 3 . Kisliuk, R.L. and Sakami, w., J. Biol. Chem. 214, 47 (1955). Miller A. and Waelsch, H., Arch. Biochem. and Biophys. Q}, 263 (1§56). Babinowitz J.C. and Pricer, W.E., Jr., J. Am. Chem. Soc. 2§.5702 (1&56). , 'Doctcr, V.H., Patton, T.L.S and Apawara, J., Arch. Biochem. and Biophys. £2, 404 (1957 Hamill, R.L., Herrmann, R.L., Byerrum, R.U. and Fairley, J.L., Biochim.et Biophys. Acta g;, 394 (1953) Phear, Elizabeth A. and Greenberz, D.“., J. Am. Chem. Soc. 22. 3737 (1957). ~ ° Blakley’ ReLe’ .BiOcheme Jo fig, 448 (1954)e Albrecht, Alberta and Broquist, H.P. Proc. Soc. Exptl. Biol. and led. 2a, 158 (1956). O Greenberg, 6.3., Jaenicke L. and Silverman, M.,Biochim. et BiOphys. Acts $2, 589 (195%). Silverman, M.,‘Keresztes J.C., Koval, G.L., and Gardiner, Rita c., J. Biol. Chem. fig, 83 (1957). $3522; H. and Rabinowitz, J.C., J. Am. Chem. Soc. 2§, 5705 1 9 e Kisliuk, R.L. and Sakami, w., J. Am. Chem. Soc. zé, 1456 (1954). élsggnder, R. and Greenberg, D.M., J. Biol. Chem. 214, 821 1 . . . Blakley, R.L., Biochim. et Biophys. Acta g;, 654 (1957) Peters, J: and 'Greenberg, D.M., J. Biol. Chem. 236, 329 (1957). /) 20. 21. 22. 23. 24... 25. 29. 30. 31. 32. 33. Chem. 33;, 865 (195 31 Byerrun, R.U., Ball, C.D., Fldkstra, J.H. and Dewey, L.J., J. Biol. Chem. £19 633 (1954). Brown, 5.1., ”Studies on Ecthylation Reactions in Plants: The Origin of the Methyl Carbon of Nicotine Formed by Nicotiana zustica", Ph.D. Thesis, Michigan State University, 1. ' Dewey L.J. Byerrum, R.U. and Ball, C.D., J. Am. Chem. SOCe ’ 39§7 (1954)e Hingler,R.L., ”The Eetabolic Incorporation of Formaldehyde Into the Nicotine N-methyl Group in Nicotiana rustica”, Ph. D. Thesis, Michigan State University, 1935. Mitoma, C., and Greenberg, D.M., J. Biol. Chem. Lié, 599 (1952). 'Zakrzewski, S.F..and Nichol, C.A., J. Biol. Chem. 21}, 697 (1955). Frontera-Aymat, Maria 3., "A Study of Pyruvate metabolism in the Tobacco Plant“, M.S. Thesis, hichigan State Univer- Yoe,'J.H. and Reid,»L.C.,.Ind. and.Eng. Chem., Anal. Ed. 11, 233 (1941). KarestZtesy’ JeCe and Silverman, Me, Je 8101. Chfime 18}, 473 (1950). Wieland, 0.P., Hutchings, B.L., and Williams, J.H., Arch. Biochem. and Biophys. 59,205 (1952). SEUerlicn, EeEe, Je BiOle Chem. 1?5’ 337 (1952)e Doctor, V.M. and Couch, J.R., J. Biol. Chem. 230, 223 (1953). Silverman, ¥., Keresztesy, J.C. and Koval, G.J., J. Biol. ChOme all, 53 (1954)e Usdin, E., Phillipsé)Phoebe M. and Toennies, 6., J. Biol. Cosulich, Donna 3., Both, Barbara, Smith, J.M.,Jr., Hultqnist, H.E., and Parker, R.P., J. Am. Chem. Soc. 2i, $252 (1952). , J. Am. Chem. Soc. 21, 5006 (1951). 34b 35. 36. 37. 38. 39. 40. Greenberg, G.R. and Jaenicke, L., Biochim. et Biophys. Acta 17, 589 (1955)e O'Dell, B.L., Vandenbelt J.M., Bloom 3.8. and Pfiffner, J.J., J. Am. Chem. Soc. 52, 250 (19475. Kisliuk, R.L., J. Biol. Chem. ggz, 805 (1957). Kursanov, 1., Biokhimiya ;, 269 (1936). fiakgzewski,'S.F. and Nichol, 0.1., J..Biol. Chem. 205, 361 19 3 . Jaenicke, L., Biochim. et BiOphys. Acta 12, 588 (1955). Osborn,'M.J., Vercamer, E.N., Talbert,'P.T., and Huennekens, F.h., J. Am. Chem. Soc. 22, 6565 (1957). May, Margie, Bardos, T.J., Berger, F.L-, Lansford, Myra, fiavel, Joanne, Sutherland, G.L. and Shive, W., J. Am. 0 Chem. Soc. 21, 3067 (1951 Cosulich, Donna B. and Smith, J.M., Jr., J. Am. Chem. Soc. APPENDIX APPENDIX I a: VALUES OF "FOLIO ACID" COMPOUNDS REPORTED IN THE LITERATURE om ound 5:319? Solvent‘I Refeggnce Pteroylglutamic acid 37 A Zakrsewski (38) 36 a: Zakrsewski (23) 24 C Jaenicke (39) 29 D Greenberf (l) 28 E Wieland 27) Leucovorin 74 B~ Zakrzewski (38) 74,86 B Zakrsewski (23) 62 C Jaenicke (39) 65 D Greenberf (l) 71 E Wieland 27) Rs-Nlo-methylene- tetrahydropteroyl- giatamic acid 25 G Osborn (40) H -formyltetra- hydropteroylglutamic acid 54 C Jaenicke (39) llo-formyldihydro- pteroylglutamic acid 58 C Greenberg (11) filo-formylpteroyl- glutamic acid 76 A Greenberg (ll) 78 C Jaenicke (39) 55 D Greenber: (1) 56 P Silverman (30) filo-methylpteroyl- glutamic acid 75 A Zakrsewski (38) retrahydropteroyl- glutamic acid 12,25,48 A Blakley (16) 48 ‘ c Jaenicke (39) 13 G Osborn (40) Dihydropteroyl- glutamic acid 10,40,73 A Blakley (16) ”several below 40" D Greenberg (l) *Solvents are designated as follows: A, 0.1 H K2HPO4; B 0.1 I phosphate buffer, pH 7.0;(C, 12% NazHPO with 0.2% ethy ene- diaminetetraacetate; D 5% K2HPO4 with isoamyl alcohol; E 5% NagHPO4 with isoamyl aicohol; F, lii NagHP04olZHaos G, 0. H phosphate buffer, pH 8.0 on 01 .uopmg coupes nemafipmfin cfl Cano>oosmq om onsmflm nofiaupmflv CH nfiom ofismusawaxonoua .fi enamfim IE qfi nouns.” gm; . . own 00m 0m 0 N 0.. o mflnfilJlE-mli 1141ou- . J W liul-Tfil-J _ amm .u o f .1 . OHaI IL O “P v 3d O Q’ l r o ‘f‘ Issstusai quad J 8.- 2.... 4 om... L 00? u 8? L wgdemadz H7213 Q72 mQZDOQSOO mozmmmmmm mo $3.0QO ZOHBmmomm< quofl>¢mHAD HH NHozmmmd .o .l I I I save oanoanooocxn 2 H30 use .uu oeexooean esfieom s H.o ca .efiom whom aflompsawamocepaonuxnmuuoa oe-onsmfim canoanooncxn 2 H00 a“ :fino>oosmq om mnsmfim is ca camcmfi m>m3 on 00m 0mm now own, - 0mm com nmw Com uowm M a d A a 1 a all J a — a a \ 33 J- \ 3.1wsu-11 Us '\ .\ 09! I 00m.r HO I. II I. .8910»... .Hmums mum-H I. .I I. olcaloiv. prmg iaaomee cam . eaom cenoaneoeean : Hoo emaaasmfie cam . chow oeuoehoocesn : H.o . enamonumn EsfiuOm E Hoo ca 03» uceEHooaxo .IIIl oufixouums ssfiuom 5 H00 Ca enhnenamsuou 89C H8332» puma pneumonosam .0 0.3m; + 36m 3533“masonopaooomnmnpea om 0.3m?” {E Cw 53:3 833 omm oom 0mm com oem - 0mm . 00m 0mm omm can 4 _ 4 J ‘ 4 m _ A — a « _ 1 _ q u A _ r L «as coa- 1U 0' 8' I noses eofiafiomae ca mm. mm 24mm .oounu ucoaaovoxo .Hmfinoums panda unannomn¢ .1« “some eeaaeomae ca mm. om game ...an» acne canvass .Hefioopms umam unmomenooam ow anomam . booms eoflafiomee 2“ an. em aemm .oounp uaoEHnooxo .Hmanepma woman pdoomonooam . noses eoflufiomae e“ um. um eamm ...anp some ufineoxe .Hmaalews woman pdeomoaosam on enamfim omm com owm 12 cw Sumnea m>e3 r \ 0mm q u 1 1 ‘ -lbom fl _ bdm ‘ OH... 0mm 00 omm . q _ 4qu qb 1 a « Ocmu a A A a r \. <3 “2 93 \ \. hotsstmsueha auto J . cope; eufiafismae ca Hm. om scum endow useEHneauo .Hmfineamfi unmao pom9n0m9¢ . have: eoeaaomee an me. cm game .esoc ocea- l" . ovum: veaaaumfiv cfi mm o H m Rdmm .uzom psoafinoaxe .Hmfinouma woman pamomeoosam ...r, :1wanonxo .Hmfinepma woman pceomeuosam ovum; emanasmae ca an. em game..auoo sc.s wfioomxe Jumuepms woman pavemenosam 36a anomam ow, onsmam IE CH nmeoH o>e3 omm oom omm owm oem 0mm oom omm omm oem _ — 4 J a — — d p - — d u d u a fi ‘ a _ J u x . - r C$Jv \ la \Omlr 1 _ 1 0mm ~ fl 1 9e. 'uotsstmsutaq iheo 155 00d? 9 Doom canoezooneaz z H.o . asfieom z H.o ca om. cm eommme .asoo noose aneuxe .Hmanmpms woman pneumonosam 3 0'0’0 “me p M... ......- Umaaflpman non mmflxonumn .mH unseen 0 0‘0 -0 . I... 8 Euom ofinoazooonmn lean eeauom 2 H00 :H om. m nanoaxo aamflnmpma woman uncommoosam aware emJHHomHe use 2 H00 . eofixoo eomnwm .moono some odd meemfia 18 5” numcea SEE com a 0mm oem J, 1 a _ d _ _ q A A _ srmsagxq fuse fad 'u01§ om. om. \\ oofir O 4 0 0-0-0 09lolonepms unmafiumfiu Una ovum; voaaapmwo use .!.l;l Ufioa ownoazoonwmz .u u u 38 3828.83 5 To . 83893 a .o . ages-8.3 5:39. a do fl 1.. am ssfiwom g Hoo :# om. mm eommmm awflom CH Cano> eon: M anowpmnmaenu vfiom ofiEmpusHmonmugoonns uoonoa Boom Hmfinepme useommnosam .eH enswam uwnuew Boom Hmwnopme nanomenosam .ma madman .18.GH nuw2vH e>ma omm oom 0mm com Dem - 0mm 00m 0mm 0mm oem _ . _ q q . q d _ _ a. _ a d _ n . a _ . . Uta: qUoo 15d % :3 sums ‘uors 00-0-0 pvuma 11 l l l A. we Hfipmfio one . ofiow ofiuoasoonomz : Hoo I_I I cola). popes ooaafiumfio one . oeaxooean eseuom a Hoo go we. om eommmm . eaom onooanooaean 2 H.o . meexoaea; moumneoamsoou + nfiom OHEmpsa Hmonouuonnzn eofioom 2 H00 ca mm. mm commmm .oHom Ga Capo» uenpmp Boom Hmanopms poooeooooam .oH ousmam loosea Eonm Hmfinoums pcoomooooam .de snowflm 18 cfi-nuwcea e>ez omm oom omm com oem omm oom- com com oem .‘eq_.7l.... .Jqq._q_... T o? 1 r 81. n £4 4 "1 .9l9l. ovum; ooaafiomfio one goes ceaoanoonean a Joe . oeaaonuan ssfinom S Hoo :H mm. mm commmm .o>fim pcoefi unooxo .Hmfiooums woman pneumonosah 05H onowfim o| l u .18 Ga camcoa o>m3 omm oom owN com com . -e _ q a _ _ mu. q « — C) d’ Om uorssrmsuexq quoo Jed OOH .-Da1.a.4‘l¢de all!!! ..1 MICHIGAN STATE UNIVERSITY OF AGRICULTURE AND APPLIED SCIENCE DEPARTMENT OF CHEMISTRY east mama. MICHIGAN CHEMISTRY LIBRARY Thesis c.2 Brown. Gail Denise 14.3. 1958 TAT MllIIIIIllI 3 1! IVERSITY LI |l IIIIIIIIES 5 4 082 12 Ill 3 IIIIIIIII 9 3 0