BIOCHEMICAL STUDIES IN PLANTS I THE ISOLATION CP TUMOR-ffliCWTH INHIBITORS FRCM BOLETUS ETOIIS n THE METABOLIC INCORPORATION CP FOEMALDEHIDE IKTO THE HXCOYINK N^MKTHXIL GROUP XN TOOXXAHA RUSTICA By Eobert hloyd Ringler A THESIS Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree ©f DOCTOR or PH3XOSOPHT Department of Chemistry 1955 ProQuest Number: 10008675 All rights reserved INFO RM ATION TO ALL USERS The quality o f this reproduction is dependent upon the quality o f the copy subm itted. In the unlikely event that the author did not send a com plete m anuscript and there are m issing pages, these will be noted. Also, if m aterial had to be removed, a note will indicate the deletion. uest ProQ uest 10008675 Published by ProQ uest LLC (2016). C opyright of the Dissertation is held by the Author. All rights reserved. This w ork is protected against unauthorized copying under Title 17, United States Code M icroform Edition © ProQ uest LLC. ProQ uest LLC. 789 East Eisenhow er Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ismxammma the author wishes to egress his sincere appreciation to Dr* B, U* ©yerrum whose assistance, guidance and counsel haw greatly facilitated the completion of this work, the author is also Indebted to Dr, S, H, lueais, who first ob­ served the tumor Inhibition by an extract of Boletus edulis. for his advice and counsel throughout the work"described in Fart I of this thesis. Acknowledgment is also due to Dr, 0, O, Stock and his associates for conducting the sarcoma lBO assay, the author also wishes to express his appreciation to the various other members of the Department of Chemistry who have given helpful advice from time to time. Finally the author wishes to egress his thanks to the American Cancer Society, th© American Institute of Health and the Atomic Bfoergy Commission for providing funds in support of this work. •*****«*& MN ff lv ff J4 , T ftw Qr w -JHfr a ii the author mas bom March 27, 1922 at Ghase, Michigan* Ha received M e secondary training lit Ghase RIgh School and at Ferris Institute, Big Rapids, Michigan* He served in the United States Naval Service frcan February of 19hl until September of 191*5* the author graduated from Central Michigan College, Mt* Pleasant, Michigan, In January of 1951 with a Bachelor of Arts Degree in chemistry and biology* He enrolled at Michigan State University for the spring term of 1951* During his tenure at Michigan State University he was a Special Graduate Research Assistant for three and one-half calender years, and a Graduate teaching Assistant for two quarters, Hi BIOCHEMICAL STUDIES IN PLANTS I THE ISOLATION OF TUMQR^GKQ^TH INHIBITORS FECK BOLETUS EPULIS II THE METABOLIC INCORPORATION CF FORMALDSHIDE INTO THE NICOTINE N-METHIL GKOOP IN N3DGQTIANA RUSTIC! By Robert Lloyd Ringlet M ABSTRACT Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHTLOSOFHI Department of Chemistry Tear 1955 ABSTRACT FART I A method of preparing tee purified tamor-growth inhibiting frac­ tions (fraction# W end V) from the mushrocMi Boletus edulig is described. An in vivo assay employing mouse sarcoma 160 was used throughout this mark* The activity of the various preparations varied from a (- ~) to a ♦) effect * Toxicity mas displayed by nearly all of the samples tested. In the method of preparation the mushrooms mere extracted with 0,0$ per cent sodium sulfide solution. This extract was made 30 per cent acetone and centrifuged at once. The residue was discarded, and the supernatant mas designated fraction X. Fraction X mas made a total of 70 per cent acetone and centrifuged after standing overnight. The supernatant mas discarded, and the residue mas designated fraction XX, Fraction XX mas suspended in 0,0$ per cent sodium sulfite solution and dialymed. The dlffusate (outside solution) mas discarded. The di* alysate mas designated fraction XIX* Fraction XXX mas buffered at pH 6 ,k with phosphate buffer (by adding the dry salts). The buffered fraction XXX mas chromatographed on a Domex $0 x 12 column, An activity was eluted with 0,3 U sodium chloride solution (fraction XT) and with 3.0 M sodium chloride solution (fraction V). Fraction XV was dialled and a solid product obtained from the dialysate. A solid product was also obtained from fraction V, however, it did not possess tumor inhibit­ ing activity. v Fraction I?, chloride free and in the dry state, was hydrolysed with acid and also with alkali* The hydrolysis products were studied using paper chromatography. A preliminary terminal H-group analysis was also made* €n the basis of this study It was postulated that fraction ffl contains a cyclic peptide composed of 10 amino acids and an unknown "basic substance* * An infrared spectrum tends to support the peptide nature , The "basic substance" appears to hare an absorp­ tion maximum at 276 am* In a water solution. PAST II as Fornialdehyde-G was hydroponically administered to three group© of 30 tobacco plants* the tobacco plants were of the high nicotine strain Hicotjana rustics 1.* var. humilis. After a ? day growth period the nicotine was isolated and Its radioactivity determined. The nicotine was demethylated and the methyl group counted as raethytriethylamraonium Iodide. Within the limits ©f experimental error, all ©f the radioactivity of the nicotine molecule was found to be contained in the methyl group* The postulation was made that formaldehyde or, more likely a close­ ly related compound, is an important metabolic precursor of the nicotine H-methyl group. TIBLE CF GGHIENTS CHAPTER FAGS fm i i wcaiaD0CTrai#...#*...##........ .. IX METHOD OF BIOLOGICAL ASSAT...... h Sagserlmental Details.... .... Interpretation of Assays..*.*...... ..... Discussion of Assay. XXX ISOLATION PROCEDURE i k $ 6 .... 9 Extraction* ......... Thirty Per Cent Acetone Precipitation. ... Seventy Per Cent Acetone Precipitation* .... Stability of the Activity Toward Dialysis Experimental Preparation of Fractions I, II and III.*... Discussion of th© Preparation of Fractions X, II and III Biological Assay of the Various Preparations of Fraction XU...*.... Fractionation of Fraction III Using Paper Chroia&tography Discussion of Chromatographic Procedure Extraction of Fraction III with n«*Butanol............... Discussion ©f the extraction of Fraction III with n-Butanol ..... Ion Exchange Chromatography. ............ Cation Exchange Chroiaatography-^eneral Considerations., Preparation of Ion Exchange Columns.. ... Buffering ©f Fraction IH. ... Chroiaatograpiiic Separation on Dowex SO....... Preparation of Fractions I? and V.......... Discussion of Ion Exchange P r o c e d u r e Treatment of Fraction XXI "with Acid and Cuprous Oxide... Discussion of Cuprous Oxide Experiment*................. Further Purification of Fraction IV... ... Further Purification of Fraction V................. Discussion of the Purification of Fractions IV and V,... 9 11 13 16 1? 21 Zk 26 29 30 32 33 3h 39 W> I4I US 1*6 17 I1-8 L9 S2 $2 IV STUDIES AS TO THE CHEMICAL CMPOSITIOH OF FHACTIOH IV.....* SS Acid Hydrolysis of Fraction IV. Alkaline Hydrolysis of Fraction IV. vii ...... S S SS table m cmmes * e CHAPTER PAGE Preparation ©f Fractions for Paginal IMSroap Study***.* $6 Paper Chromatographic P r o c e d u r e # 57 Reeult of Chromatographic S t u d y . .....* 62 Discussion of the ChromatographicStudy*.******......... 66 Spectroscopic Studies UsingFractionX V . 70 V0OS£^0SXOIfS*. . . . . . . 71* ........ 76 *****...♦.*.. 77 4PP1MXX*................... ............... *........ 81 PAir If m'mmrntwm* EXPRRXHBIfTAL*,... * * ..... a? .......... ... 91 of P i a n t S ' * # Preparation of Fomaldeby&c Solution®, * Determimtion of R a d i o a c t i v i t y . Uptake of Formaldehyde by the Plant®.....,........... jkhainistration of Radioactive Fomaldeliyde Xsdation and Purification of Hicotim.................. Demethylation...*. msCUSSIQH...... ..... 9k 9$ 96 97 101 ........................... 105 BmiOGRAPHf APPSMDIX......... 93 9h 100 RESULTS* .,*..... s u M t t m . . 91 ....... 106 ... ....................... vili 108 1XSX W TiBXtBS tmi PAGE fmt t X the iBomdiate Precipitation of the Active Principle by Various Concentrations of Acetcno**#» 12 IX the Bidegleal Assay of the Vazdeos Preparations of tfaotlen X X X . 25 XXX Hinfaydrin Positive %©ts m Paper Chromatogram of Fraction XXI** *.**.,**♦... **....... 2? XV Chromatographic Fractionation ©f Fraction XXX Per Cent Effect (c) U 01 m (?) L108 90 1111 n (?) 1*113 90 (*•) Acetone was therefore added slowly to the original extract until an abundant viscous precipitate was well formed. The acetone concen­ tration was found to be kO per cent (v/v). This viscous precipitate was removed by filtration, suspended in water and tested for activity (1129--). Inasmuch as activity was lost at an acetone concentration of kO per cent an experiment was carried out In which the acetone concentration was decreased still further. It was observed that a rather abundant viscous precipitate formed soon after the addition of acetone to a concen­ tration of 30 per cent (v/v). This precipitate, when removed by filtra­ tion, suspended in water and tested also showed the presence of tumor inhibiting activity (1136--) * Pus to the nature of the precipitate which formed at an acetone concentration of 30 per cent the filtration at this point was quite slow. It was therefore considered possible that if the precipitate could be 13 removed more rapidly the lose of activity night bo decreased, In experi­ ment was therefore carried oat In which the preparation was centrifuged in a Sharpies centrifuge immediately after being made 3® per cent acetone, Upon opening the Sharpies centrifuge bowl a very dark colored fibrous residue was found to be present in the lower portion of the bowl and a light colored fine residue near the upper portion* These two types of residue were separated, insofar as was possible, and suspended in water. The suspension resulting from the lighter colored material was tested as sample (L319-) and that from the darker colored residue as sample (1320-), Inasmuch as there was m indication of the loss of activity in this 30 per cent acetone residue, the above precipitation technique in which the preparation was centrifuged in the Sharpies centrifuge, was used throughout the remainder of this work. The supernatant resulting from centrifuging the 30 per cent acetone solution was designated fraction 1 , Seventy Bor Cent Acetone Precipitation Is a result of considerations mentioned below, the precipitate which forms between the limits of 30 and 70 per cent acetone was collected throughout almost all of this work. The lower limit of 30 per cent was selected from considerations mentioned In the previous section. The upper limit was chosen partially as a result of that same study, After discarding the residue which formed at an acetone concentration of 30 per cent it was desired to selectively precipitate the active principle If possible, When the ob­ servation was made that some of the activity was precipitated almost It immediately even at an acetone eoncentration of 50 per cent, it became apparent that the greatest selectivity might result if a relatively low acetone concentration were used to precipitate the activity, providing sufficient tine m s allowed for precipitation to take place. A relatively 1ow acetone concentration also Seemed to be desirable inasmuch as the possibility of the loss of activity doe to a high acetone concentration had not been Investigated to any great extent. Use, the observation had been made by Eitchie (23 ) that the precipitation of the active principle appeared to be complete at an acetone concentration below 66 per cent. With these considerations in mind, acetone was added to a fraction similar to fraction I to a total concentration of 60 per cent. The difference between the fraction used here and fraction I was that the first acetone precipitation was made with hO per cent acetone rather than 30 per cent as in fraction I. At an acetone concentration of 60 per cent a precipitate seemed to be well formed. this precipitate was removed after nine hours standing by centrifugation, suspended in water and reprecipitated by adding acetone to a concentration of 60 per cent, the precipitate was collected the second time by centrifugation after 12 hours, suspended in water and tested as sample (L121 *-). the prepara** tion was repeated with one alteration, the first precipitation was allowed to take place for six hours, instead of 12 hours as used in the previous ease, sample (11222). A preparation was also carried out in -which only one precipitation with 60 per cent acetone was used. The 15 precipitate was collected by centrifugation after only two houre stand­ ing* This precipitate was suspended in water and tested as sample CLX28****) • Several preparations were also carried out in which fraction I was made TO per cent acetone* In one of the first of these fraction X was made a total of 70 per cent acetone and the precipitate collected after two and one-half hours by filtration with auction on Whatman Ho. 2 filter paper. This residue when suspended in water was tested as sample (1135**)(retssti-). In a slightly later preparation the precipitate which formed after three hours was collected fey filtration as before. However, a second precipitate was also collected by centrifugation after allowing the filtrate from the three hour filtration to stand a total of 6U hours at 7° 0. The precipitate which was collected after three hours was not tested for biological activity at this point but was extracted with n-butanol by a technique to be described later* The aqueous layer was tested for biological activity as sample (LHi6^~). The precipitate which formed between the time Interval from three hours to 64 hours was suspended in water and tested as sample (1150-). In a third experiment, using acetone as a concentration of 70 per cent, the preparation was allowed to stand for 20 hours at 7° C, before collecting the precipitate. The precipitate was collected by centrifu­ gation, suspended in water and tested as sample (L195~~) * The biological assay doss not permit an accurate evaluation of the two methods of preparation. However, after considering Ritchie*s (23) 16 observation mentioned above and the experiment in Which the activity did net appear to be destroyed after standing in the presence of the high acetone concentration for 20 boors* it was concluded that a precipitailea with acetone at a concentration of 70 per cent might be preferred. Fraction 11 is therefore designated as the precipitate obtained by centrifugation after adding acetone to fraction X to a total concentra­ tion of 70 per cent and allowing the precipitation to take place over* night at ?0 0 * Stability of the Activity foward Malyais With but one exception* the activity was found to be non-dialyaable. Eitchle (23 ) also observed the stability toward dialysis. In the one experiment in which activity night have been present in the diffusate (outside solution)* the preparation of fraction II was made at the Upjohn Company at KaXamaaoo, Michigan using $ kg. of mushrooms. An exact duplicate experiment was never carried out. However, two experiments were carried out to determine the loss of activity due to dialysis of fraction II when dissolved in 0 .0 5 pa* cent sodium sulfite solution* In the first of these fraction II from 200 g. of mushrooms was dissolved in the sodium sulfite solution and dialyzed against 2200 ml. aliquots of distilled water. An aliquot of the first 2200 ml. of diffusate was tested as sample (L38O-). In the second experiment fraction XX from 300 g. of mushrooms was used and the first three ali­ quots of the diffusate collected, the diffusate was concentrated to a volume of 75 ml* on the revolving concentrator described by Craig et al. (27 )* the tewpemture of the sample was maintained at 25° C. or below 17 during the 2J* hour concentration procedure. The 75 ml* concentrate of the diffusate was teeted as sample (L389*) * In both of the experiments just described the dialysate (inside solution) contained activity (L381-+) and (l*388~+) * Zn all future experiments fraction IX was Suspended in 0.05 per cent sodium sulfite solution and the mixture dialyzed. The dialysate (inside solution) was designated as fraction III* Experimental Preparation of Fractions I* H and H X As a result of the experiments described in the previous sections the experimental procedure outlined in Figure 1 was used for the preparation of fractions I, II and XXX* during the experimental develop* ment of this method of preparation quite small amounts (15 to 30 g.) of mushrooms were used for each experiment* However, as the development ©f the method progressed larger amounts of the crude material were used to eliminate unnecessary repetition of the actual operations involved. Therefore in the experimental procedure which follows the volumes and weights of the various fractions are based on X kg, of crude starting material, A relatively large amount of fraction II resulted from this amount of starting material and it was used for further work on the purification of the active principle, Xn this method of preparation all operations except the actual centri© fuging were carried out at 7 C, using precooled solutions and equipment . Ten liters of 0.05 per cent sodium sulfite solution were placed in a large glass cylinder about 30 cm, in diameter and 1*6 cm. high, having 18 FIGURE 1 PREPARATION OF FRACTIONS I, II and III Crude muahrooin (not ground) .0 1. Extract with QmO$% aqueous Na3S03 solution (is10 w/w) at 7 C, 2* Filter through cheese cloth. Filtrate Residue 1* Extract with 0.0£$ aq* NaaS03 sol. (1*5) at 7°C 2. Filter through cheese cloth. Combined filtrates 1. Hake 30% acetone at 7°C» 2. Centrifuge in Sharpies centrifuge. Residue (discarded) 1 1 Supernatant Fraction I 1. Add acetone at 7°C . to a total concentration of 70%, 2. Allow to stand overnight at ? C, 3. Centrifuge in Sharpies centrifuge* Supernatant (discarded) Residue (discarded) Residue Fraction II 1. Suspend in Ha3SOs solution at ?°C* 2* Bialyse 2I4 hours. I---------------------Diffusate (discarded) “ I Dialysate Fraction III 19 a volume of about 2k 1. The cylinder van equipped with a stainless steel stirrer attached to an electric stirring motor. She kilogram of mushrooms were added and the preparation stirred vigorously for 15 minutes. The extraction mixture was then filtered through three layers of cheese cloth into a 10 gal. milk can* the cheese cloth, containing the residue, was kneaded to obtain as much filtrate as possible, the residue was transferred back to the large glass cylinder and re-extracted by the same technique using 5 1* of the sodium sulfite solution, this extract was also filtered Into the milk can. the total volume of the combined extract was 12 to 13 1. Acetone was added to the combined extract with stirring to a con­ centration of JO per cent (v/v) and the preparation centrifuged at once in a Sharpies centrifuge using a polyethylene liner In the centrifuge bowl, this liner facilitated the easy removal of the residue which formed in the bowl. The bowl and liner assembly were parecooled before being used* However, the centrifuge was not located in a cold room. The flow of liquid through the centrifuge was adjusted, by mans of a screw clamp on the feed line, such that a dear supernatant resulted. At the conclusion of the centrifugation the supernatant (fraction 1) * o \ was immediately returned to the cold room (7 CJ. St usually required about two hours to run through the preparation up to the point where tin above supernatant was returned to the cold room, the residue contained in the centrifuge bowL was discarded* Acetone was added to the super­ natant to a total concentration of 70 per cent with stirring. The prep­ aration was then allowed to stand overnight at 7°C* 20 The following morning the preparation was again centrifuged in the Sharpies centrifuge ualng the technique described above* 7m this centrifugation the Majority of the supernatant was decanted from the precipitate which had famed on standing. Using this technique a more rapid flow rate could be used throughout the first portion of the centri­ fugation. Immediately upon completion of the centrifugation the polyethylene liner, containing fraction II, was removed from the centrifuge bowl and dropped into a 1 1 , graduated cylinder containing 1 1 , of 0 ,0 $ per cent sodium sulfite solution, the solution was stirred with a stirrer made by sealing the ends of a piece of polyethylene tubing. When turned slowly, by means of an electric stirring motor, this stirrer acted with somewhat of a whip action and was quite effective In accomplishing the suspension of the residue. After stirring for about two hour® the solution plus any solid material still adhering to the polyethylene liner was transferred to a Ticking casing and dlalysed against about 10 changes of distilled water (total volume about $0 1.5 for 2h hours at 7 C, the dialysis apparatus used was of the *rotating external liquid* type described by UJang et &1.(28). Using this apparatus the dialysis membrane is immersed in a cylinder eccentrically to facilitate agitation, with consequent shortened equilibrium, the cylinder was rotated by a fractional horse power motor at a speed of about $0 rp®. At the end of this dialysis period complete solution apparently re­ sulted inasmuch as no visible residue was obtained by filtering the 21 dialysate through a coarse sintered glass funnel. The dialysate, designated fraction tJX9 varied in volume from about 1200 ml, to about 1500 ml* in the various preparations. This solution was divided into aliquots of a site depending on the expected future manipulations and stored in the deep t m m e until used. Fraction i n contains about 22 mg, of solid material per gram of crude mushroom used in its preparation* This value mas obtained by talcing duplicate 10 ml, aliquots of fraction IH and drying them to constant weight at 10^°C, From the crude mushroom equivalence of fraction I H in terms of g,/sl, and the dry weight of the residue, the milligram of dry matter obtained per gram of crude mushroom was calcu­ lated * Discussion of the Preparation of Fractions I, XI, and XXX The original extraction presents no operational difficulties. Some small bits of mushroom did go through the cheese cloth filter. However, these are conveniently removed at a later step (the centrifugation of the 30 per cent acetone solution). The residue remaining after the second extraction has never been extracted a third time to determine the completeness of the extraction procedure. However, lucas (22) demon­ strated that activity was extracted after merely soaking the mushroom pieces, without stirring, for a period of only three minutes* is a re­ sult of this observation it would seem that the extraction should be essentially comple te when carried out as described in the previous section. 22 The nature of the subatanee(s) responsible for the extensive dis­ coloration observed during the extraction and especially during exposure of fraction XX to air has not been investigated. Host mushrotan species do contain polyphenol oxidases (29530)* However, Boletus edulia apparently produeea a very snail amount of these oxidases (31 ). foinovitch ©t *1 , (32 ) have studied %hs inhibition of the oxidases present in the mushroom Ajgarlcua ©ampestris and found sulfur dioxide la combinations with a small amount of thiamin5 nicotinic acid, cysteine or glutathione to inhibit oxidase activity. Ascorbic acid m s also investigated and found to be less effective than either thiamin or nico­ tinic add. Voinovitch (33) also observed a non enzymatic browning to take place in autoclaved mushroom juice on exposure to dr, 3s found the addition of sulfite or acidifying the solution to below pH 5.2 retarded this discoloration, Orabor et a!,. (3I4) reported the inactiva­ tion of polyphenol oxidases of Agaricus camp©stria with ultrasonic waves. Kuttner and Wagreich (35 ) have studied the inhibition of catecholase activity using an enzyme preparation from the mushroom fsalliota campestris. these investigators studied the inhibition of this enzyme system by kO organic compounds. Among others, acetone was found to inhibit the enzyme activity to the extent of J48 per cent at a concentration of 3 .5 mole/1 . Inasmuch as the 30 per cent acetone and the 10 per cent acetone solutions used in the preparation of fraction H do not discolor appreci­ ably even on long standing It might be inferred that the discoloration is at least partially due to an enzymatic oxidase activity. 23 If a lore definite correlation between the observed discoloration in fraction XI and a decrease in tumor inhibiting activity is aide In a future study, further investigation of the inhibitors mentioned above would seem to be warranted, there is ©till the possibility of some small lose of activity in the precipitate which forme at an acetone concentration of 10 per cent. However, inasmuch a© the precipitate doe© not appear to form much below an acetone concentration of 30 per cent, the purification accomplished by title step would seem to warrant a m t H loss of activity if one doe© exist. Fraction I was a veiy clear amber colored solution, It immediately became opaque the addition of acetone to a concentration of 70 per cent* the overnight period during which precipitation was allowed to take place might be longer than was necessary inasmuch ft© activity was obtained in sample© 113$ and &1U6 after ft m m h shorter period. Also, the failure of activity to be detected in sample H$0 suggests tliat precipitation of the activity was complete after three hours standing at 7° G m Tim longer precipitation period was used for an operational reason* It was desired to dialyxe fraction J3t during the day so that the diffusftte could be changed frequently during the first portion of the dialysis. therefore, the preparations were normally started during the Iftte afternoon and the precipitation in the 70 per cent acetone solution allowed to take place overnight* Fraction H was ft medium tan color when first obtained; however, it darkens on exposure to air and the resulting sodium sulfite solution 2h ©f fraction XX was a wa*y dark color. It would be interesting to ornery out a preparation In which this fraction was kept under an inert atmosphere and to compare thetoa&eity and activity of the resulting fraction with one carried oat without m e precaution. The possibility that the supernatant correspondingto fraction II contains additional activity has not been investigated. If a particu­ larly quantitative recovery of the activity were desired this supernatant night be concentrated at a low temperature and reprecipitatedl with acetone at a concentration of 70 per cent after removal of the precipitate which might t o m at 30 per cent. Inasmuch as tbs supernatant would be ejected to contain the majority of the lew molecular weight compounds which were extracted a concentration and reprecipiiation at this point might not accomplish much in the way of a purification of the active principle. The dialysis step described above was carried out quite empirically since no convenient method ©f detecting a **complete" dialysis was avail­ able. The major portion of any low molecular weight compounds either precipitated or coprecipitated In fraction H, by acetone at a concentra­ tion of 70 per cent, would seem to be removed by the 21* hours dialysis technique used. Assay of the Various Preparations of fraction III Table IX contains a list of the various preparations of fraction XIX which were assayed for biological activity. Inasmuch a® these samples were tested at various dilutions, possibly due to different toxicity levels, column (b) in Table II is an expression of the number of grams of crude mushroom represented by the daily mouse dose. 25 TABLE XX the bxolooxgal assh or tm wsxoas m Sample Niratber {»> Crude Huebrocsn Squtvalenee (b) grains rmssmm m Effect in Ttaaor Assay (e) 1*92* 0*13 7 L292*»b c 1*293 0 .0k «*•* 0.22 1293 0 .0k t + «s»-4* L322d cut T L322b,d 1.361 0.09 £388 0 .O8 A3 0 .0k £393 0.05 a* ♦ ♦ * a.4* ♦ u,m 0 .1 2 ♦ ■awe LU28 0 .2 0 t L2*28b 0.05 ? ttmwisxms Ttunor Dlfiuneter Tr0ated/0 onti*els (d> CB. 0.1*9/0.98 1 .03/1.02 0 .56/1.11 0*36/0.89 0.51A.02 O.59/0.9U 0.73/9.98 * Fraction XX dissolved in phosphate buffer pH 6.1*. b Katest. ® Original extraction 1»15, no second extraction. Ho sodium sulfite solution used. 26 By averaging the values in column (b) for those samples which gave tumor inhibition (fable H) It can be seen that the average crude mush-** room equivalence is about 0,07 g* Considering that about 22 mg, of solid material man obtained in fraction H I per gram of crude mushroom, a daily mouse dose of about 1 ,5 mg# of dry matter can be calculated. Since the daily dose is ordinarily reported in terms of milligram® of sample per kilogram mouse per day (»g,/kg,/day) this value becomes 75 mg,/kg./day (average mouse weight 20 g, or 1 /5 0 kg.) Fraction H I has also been tested against the Murphy-Sturra lymphs* sarcoma and found to be inhibitory (36), Fractionation of fraction I H haing Paper Ghromotogranhy In an attempt to gain some insight into the chemical composition of fraction HI, a one dimensional, chromatographic separation on Whatman Ho. 1 filter paper was attempted. The chromatograms warm developed using the solvent n-butanol, ethanol, water in the volume ratio of h#l*l (37) by the ascending technique. A glass cylinder about 1*5 cm. in height and 15 cm, in diameter was used as the chromatographic chamber. Ifter de­ veloping the chromatograms overnight at room temperature they were removed from the chamber and dried at room temperature. Three different means were used to detect the spots on the various chromatograms. The first of these was a reagent consisting of a 0 .2 per cent solution of ninhydrin (tidketohydrinden© hydrate) in water saturated n-butanol (37), A second reagent was prepared by dissolving 0.53 g* aniline and 1 ,6 6 g« phthalic anhydride in sufficient water saturated n-butanol to make 100 ml, of 27 solution (37). The third means ©f detecting the spots consisted of merely observing the chromatograms under ultra-violet light in the dark rooai (37)* One chromatogram ate sprayed with the ninfey&rin reagent and a second with the aniline-phthalic anhydride reagent * These too chroma* tograms sere then placed in a forced draft oven at about 95° 0* for approximately 15 minutes* Seven Mnhydrla positive spots appeared on the chromatogram nfcieh bad been sprayed with this reagent. These spots sere numbered 1 through 7 and sill be referred to in future discussions by this number* The seven spots and their U£ values are given in Table III* Spots number 6 and 7 sere very faint on the chromatogram* TiSIJS m sirara positive spots on m & m c m m m m m w pbxction %>©t Number % Value 1 0.00 2 0.03 3 0.10 i* 0.15 5 0.33 6 ©JA 7 0.56 m 28 two spots appeared on the clirowatogram which had been sprayed with the antline-phthallc anhydride reagent, they war® designated as carbohydrate spots X, &£ 0 ,1 5 and a, % 0*29* *** ®P©ta were also visible under ultra-violet light, These were designated as 0* ?* spots .'X, 0*00 and 2, Bf 0*56. A brown spot msalao observed at the origin (Sf 0*00) of the un~ sprayed clufowategraro under ordinary sunlight. As a result of the observation that at least eleven spots could be detected on a paper chrcsnatogram of fraction XXX an attempt was made to further purify tills fraction using a ieciiniqu© based on this observation. For this eaperimont fraction XXX was applied as a band about 1 cm. wide near one edge of a sheet (1$ 1/h x 22 1/2 Inches) of Whatman So* 1 filter paper. A total of 8 ml. of solution, equivalent to about 3 g* of crude mushroom, were applied to three sheets of filter paper by means of a pipette drawn out to a fine capillary. The papers were dried periodic­ ally between applications with an infrared lamp at very low heat. The chromatograms were developed with the solvent mentioned above In a Chromatocab (manufactured by University Apparatus Company, Berkeley, California) for about 12 hours using the ascending technique. After being developed and dried a narrow strip, about 1 cm. wide, was cut vertically from each edge and from the center of each chromatogram. The two strips cut from the edges of the sheets were treated with the ninhydrin reagent as described in the previous section* The middle strip was treated with the anllins-phthalic anhydride reagent* The bands made visible by ultra-violet light were marked on the remainder of each 29 chromatogram* Then, using the narrow strips as guides the various bands made visible by the two reagents were narked on the remainder of each cinromatogram* It was observed that U* Vm spot no* 2 was not present, immmr, a secondD* V* spot, designated D. V*spol 3 , 0*19 was visible under ultra-violet light. The chroiaatograifis were then cut into five horiaontal sections, each section containing one or more of the various bands. Each section was eluted with water and the eluate from corresponding sections cojsbined* The resulting five Naples were tested for biological activity* The toner inhibiting effect as well as the various spots which would be contained in each sample are shown in Table XT* trnm w Gm c&m m &M m G w m m to m ttm or w m m m x u m w m tm papek eu 11 Chromatographic %ots Present 1159 nin* 1 1160 nin* 2,3,2* & carbo* 1 1161 ?♦ 3 Effect in Tumor Assay + 4*W* -«* 1162 nin* 5 - 1163 nin* 6 - Discussion of Shrcaiatoaraphic Procedure Xt is evident from Table XV that the activity did not migrate in the solvent used to develop these chromatograms* Some purification might 30 have been accomplished ky procedure. However, inasmuch as the activity did not migrate, it mas considered questionable if the purifi­ cation merited the effort involved Perhaps other solvents should have been investigated. One such possibility mould be a solvent composed of acetone and mater. Inasmuch as the activity is soluble in mater but relatively insoluble in a water solution containing a high concentration of acetone it might be possible to select a concentration of acetone and mater which mould cause the activity to migrate as a moll defined spot. Held (38 ) has reported the resolution of a protein fraction in a mater acetone solvent on Whatman He. 1 filter paper. Eently and Whitehead (39) have reported the resolu­ tion of amino acid mixtures using an acetone mater solvent and also using m acetone 0 ,5 per cent aqueous urea solvent. Such a technique, if applicable, might offer a convenient method of assaying for the biological activity providing a correlation between a given spot and the tumor inhibiting activity could be demonstrated. Extraction of fraction III With n-ButanoI Inasmuch as fraction XU mas found to be a rather impure fraction in the chromatographic experiment mentioned above an attempt was made to further fractionate it by means of its distribution between n-butanol and water. A solvent system of neutral pH was chosen inasmuch as Bitehi© (23 ) had shown the activity to be destroyed if the pH of his preparation was altered for a short period of time (ea. 2 hr.) and then neutralised back to the original pH of 5>*1* 31 The extraction or fraction III with n-butanol was carried out by four different techniques, The extraction was carried out in separatory funnels both at room temperature and in the cold room (7°C.)* In both of these ©xperbsenta a considerable length of time m s required for equilibrium to be attained. In the last experiment carried out in the cold room, the preparation m s allowed to stand for aperiod of three days with obly poor separation of the two layers* In flea attest to facilitate the attainment of equilibrium a small amount of sodium chloride and also ethyl ether was added to the mixture without significant success. The technique used resulted in the preparation of three fractions. After a given number of extractions the combined aqueous layer was con­ centrated to dryness in vacuo at a temperature of less than $0° C* The residue, suspended In water is designated fraction A* The combined organic layer was also concentrated ig vacuo to a small volume at which time a precipitate was Observed* This precipitate was removed by fil­ tration and suspended in water and was designated fraction B. The filtrate was concentrated to dryness Ig vacuo« and was called fraction C* Table T shows the resulting activity of the various fractions when assayed in the tumor test* From the result shown in Table V it was concluded that the activity present in fractions A and B might be due to the poor separation of the two layers during the extraction procedure. An attempt was therefore made to extract fraction III with n-butanol in a liquid liquid extraction* In one experiment the extraction was done at atmospheric pressure. In a second experiment the extraction was carried out under reduced pressure 32 ■ irmM v vmmmwzm w m wmtmm m Bbqperiment Bumber Number of Extractions i* Fraction A Fraction B Fraction e (ULia~) a* iS' ■■■ 3b ■ISk kh wmumm with n ^ m m o h (&1U6£-) (I>391-+) (me«)e (U57~)C (M55*) (L390ii) (I.392-) * detracted at room tea^serature. ® Extracted at ?%* Sample dialled before being tested for activity. (water aspirator) * In each of these experiments the aqueous and organic pbaess were treated in the same laauner as described earlier. However, all samples proved to be negative on assaying fear biological activity. Discussion of the detraction of Emotion lH wlth n^Butaapl At the tine this work was carried out essentially nothing was known as to the chemical identity of the substance responsible for the tumor inhibition. The choice of butanol*waier as a solvent system was there­ fore quite arbitrary. This solvent system was used by Bakin (M>) for the fractionation of protein hydrolysates, however, a number ©f other solvent possibilities do exist (Itl). Craig and Craig (la) list a number of factors which should be ©onsddered when the choice of a solvent system Is to be made. The fact that the particular solvent system chosen forms 33 a*t omulsio»# apparently due to a. surface active agent present in fraction HI, renders the choice unsatisfaetory. Craig and Craig Effect U) LX75 Bowsx 1 x 8 &» ta?6 mi me m m x 2 x 10 Bowex SO x 8 + 4- L179 Hone Oomx SO x 1 4* From the results show* in Table ¥T it can be seen that the active principle was apparently removed from sample 1178 by the treatment with Bases: JO xl* On the assumption that the activity was adsorbed by the cation exchange resin, farther study was undertake to detemine the feasibility of using Bowex SO as a means of further purifying fraction HI. from the standpoint of practical considerations there are at least four variables to be noted when selecting the most desirable ion exchange 35 procedure for a specific problem* those may be referred to as the elutics* teelmlque, degree of cross linkage of the resin, mesh size of the resin and ih© structure of the resin bed* the type of exchanger, e*g., strong or soak acid, is also a variable. However, in the preliminary experiment mentioned above the strongly acid Dowex §0, salfonic sold exchanger, apparently adsorbed the active principle* Therefore, farther considerations were based bn the us© of tide resin* The technique of ion exchange may be thought of as a two step process (U2 ,h3>* The first step is adsorption by the ion exchange resin* The second step la the elution of the adsorbed ions from the resin, faring the adsorption step the conditions should be such that the affini­ ties of the resin for the lone to be adsorbed are made maximal* This Is ordinarily accomplished by suitable pH adjustment (b£> * Boring the elution step conditions should be such that the affinities of the resin for the adsorbed ions are mad© minimal. This is ordinarily accomplished in one of two ways (lit), either by suitable pH adjustment or by ionic strength adjustment* Elution by pi adjustment is achieved by altering the charge, usually a decrease, of the adsorbed Ions by adjustment of the pH of the eluent {incoming solvent}* Elution by ionic strength adjustment is accomplished by increasing the ionic strength of the eluent to the point where the concentration of the competing ions displace the adsorbed lens by mass action* Elution by pH adjustment, frequently referred to as elution analy­ sis # is frequently the method of choice especially in biochemical mix­ ture© (1*2). This method possesses the advantage of producing an eluate 36 (outgoing solvent) of lover Ionic strength and thus greater flexibility mith respect to subsequent chemical step*. KLution by ionic strength adjustment, also referred to as displace** meat ©hroraotography» utilise* tl^maximum adsorptive capacity of the Ion ssnhang© column (&!*), a feature particularly desirable in preparative mork. being this method of elution ^tailing" ordinarily does not Occur* Hemever, the bands are frequently close together (b$), Of the tm© methods of elution, elution analysis tends to posses* the greater resolving poser {MK Insofar as the farther fractionation of fraction H I mas concerned, elution by increasing the lonle strength of the eluent seemed to be the method of choice for several reasons, The active principle m s appar­ ently unstable to appreciable pE adjustment ill) , Bus to our assay it mas desired to collect the active principle in a minimum volume of eluate and a method easily adaptable to a preparative scale mas desired, A sodium chloride solution mas therefore chosen as the eluent* Aside from being readily available, th© presence of a small amount, physiological or belom, of medium chloride in the samples when tested for biological activity mould not be expected to he toxic to the mouse* The concentration mas chosen quite arbitrarily. A very concentrated solution mould tend to elute the activity in a minimum volume of eluate . However, inasmuch as the active principle mas apparently a macro molecule, too high an ionic strength might cause alteration of the molecule * Conversely, a very dilute solution mould be desired from the standpoint I of stability but mould be undesirable from the standpoint of dilution of 3? the motive principle in the eluate , A 3 M eolation, approximately a half saturated eolation, was therefore selected as one possibility. In the event the active principle mas loosely bound to the resin a wore dilute eluent would tend to render the separation more selective with respect to the other eonstituiients of fraction HI* Tlierefore, 0,3 H sodium chloride m s used at the first eluent, after sashing the column with water, to be followed by the 3 H solution, A second consideration in setting up an experimental Ion exchange procedure m s the choice of cross-linkage to be used. The resin crosslinkage In the case of Sow»x 50 in..defined as the per cent of divinyl benzene added to the vinyl benssne in tije polymerisation of this type of resin (h?)* the Bow resins the degree of eress-linkag© is designated by the rosaeral following the nmm of the resin e.g. 0owx §0 a W is a IS per cent cross linked resin, A resin of a very high degree of crosslinkage undergoes a minimum volume change with a change in ionic strength of the external resin phase (U6 ,h8 ,iih). On the other hand a resin of a low degree of cross-linkage undergoes a large volume change under these same conditions. Equilibrium between the external resin phase and the resin phase, is attained most rapidly with a low degree of cross-linkage (l*8,h9)* For column work a high degree of cross-linkage would be preferred inasmuch m a minimum volume change of the resin bed is desired, However, with reference to the tumor inhibiting activity, a low degree of crosslinkage would be desired Inasmuch as such a resin would be store porous and would approach an equilibrium between the external resin phase and the resin phase more rapidly . Ordinarily a resin with the highest 36 degree of cress linkage compatible with the separation to be achieved should be used (J46). Inasmuch asBowex 50 at12 was available la the laboratory it was selected, 'A ISper cent cross-linkage ndght be 00a* sidered a high degree of croes-llnkage inasmuch as Moore and Stein ($1) have recently used a fepea* cent cross-linkage for an amino acid and peptide separation* fbe size of the resin particles is also to be considered, however, It Is probablysemawhatlesa critical than the considerations mentioned previously. Boses: 50 i© available in a variety of mesh sizes including a colloidal sis© , The exeiiange capacity'of a resin'bed increases as the particle size decreases (52 ). Also, elution appears to be more rapid with a finer mesh resin (53 ) and the resulting elution Mp©aks" tend to be more sharp (h6)» However, the use of a fine mesh resin pro-* daces s decreased flow rats ($M * From the standpoint of the present considerations the flow rate was thought to be quite important inasmuch as large columns were to b® used at m m temperature. A rather coarse resin, 100-200 mesh, was therefore selected* The shape of the resin bed influences the flow rate and the resolv­ ing power of the colusm {$k) * This factor has vary Httla influence m the capacity of the column per unit amount of resin <5h> • Generally speaking the larger the ratio of the cross section area to the length of the resin bed the greater the capacity of the resin bed (55). Con­ versely, the smaller the ratio the greater the resolving power of the resin bed. 39 the four columns to be described in th® next section, the two smile? ones were purchased coimnerei&Ily. The first large column con­ structed (resin bed ? x 80 cm.) has a cross section area to length ratio of about 0,*>, The ratio was increased from tbat of the smaller columns inasmuch as a largo preparative column was desired. However, in the fourth (resin bod 5 % 8? cm,) this ratio m s decreased to about 0.2 to gain a greater resolving power* The flow rate perhaps is a fifth point to be considered inasmuch as the more rapid the flow rate the smaller will be the approach to equilibrium between the resin phase and the external resin phase at each section of the resin bed, Howswr , as mentioned previously the flow rate Is detensined to some extent by the choice of resin and the dimensions of the resin bed. The flow rate used might be considered quite rapid considering the active principle was believed to be a macro molecule. However, the time factor due to the instability of the mole­ cule was also considered in this connection. With these considerations in mind the chromatographic procedure described below was used for the further fractionation of fraction HI. Preparation of Ion Exchange Columns The Dewex 50 x 12, 100-200 mesh was purchased from the Bow Chemical Company, Midland, Michigan, The resin, as received, was slurried with a large volume of water and allowed to settle until the larger particles settled out. The supernatant, containing the line material, was decanted and discarded. The process was repeated until a homogeneous sample 1*0 resulted, 4s determined by uniform sedimentation and the absence of fins nabsrial suspended in the supernatant. The resin was than slurried with 2 S by&roei&oric acid and filtered in a large coarse sintered glass funnel * The washing with hydrochloric acid was continued until the re­ sulting effluent was free of the yellow colored material which is removed by this treatment* The resin was washed next with distilled water until the effluent was neutral and than with 2 H sodium hydroxide until the effluent was strongly alkaline* After washing with distilled water until neutral again the cycle, acid-water-base-water, was repeated once more* The columns were filled by slurring the resin with about two volumes of distilled water* A large funnel was attached to the top of the column by means of a robber stopper and the column filled with distilled water to the level of the bottom of the funnel* The slurry was then Introduced into the funnel, car® being taken that m air bubbles were trapped in the system* Sufficient resin was used in making the slurry that one filling of the funnel would fiH the column with resin* Columns prepared in this manner were free of sedimentation bands Which otherwise result due to the non-uniform particles wise of the resin* After the columns were filled with resin they were recycled through the above mentioned acid-base cycle and then washed with distilled water until no color was produced in the effluent upon the addition of pheiKxlphthalein. The columns were then considered ready for use* Buffering of Fracticn lll The various preparations of fraction I U ordinarily have a pH of 6.6(~ 0*2)* Inasmuch as the activity in fraction HI, without the U1 addition of 'baffoi*, apparently w o adooxtoed da Bowk $0 in the prelinl** nary mentioned above it wight be concluded that the tumor inhibiting substance was in a cationic form at this pH* It was there* fere them^it desirable to add a buff©r to fraction III to insure pH stability daring the adsorption step of the chromatographic separation. Fraction l H was therefore buffered at pi 6*h by adding m m basic potassium phosphate and di basic potassium phosphate as the dry salts immediately before chromatographing* The pH of the resulting solution was checked with a pH meter in all cases. In the course of this work four different sised columns sere used. The preliminary work sas done on ts© small columns. However, once to technique was worked out larger preparative columns sere used. Throughout all of this work the solvent changes were wade abruptly. Before a new solvent (eluent) was a&sitted into the column the previous eluent was developed to within a few millimeters of the top of the resin. This technique was used inasmuch as it was desired to collect the activity in a very minimum volume of eluate. This was especially true in the early work ton the stability of the resulting fractions to concentration was unknown. The first column used contained £ x £1 cm. (2 cm, diameter) of resin in the sodium form. Four milliliters of fraction III was admitted to the top of the column and allowed to pass onto the column to the upper level of the resin bed. The column was then developed with distilled 1*2 water until the eluate fis essentially free of colored xaaterial. Two 90 ml, fractions of ©luate were collected, using distilled eater as the eluent, (L233- and L23&-) . The eluent was then changed to 0*3 H sodium chloride and three 90 ml* fractions collected, 1236* m d 123?-). three 90 ml. fractions were then collected using 3*0 H sodium chloride as the eluent* These sere tested for biological activity after dialysis to remove the sodium chloride, (&23S&*, 1239- and 12l*Q~>. VPour n&lli^ liters of the same buffered sample of fraction H I was diluted to 90 ml. with distilled water and tested as sample (12i*li~) to serve as a control. It should be noted in the shove exporlsient that if the elution with 0.3 K sodium chloride solution had been continued Slightly longer the activity might have been recovered in the eluate from the 0.3 H sodium chloride solution rather than in that from the 3,0 H sodium chloride solution,. in the following experiment a slightly larger column (3 x 21 cm.) was used, along with a 1*0 ml. sample of fraction HI. fids volume of fraction HI proved to be too large inasmuch as activity was recovered in the first aliquot of eluate collected from the column, fhe remainder of the activity was found to be present in the sluate resulting from the 0,3 M sodium chloride solution (L2l*9~**}. At this point a large column (? x @0 cm.) was constructed with the intension of carrying out the separation on a large preparative scale. In this experiment 700 mL. of fraction III buffered at pH 6.U was placed on the column, Water was then used to develop the column until the eluate was essentially colorless, six 1 liter fractions having been 1»3 collected. Throe 1 liter fractions tiers then collected using 0.3 M ■odium chloride as the eluent, followed by four 1 liter fractions in iddeh 3.® M sodltita chloride was used as the eluent, An aliquot of each fraction m s tested for biological activity. Only the third fraction resulting from the 0.3 H sodium chlorldo eluent sea found to Contain activity (1*308— )* A duplicate experiment was carried cut and la this case the corresponding fraction was the only o m found to contain activity ( 317— ). This large column was accidently broken, probably due to the ex­ pansion of the resin during the regeneration of the column* The ordinary technique used for regenerating the resin was to pass 2 H sodium hydroxid® through the column until the eluate was strongly alkaline to insure the complete regeneration of the resin in the sodium form* At this point the resin has the minimum velum* in the cycle used* Therefore when eater was introduced to sash the resin bed until neutral to phenolpbthaleia a rather large expansion took places As a result of this experience a eel-* m m sac designed with a sintered glass disc at the top as sell as at the bottom, A ground glass joint was inserted near the upper disc for filling the column. The technique for regenerating the resin at the end of a run was also altered in the following way. After the last fraction resulting from the 3*0 H sodium chloride eluent was collected water was forced through the column in the reverse direction. This forced the resin up against the upper disc, The sodium hydroxide solu­ tion was then admitted, also in the reverse direction, 4t such a rate that the resin particles would slowly fall under the force of gravity hk a* the sodium hydroxide solution flowed up though the column. This technique served the added purpose of collecting tiie finer reelm partieles in the upper portion of the column time ellniiwrfcimg the possibility of their clogging the loser disc during an actual run. The sodium hydroxide solution m » passed through the column until a strongly Alkaline eluate resulted* The column see then sashed with distilled water in the ordinary maimer* The newly designed column contained (5 x 8? cm,) of resin, A l&$ ml. sample of fraction 2 H was placed on the column which m s then developed with the solvents described earlier. Fractions of the eluate were collected at four Mnute intervals using an automatic fraction collector, A total of 60 fractions of approximately 100 ml, were collected. The first fifteen fractions collected, using distilled water as the eluent, contained evidence of activity. A total of 3710 ml. of distilled water was passed through the column before the eluate became essentially colorless* Fifteen fractions were collected using 0.2? H sodium chloride solution as the eluent. The total volume of 0.27 M sodium chloride solution used was lkl9 ml. These fifteen samples were combined, in groups of two, except that the last three were combined and assayed for biological activity. The biological activity was found to be contained in the eluat© between 61? ml, and 101*2 ml. (133?-*, and 1*338^**). Fifteen fractions were also collected using 2.? M sodium chloride solu­ tion as the eluent. Total volume of 2.7 M sodium chloride solution 172h ml. These fractions were combined in the same manner as the previous fifteen samples were and assayed for biological activity after dialysis, hS Activity m i found to be contained In the duate between the limits ol 1006 ml* and 1169 ml, sad X*3h7J1}. It should be noted that these two activities were separated by about lldO ml. of ©Xante, In future discussions the activity contained In the eluate result­ ing from the 0 J I (ca) eluent is designated as faction 1ST sad that resulting from the 3.0 (©a.) eluent as fraction V. The following chromatographic procedure was used in the preparation Of all future samples of fractions IV and V. The same column, contain­ ing C5 x 6? cm.) of Bowex $0 % 12 in the sodium form* described previously was used, The technique of preparing the resin, filling the column as well as the technique for regenerating this column were all as described earlier. Thro© hundred milliliters of fraction XXX were buffered at pH 60h by the addition of 0.6>9 g, of mono basic potassium phosphate and 1,526 g. of di basic potassium phosphate as the dry salts. The buffered sample of fraction III was placed on the column and allowed to drain to the upper level of the resin bed under the force of gravity. This procedure usually required about 30 to 1*5 minutes, The column m s then developed under a positive pressure of about 19 cm, of mercury using the solvents described below. Under these con­ ditions about 15 ml, of eluate m s collected per minute. This corres­ ponds to & flow rate of Q.77 cm, per mia, on a column 5 cm, in diameter. m Not© tumor diameter. 46 4 total of 294© ml * ©f distilled water m s passed through the column and tlae entire elwat© diaeaydod* The distilled water was do* voloped to i&tfcifc a f©w;adXXimetars of the upper level ©fihe resin bad before the admieaion ©f the Second solvent . the second solvent used m s 15©Q *&» of ©.©©?) IT sodium chloride solution, The first ZOO ail. of this eluate were discarded. the next 120© ml. were collected as faction If, the last 100 al, were discarded. This solvent m s also developed to within a few millimeters of the resin bed before admitting the third Solvent, The third solvent used was 15©© s&, of 2.65 (-0*05) H sodium chloride solution, the first 600 ml. of this eluatc were diyarded. The next 9©© v&* were collected as fraction ?+ Blacuaaioa of Ion Ifochangs frecednre. This procedure presents no particular difficulties. However, certain aspects of it might b© iznrestigated further. The 12 per cent cross** linkage is perhaps quit© high. A lower cross-linkage might be investigated. Moore and Stein (Si) report the use of Bemx 5© x h for the resolution of peptides containing up to 8 or l© amino acid residues and suggest Bowex 5© x 2 for larger peptides. in is indicated by the next section, tho activity is apparently more stable to acid than it was originally believed t© be. This might suggest the possibility of buffering fraction III at a lower pH which would tend to increase the cationic nature of the active principles and thus their ability to be adsorbed by the resin. If such a technique proved to be hi feasible a Iftygw quantity ©f fraction XIX could be applied to the eolwsnu the possibility ©f concsntratJuag fraction XXX before placing it on the column might also be considered* A smaller volume ©X sample aolutism would tend to Improve the rcsoluticm due to the activity being adsorbed in a narrower band near the top of the column* It would also be desirable to collect a taller volisae of eluate representing fraction X? and also fraction V* the empirical method used necessitates' the- ooHeetion of a very wide band of eluate for each fraction since the active principles would not be expected to be eluted in exactly the same volume of ©luat© each time* Such a technique introduces the possibility ©f other substances also being contained in the sXu&is collected inasmuch m tbs elution bands would be expected to be very close together using the elution technique described in the prev­ ious section* treatment of Fraction XIX with Acid and Cuprous Oxide In an early study of ih® hydrolysis products of fraction IV using paper chromatography some indication was given that this fraction might contain cystine. This later proved not to be the case * However# in the intervening period m experiment patterned after the precipitation of gXutatiiion© (56) was carried out* For this experiment 63 si. of fraction XXX was wanned to 50 0, and $ S ml* of a 3 H sulfuric acid solution added with stirring* to this solution was added a suspension of cuprous ©add©, prepared by boiling h$ 2© of Benedict*s solution with an excess of gLucoa© wad removing t o precipitate of e f m oxide % immfcrifugation, The mixture was allowed to stand for four hours to the refrigerator* After the four hour period a considerable M i of precipitate was observed to hare settled out* The precipitate was removed hr centrifugation, the super** oatant being designated as fraction B. The precipitate, after washing with water, was suspended in 5© si* of distilled water and saturated with hydrogen sulfide gas* The precipitate of cuprous sulfide was rewoeed fey centrifugation and discarded. The supernatant was designated as fraction E* Fraction 1 was aerated with nitrogen gas to remove any remaining hydrogen sulfide and tested as sample (Liil2-) after adjusting the pH to 6.1* with very dilute sodium hydroxide solution. Fraction D mas adjusted to pH 6 ,h with very dilute sodium hydroxide solution and tested for biological activity as sample (Mil©**} (retest^*). In a duplicate experiment an aliquot of fraction 0 was tested for biological activity after neutralisation (Hl9^~). A second aliquot of fraction B was dialysod and to dialyoat© tested as sample (Lh2W ) * Discussion of Cuprous ©add® Eapei^east Although the active principle was not precipitated by the cuprous oxide as might have bees expected if a sulfiiydral compound had been present the experiment provided some wortiwwhiXe information. fb® active principle was apparently stable to to acidity used in this experiment. This is in disagreement with to observation made by aitchle (23), However, the discrepancy is possibly explained by to k9 technique used to neutralize fraction D. Bitehie (23 ) reports having Acidified preparation to pH 2.0 with hydrochloric acid and after too tears neutralising the Maple to pH 5*1 with 10 per cent sodium hydroxide solution with a subsequent loss of tumor inhibiting activity, la the preparations just described fraction D vas alleged to remain at an acidity of less than pH 1 for fear tears. However, the preparation nmm then carefully neutralised to pH 6 *1* with a very dilute solution of sodium hydroxide, the pH 6»h see selected since the pH of fraction I H is ordinarily 6*1* or slightly above* In Bitcbiete case the loser pH mlgjbt possibly have resulted in alteration of the active principle before tte preparation was tested for biological activity. A second observation can also be made as a result of the experiment described above if samples (Lt21~*) is cc^parsd with sample (LllS-**) (Table XX). These t«© samples sere prepared at comparable dilutions* Honever5 sample Lh!5 was diluted 1«5 due to its toxicity in the mouse teat and resulted in a (-£-) effect. Sample 11*21 was not diluted making a larger dose possible with a subsequent (&0 effect. This observation would seem to indicate that some of the toxicity of fraction XXX m m either removed or destroyed by the treatment with cuprous oxide in tte acidified solution. Further purification of Fraction X? It was thought desirable at this point to attempt to recover a solid product from tte fraction eluted with 0.3 M sodium chloride solution which still retained biological activity. 50 Fraction 17 was therefore dlalyaad in ¥±sking casing on the rot&t* ing external liquid type dlalyxer described earlier. The dialysis was carried oat against frequent changes of distilled water at ?°C* The diffusate was discarded in all eases, The dialysate varied in velum© from 1230 to 1255 ml* in the seven eacperiraenis carried oat* The dialysai© was concentrated in vacuo on the revolving concen­ trator described by Craig «t al* (2?) using a water aspirator. This apparatus is well suited to the concentration of biological materials for at least two reasons* The sasapl© container, an ordinary round bottom flask, is made to revolve thus increasing the surface area of the sample to essentially the surface area of the flask used* This facilitates a very rapid rate of evaporation even at relatively' low temperatures. Secondly it is not necessary to pass air or some inert gas through the solution to prevent bumping* Is these e^eriments the sample solution was maintained at a temperature not greater than 29° $ * The concentration to a small volume (3-IS ml.) requiredabout four to five hours. The concentrate was then dialysed again to remove the last traces of chloride ion as indicated by the addition of silver nitrate solution to an aliquot of the dialysate. Three different techniques were used in the attempt to recover an active solid product from the chloride free dialysate, Xm the first experiment the m m ®le was concentrated to a volume of 3 ml. and dialyced free of chloride ions. To this chloride free dialysate 15 sdL. ©f acetons were added and precipitation allowed to take place overnight at 1°C0 About 1-2 mg* of dry residue was recovered by $1 centrifugation followed by aiding in a vacuum desiccator for two hours, $he entire product was dissolved in 80 sail, of physiological saline {at Sloan-Kettering Institute) and tested for biological activity (I4©0?) {retest dll, pretest dll, 1*2^-), tn tfee second eaqperiment the clsloride free dialysate was concen­ trated to dryness by the technique described previously, About H mg. of solid material was recovered* Of this material, 8,1 rag, were dis­ solved la physiological saline (at Sloan-Eettering Institute) and tested for biological activity (1^06^-) , B* the third eajperiment the chloride free dialysate from two rums on the ion exchange column was frozen and lyophiliaed for 2it hours, the Xyophilization apparatus was constructed by submerging a !>Q0 ml, throe necked, roaad bottom flask in a ethyl alcohol solid carbon dioxide bath to a level about 1 inch above the lower end of the nodes, By means of ground #&as Joints one neck wm attached to a 50 ml* round bottom flask at an angle of 9& degrees, the 5© ml* round bottom flask contained the freasn sample. A stopcock was placed in the middle neck. The third neck was connected by means of a glass adaptor and a short length of robber tubing to a second "dry ice trap* which was in turn connected to a vacuum pump. This apparatus has the advantage of being constructed almost entirely of standard laboratory equipment. It is also quite versatile inasmuch as several samples can be attached to the one neck of the three necked flask by means of suitable adaptors, being the lyophillzation technique Just described about 86 mg* of solid material was obtained. Tide product was not dried in a mm stored in the deep frees*, Fourteen end one-half milligrams of this predict mm eent to Sloaa~Kett«ring Institute, desiccator but however, the results ef the assay were not completed when this thesis ess Witten {fiWbO n© result), Further Furiflcstion of Fraction V in attest see also made to recover a solid product from the free* tiom eluted from the column withJ.G M sodium chloride solution, The fraction was dialyzed and concentrated to a m m 11 wolwme by the seme technique described for fraction IV, in ©is© experiment 15 ml, of acetone sere added to 3 ml, of concen­ trate which had been dialled free of chloride ion. After standing overnight the precipitate was collected by centrifugation and dried In a vacuum desiccator, The entire product (ca. 19 mg,) was dissolved in physiological saline (at SIoan-Kettering Institute) and tested for bio­ logical activity (l4*03~). In a second exper&naent the chloride free dialysate was concentrated to dryness• Only about 3 mg, of product was recovered. In a repeat experiment, in which the last dialysis was emitted, 32 mg. of product was obtained, the two preducts <3 mg, and 32 mg,) were combined and 8*5 mg* used for the biological assay (Li$7~). H© attempt has boon made to lyophHize the chloride free dialysate from fraction V, Ufipjjssien of ,the IHzrification of Fractions Iff and V If sample I»hOO, in which tha ciilorid© free dialysate was precipitated with acetone, is compared to sample U 4O6 , in which the chloride free S3 dAalysat© was concentrated to dryness, several observations can bo ■■■SwWP^f. Sample IhOO m b dissolved in 80 ml, mi physiological saline and used In the first test at a doe© of 0,6 ml, (see Appendix) twice daily, la tense of dry material this corresponds to approximately 1,5 mg,/kg,/day. In the two retests the above solution was diluted 1*2 and a total of approximately 1 ml, given daily, At the final dilution 1 nil, corresponds to About 0,4 mg ,/kg./day in iemr of dry material. In the ease of ample 1406 a daily dose, in terms of dry material, of 6 rag./kg./day can be calculated. the wide difference in the tolerated daily dosage would suggest that sample 1*406 was much less toxic than sample 1400, At the dosage given sample 1406 also gave the greater Inhibition (see tumor diameterAppendix). From these considerations it would mwn that the technique of con** centratimg the chloride free dialysate to dryness was to be preferred over the technique of precipitation with acetone. Although the result® of the assay of sasiGple 1440 are not available this sample might be expected to be the least toxic of the three since the lyophiliaation would seem to be preferred to concentrating the fraction to dryness due to the temperature at which the operation is carried out. The explanation for the loss of activity in both sample 1403 and sample I40T is unknown. Apparently the activity present in fraction V 5k It not sufficiently stable to withstand the isolation procedure used, kyophllissation of this chloride free dialysate might be considered since the conditions would seem to be more desirabla, omnm xv B&mtK S A3 TO THE CHEMICAL CCKPGSITIGB CT FRACTION XV Acid Hydrolysis of Fraction XV About 1 lag. of fraction XV ^ dissolved in 0.1 ml* of 6 K hydro­ chloric acid and the solution sealed in a capillaay tube* The tube was placed in an oven at A05°C* for 12 hours (57). After cooling the con~ tents of the tube a®re placed on a 2 inch natch glass mad® of poly­ ethylene (57) and evaporated to dryness in a vacuum desiccator* The residue mas dissolved in about 0*2 ml* of distilled eater and evaporated to dryness a second tine * The resulting residue, essentially free of hydrochloric acid, was dissolved in about 60 ml* of a 10 per cent aqueous solution of isopropyl alcohol which Block (58) has reported to be a good preservative for an amino acid mixture* A duplicate hydrolysis was also carried out using about 2 mg* of fraction X? and a correspondingly increased quantity of the other materials* These two hydrolysates dissolved in the 1© per cent aqueous isopropyl alcohol solution sere used for the chromatographic study to be described later* Alkaline Hydrolysis of FractionlV The hydrolysis technique used is described by Block (58) * About 10 mg* of fraction IV was dissolved in 10 ml* of Ih per cent barium 55 56 hy$roxid© solution* The resulting solution was heated in an oil bath at about 125® G* under reflux for 20 hours* The bydrolysste, after cooling m m made very sll#itly acid (to litmus paper) with 1 K sulfuric acid and the precipitate of barium sulfate removed by nitration* The barium sulfate precipitate was mashed sitb about $0 sal* of hot eater, containing a few drops of acetic acid* and the washings combined with Idle original filtrate* The combined solution saa concentrated to dryness on the revolving concentrator and the resulting residue desiccated overnight over calcium chloride. The residue was then suspended in about 0*5 xl* of a 10 per cent aqueous solution of isopropyl alcohol* This solution was used for a chromatographic study to be described In a later section and is referred to as the alkaline hydrolysate of fraction IT, ^ration of Fractions for Terminal The method of preparing the terminal dinitrophenyl derivative of peptides described by Sanger (59) was used. About 3 mg, of fraction T9 was suspended in 0 ,3 ml, of a 1 per cent aqueous solution of trimethylamine contained in a 1 ml* volumetric flask. To this solution m m added approximately 50 mg, of l-fluoro-S,h~dinitroben&ens (IUFB) (purchased from the Aldrich Chemical Company, Milwaukee, Wisconsin) contained in 0*6 ml* of ethanol. The mixture was shaken mechanically for two hours* After being shaken the mixture was diluted with about 10 drops of the 1 per cent trimethylamine solution and then extracted with four 0*75 ml* portions of ethyl ether* The organic layer, containing the unreacted $7 iras discarded * The aqueous layer was evaporated to dryness In a vacuum desiccator. The residue from the evaporation of the aqueous layer m e dissolved in Sheet 15 drops of constant helling hydrochloric add solution and Sealed in a eapiHary tube, The sealed tube m s planed in an even at 1©5° ©« for eight hours* After cooling the tube m s opened and its contents diluted about twice with distilled water, The hydrolysat® m s then extracted with three 0*75 ml* portions of ethyl ether. The cook bined organic layer, containing most of the dinitrophenyl amino acids (BHPM) was evaporated to dryness on the steam bath. This fraction ms designated as the "HJPAA* fraction, however, it m s not used for further study {see discussion), The aqueous layer m s evaporated to dryness in a vacuum desiccator and the residue suspended in water and evaporated to dryness a second time. The resulting residue, containing the unre­ acted amino acids for the most part, m s suspended in a small volume of distilled water and designated as the "non TMTAk** fraction. In a duplicate experiment f mg, of fraction I? was used with a corresponding increase in the relative quantities of the other materials. The "non 8BPM* fraction m s used for a chromatographic study to be described in a later section. Paper Chromatographic Prefectures A ciuroroatographic procedure very similar to that described by Sanger (60) was used to chromatograph fraction IV, the acid hydrolysate of fraction IV, the alkaline hydrolysate of fraction IP and the "non HBPAA* fraction. 56 Sheet* of Whatman Ho* 1 filter paper approximately 17 inches square were used. The sample to he chromatographed was applied at one corner of the paper h inches free either edge. A reference spot, for compara­ tive purposes, was also applied such that it was k inches from the bottom edge of the paper in the direction of the first solvent and 2.5 inches from the bottom edge of the paper in the direction ofthe second solvent. Beth the sample solution and the reference solution were applied in 5 ul. aliquots by means of a micro pipette. The spots were dried by mean* of a low stream of air sold an infra-red lamp between applications. The chromatograms were developed % the ascending technique using 12 chromatograms per r m in the Ohromatocab mentioned previously. Before being developed the chromatograms were equilibrated for 16 hours with the vapor phase of a solution composed of 0,125 g* of potassium cyanide, 5*2 ml* ©f concentrated ammonium hydroadd© (28.9$ $S3) and sufficient distilled water to make $00 ml. of solution. After the equilibration period a solution of phenol saturated with distilled water was added to each trough and development allowed to tabs place at room temperature for 2k hours. After the 2h hours development with the first solvent the chromatograms were removed from the cabinet and dried. Two methods were used for drying the chromatograms at this point. The first method consisted in drying them In a forced draft chromato­ graphic oven at about 1*0-50° C. for eight hours and then overnight with only the forced draft on. In the second method the chromatograms were dried In the forced draft oven with no heat on for about one-half hour 59 and them washed with ethyl ether. They were then dried in the even wltt only the forced draft on for about six hours. So difference in tt# final result was observed between the two methods. Before being developed with the second solvent the chromatograms were equilibrated for 18 hours with the aqueous layer resulting from the ceabination of Ji©© ml. of n-butanol, 500 ml. of distilled water and 100 ml. of glacial acetic acid. This solvent was made up 1*8 hours before it was to be used. After the equilibration period the chromatograms were developed with the organic phase from the above bwtanol-wateracetic acid solvent for 2b hours. After developing with the second solvent the chromatograms were dried in the ehromatographic oven with only the forced draft on for about ©no-half hour. The following techniques were used for detecting spots on the various ehrematogrwms* (a) The chromatogram was sprayed with a 0*2 per cent solution of ninhydrin in a water saturated solution of n-butanol followed by heating at about 90-95° C. for about 15 minutes (3?). (b) The chr©a»t©gram was sprayed with a solution composed of 0.93 g. of aniline, 1.66 g. of phthalic anhydride and n-butanol saturated with distilled water sufficient to make 100 ml, of solution, After being sprayed the chromatogram m e heated for about 15 minutes at about 10O° C. (37), (e) The chromatogram was sprayed with a 0.0k per cent solution of bromeresol green in ethanol (37) - 60 (d) The ofcxmatograa m e viewod under ultra-violet light In the darkroom C37) * A MtneraUght Hodel SI Manufactured by Ultra-fiolet Products$ Inc., South Pasadena, California sea used as the source of ultra-violet light. The spots made visible by the above techniques were marked by means of a small dot In the center of the region of greatest color intensity. Sines a solution of the amino acid leucine mas used as the reference solution the reference spot mas made visible on those chromatograms not otherwise treated with the ninhydrin reagent by treating the chromatogram with this reagent as described above after previously hairing subjected It to one of the other techniques for detecting spots. Inasmuch as a solution of leucine was used as the reference solution a *reference leucine* (8^) value mas calculated for purposes of compari­ son. The R$, value was defined as the ratio of the distance a given sub­ stance migrated to the distance leucine migrated in the same solvent system. The distances were measured to the nearest millimeter with an ordinary ruler. The chromatographic procedure just described m s designated pro­ cedure A. k second chromatographic procedure m s used to detect the presence of phenylalanine, leucine or isoleucine in the acid hydrolysate of fraction If and also in the "non DUPM® fraction since these amino acids were not resolved by procedure A. This second chromatographic procedure m s also used to chromatograph the alkaline hydrolysate of fraction If. 61 For this procedure sheets of Whatman Ho* 1 filter paper 7 x 22 inches wore used. The gessittl technique used hero Is described by MeFarren (61)* The papers were buffered by dipping them In borate buffer at pH 8*i* end drying at room temperature * Fire samples were applied to each chromatogram. After applying the eastplee by the technique described for procedure A the ehroaatogra®® were equilibrated with the vapor phase resulting from saturating the borate buffer with a 1*1 (v/v) solution of beaayl alcohol n-butyl alcohol in a 12 ac 2k inch glass cylinder* After an 18 hours equilibration period the chromatograms were developed by the decending technique using a solvent composed of a 1*1 solution of benzyl alcohol n~butyl alcohol saturated with the borate buffer. Development was allowed to tabs place for 2b hours. After developing, the chromatograms were dried in a forced draft oven with no heat on and sprayed with a 0*2 per cent solution of nin~ hydria in n-butanol saturated with a 2 per cent acetic acid, They were then heated for about 15 minutes at 90*»95°C. For comparative purposes known samples of leucine, isoleucine and phenylalanine were run bn the same chromatogram with the acid hydrolysate of fraction 1? and the wnon MPAA* fraction. M authentic sample of tryptophan was used for comparative purposes m the chromatogram of the alkaline iiydrolysate of fraction IF. The distances migrated by the known ^Inn acids were compared with the distance which the various ninhydrin positive constituents of the unknown samples migrated. 62 The chroiaat©graphic procedure $mt described m m designated as procedure B. Beeult of Bhramato&raphlc Study Fraction 17 m m chromatographed by procedure A and th© chromatogram treated with th© niidJydrin reagent described under detecting reagents tor procedure A. Ho ninhydrin spots resulted* The add hydrolysate of fraction 17 m s also chromatographed using procedure A* Tmlv© ninhydrin spots and one blue bromcresoX green spot resulted. The broiacresol green spot appeared to coincide with one of the ninhydrin spots, however, this ninhydrin spot m s observed to has® a very reddish color and to be quite faint making the evaluation of its values very difficult. The other ninhydrin spots sere of the typi­ cal blue-red color. Ho spots were detected with the aniline-phtiialAc anhydride reagent or under ultra-violet light. The % value of the 12 spots in each advent is given In Table VH. The spots are numbered in the increasing order of their % value in solvent no. 1 for purposes of future discussion. Spot no. 2 Is the spot which produced a blue color with the bromcresol green reagent and the &£ values for this spot in Table TOC are calculated on the basis of the spot given with this reagent. Table 7XX1 contains the values of 19 known compounds which mr© chromatographed by procedure A and the spots detected with the ninhydrin reagent. The fourth column in this table contains the number of determin­ ations which were used to determine the values given in this table. 63 t m m n x t m Rf y i m m w t m w m m x s x & m m & m s or m m m m iv BL Hximber Solvsnt So. i TallM siolVent So. ^ 1 0.19 0 .2 2 2 0 .2 1 0.16 3 0 .3 2 0.30 b o.t*5 0.22 5 0.S3L 0 . 21* 6 0.61 0.31 7 0.69 0.35 s 0.75 0.52 0.91 0.61 10 0.95 0 . 12* nb 1.03 0 . 1*2* 12 l.o lf 0.22 a Breroerosol green spot. 1(5 Very faint spot. 61* tmmnxz the \ rmjm In solvent no* 1 and theB^ value for aspartic acid (Table VIII) in this solvent do not agree as well as would be desired* However, since a new spot corresponding to aspartic acid did not result on the chromatogram on which the nine amino acids listed in Table IX and the acid hydrolysate of fraction IV were 69 applied, spot no. 1 is boXioved to bo asportio add* The discrepancy in vain® Is possibly due to th® nearness of spot no* 2 since this spot also produces a color with nlnhydrim making the location of the center of spot no* 1 somewhat difficult* From the data shown In Table® T O and VTII It may be concluded that fraction I? contains a peptide which on acid hydrolysis gives rise to th® amine acid® shown In Table IX plus a '"basic11 substance (spot no* 2) and leucine* Tryptophan is believed to be absent. Inasmuch as the amino acids appear to be present in th© acid hydrolysate of fraction IV In about th® same molar ratio, as Judged from the color intensity of their nlnhydrin spots on the chromatogram, a rough estimate of a minimum mole­ cular weight of shout 1500 might be made. It might also be concluded that this peptide 1® a cyclic one for the following reasons* The chromatogram of fraction IV did not produce a ninhydrin spot. lysine appears to be the only amino acid with a terminal amino group as Judged from the absence of spot no* 10 from the chromatogram of the "non I3NPAA" fraction* The presence of the yellow spot (B^ values 1*10 and G.SW on the chromatogram which also proved to be ninhydrin positive might suggest that this spot Is th© H® DUP derivative of lysine making a cyclic structure seem quite likely. A study of th© second new spot (B^ values 1.IG and 0.92) on th® chromatogram of the "non CMPM* fraction lias not been mad®. However, since this spot Is very faint it is considered possible that it repre­ sents a small molecular weight peptide inasmuch as the hydrolysis in th© sealed tube was only for a period of eight hours. 70 A number of possibilities for further study would sees to be sug­ gested by this work* The H BHF derivative of lysine should perhaps be prepared (63 ) and chromatographed by procedure A* Also the "B8PAA" fraction should be studied, possibly by the method of Blackburn and heather (6t) involving paper chromatography. The alkaline hydrolysis should also be repeated and the hydrolysis products identified. An early experiment should also be conducted to determine the nature of spot no. t (Table TU) . Since this spot has such a loir Rj, value when chromatographed by procedure A, other Solvent systems should be investigated, Spot no, 2 has been eluted (to be dsserlbed later) from a chromatogram developed by procedure A, However, this spot is so close to the aspartic acid spot that this amino acid almost certainly contaminates the duets* It might also be desirable to determine which isomer of the individual amino acids is present in the acid hydrolysate of fraction 17. Such a determination has been made by spraying a chromatogram of the amino aelds in question with a B-amtse acid oaddas© preparation (65), If the amino acid sequence is to be determined the method of Sanger and Thompson (59) might be suggested. Spectroscopic Studies Being fraction 17 Throughout the course of this work several attempts have been made to correlate the tumor inhibiting activity with the absorption spectrum of the various preparations. However, such a correlation apparently has not been successful. 71 An aqueous solution of fraction 1?, which was prepared in the dry state by lyophilication was used to obtain an absorption spectrum in the region from 2b© mu. to h©0 mu. the measurements were made at 5 mu, intervals, except in the 25© to 29© mu. region where they were made at 2 mu. intervals, on a model M Bectesan spectrophotometer* An absorption maximum was found to occur at about 276 mu* and a minimum at about 260 mu* 0a several occasions the absorption in the 23© to kOO mu. region was measured using the various eluates f r m tb© Bewex 5© columns described previously* Inasmuch as these fractions were all very dilute no significant absorption wpeaks* were observed* However, as a result of the above maximum observed at 276 mu., the optical density of the various fractions collected from the ion exchange colvam might be measured at 276 mu. and the resulting absorption (optical density) plotted against the volume of eluate collected at each fraction, ©sing this technique a correlation could be made between those fractions which exhibit a maximum biological activity and those exhibiting a maximum absorption at 276 mu* If the two Mp®aks* coincide a correlation might exist between the physical measurement and the biological measurement* If the two "peaks# do not coincide fraction IV must represent an impure fraction. In another experiment the area represented by spot no. 2 (fable VII) was out out from three chromatograms developed by procedure A* fhe spot was eluted with about 3© ml* of distilled water and the eluate evaporated to dryness, on the revolving concentrator9 and the residue desiccated 72 overnight * After desiccation the residue was dissolved la a minimum volume of water, about 3 ml., and the absorption measured in the 2k0 to too mu* region. An absorption maximum was found to be present at about 276 mu. and a minimum at about 26$ mu* Due to the nearness ©f the aspartic acid spot (spot no. 1, Table VII) on the chromatogram the eluate lust described quite possibly was contaminated with this amino acid. However, since the absorption spectrum of aspartic acid does not show an absorption maximum at 276 mu* (66) it would appear that the absorption at this wave length is das to the "basic” substance (spot no* 2, Table VII). An aliquot of fraction IV which was prepared in the dry state by lyophilization was also used to prepare a nujol mull on sodium chloride windows. The absorption spectrum was determined using the Perkin-Elmer spectrophotometer model 21 in the region from 2 u. to about 15 u. A photographic reproduction of tbs resulting spectrum is presented as Figure 2. Although the spectrum in itself does not appear to present too much information it was felt that it might be of value for compara­ tive purposes and for correlation with additional chemical studies which might be conducted in the future. The spectrum would seem to be in agreement with the peptide structure postulated for fraction IV Inasmuch as it appears to contain the typical bend at 3.05 u. and also the amide I and amide II band at about 6*1 and 6.5 a, respectively. A number of other weak absorption bands can be observed, which if examined by an experienced spectroscopist might help to corroborate the purposed amino acid composition of fraction IV* 73 FIGURE 2. 1 THE ABSORPTION SPECTRUM OF FRACTION IV IN THE INFRARED REGION WAVBLBNOTH 8 MICRONS 9 4 9 0 7 CHAPTER V COKCiasiOHS referring to the assay results for the various samples (see Appendix) It ©an he seen that nearly all of the samples tested mire toxic to the nice as Judged by the weight less of th® treated mice as compared t© the controls and also by the number of deaths. It would also appear that the toxicity of fraction If (samples U 4OO and U 4O6) was approxi­ mately the same as the toxicity of fraction H I (see Table XI and the Appendix) * However, this might not actually be the case since the maximum tolerated dose was given In each case. If the toxicity is compared to that of azaserlne and amethopterin, two of the most potent inhibitors of sarcoma 180 f (25 ) a comparable toxicity can be seen. Azaasrine at a dose of 5 mg,/kg,/day resulted In a weight change of {-!i.0/~0.5) and in a second test of (-U.5/-1.5). One or two deaths occurred during the first test, Amethopterin at a dose of 1 .5 mg./kg./day resulted in a weight change of (-2 .5/-1 .0 ) and in a weight change of {-3.0/-G.5) at a dose of 2.0 mg./kg./day. I» the second test one ear two deaths occurred. At these dosages both ©f these compounds exhibit a greater Inhibition than was exhibited by the fractions prepared in this work. The tumor diameters in the two tests for asassrlne were (0.30/0.96) and(0,27/1.22). For aroethopterin they were (0,h5/0.89) and (0.22/0.92). 7h 75 It would therefore seem desirable to attempt to selectively hydrolyse the peptide structure (fraction UT) to obtain a biologically active residue. Hitchie (23 ) attempted to detoxify one of his preparations by treatment with trypsin. Although the results were inconclusive farther study on « mere purified fraction (fraction 1?) should be undertaken. In the course of this work a considerable purification of one active principle (fraction IV) has been described end evidence presented to indicate that this fraction contains a cyclic peptide composed of ten amine acids and a "basic* substance. The purification of a second active fraction (fraction T) was also described, however, attempts to obtain a solid product containing activity fv m this fraction were unsuccessful. We studies as to the chemical composition of fraction ? were carried out* It would seen desirable at this point to study the sedimentation behavior of both fraction axel fraction V, especially fraction I?# in the ultracentrifuge. Such a study would be expected to supply inform­ ation ms to the purity of these fractions and might also be used to obtain a molecular weight measurement* A H of the assay mark with sarcoma 100 was carried out on a routine basis* It would be interesting to investigate the tumor inhibition somewhat further and also to screen the activity against other tumors* 76 mmmi 1* A method for th© preparation of two tumor inhibiting fractions from th® mushroom Boletus ©dulls was described. 2* Evidence indicating that one of the tumor inhibitors is a cyclic peptide containing 10 senin© acids and a "basic” substance was pre­ sented* 3* Numerous suggestions for further study were presented 77 BXBLXGGRAFHf 1 . Woglom, W. H., "Approaches to Tumor Chemotherapy," A .A .A.S., p. 1 , 2* Greensteln, J. P"Biochemistry of Cancer,11 1st, ed. p. 163ff, Academic Press, Hew fork, (I9b7). 3* Greenstein, J. P., "Biochemistry of Cancer," 2nd. ed. p. 276ff, Academic Press, Heir fork, (195b)* k. 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A. klBU© (1955). 22. Lucas, I* H., Unpublished data. 23. Ritchie, A. E ., "Studies in the Isolation of an Anti-tumor Growth Factor from Boletus edulis.* Master of Science Thesis, Michigan State University, (l95h). 2U. Stock, 0. C., Am. J. Med., 6, 658 (1950). 25. Clark®, B. A., Cancer Research, Supplement Ho. 3, Ik (1955). 26. Stock, G. G., Adv. CancerResearch, 2, 1*25 (195k). 27. Craig, L. C., Gregory, J. P. and Rausman, ¥., Anal. Chem., 22, 1U62 (1950). 28. Djang, S. S. T*, Ball, 0. B. and Lillevik, H. A., Arch. Biochem. and Biophys., k£, 165 (1952). 29. Colin, H. and Legrand, G., Corapt. rend. 211. 1*50 (191*0). 30. Kelson, J. H. and Dawson, C. R., Advances in Enz^nol., k, 99(l9kl). 31. Lendeberg, G., Physiol. Flantarum, 1, 196 &9k8)$ C. A. 1*2, 7839g (19k8). 79 32. Voinovitch, I., Cheftol, Hv Durocher, J. and Kahane, E., Cowpt, read., j>28, X823 (1949). Voinovitch, I„ Compt. rend., 2J1 , 16? (1950)* X5in°Cl952j r*' 8^3L1* soc. chk, biol., 337 (1951)} C. A. 1*6, 33* Voinovitch, I., Bull. soc. chim. biol., 33 , 1414 (1951>| C. A. 46, 7l80f (1952). 34. Grabar, P., Voinovitch, I. and Prudhomm©, R. 0 ., Biochim. ©t Biophys. Acta, 412 (1949). 35. Kuttner, R. and Wagneich, B*, Arch. Biochem. and Biophys., 43, 80 (X953)* 36 . Petering, H. 0 ., Unpublished data. 37. Berry, H. K., Sutton, H. h*, Gain, X>. and Berry, J. S., "Biochemical Institute Studies IV," Ho. 5X09 p. 22, University of Texas, Austin, Texas (X95X>. 38 . Reid, W. ¥., Hature, 166. 569 (1950). 39. Bentley, H. R. and Whitehead, A. K,, Biochem. J*, 46, 341(1950). 40. Bakin, B. B., Biochem. J., 12, 290 (1918); J. Biol. Ghem., 44,499 (1920). 41. Craig, X*. C. and Craig, B., "Technique of Organic Chemistry,” Vol. HI, p. 171, Interscience Publishers, New York (1950). 42. Cohen, W. S ., Ann. H. Y. Acad. Sei., JJ, 204 (1953). 43. Samuelson, 0., "Ion Exchangers in Analytical Chemistry,”p. 45, John Wiley & Sons, New York, (1953). 44. Partridge, S. M., Discussions Faraday Soc. No* 7, 296 (1949). 45. Samuelaon, 0., og. J&££** P* 63. 46. Moore, S. and Stein, W. H., J. Biol. Chem., 192, 663 (1951). 47. Bow Chemical Company, Midland, Michigan, Personal Communication, 48. Samuelson, 0., op. cit., p. 13- 80 b9, Bergs**, <1. l „ Aim. H. I. Aesd. Set., ,g£, M g (1953). SO. Bunin, K. and Myers, R. a.. "Ion Exchange Reeine," p. 61, Join m * y A Sons, H. *--- ‘ 51. Mocrre, S. and 3tein, W. H., J. Biol. Chen., 211. 893 (1951*). 52. Saaraelsen, 0., «%. pit., p. SS. 53. Sosraelson, 0 „ alt., p. 66. 5b, Sawuolson, 0., flit., p. 83. 55. Schubert, J., "Ion Exchange,” p. (191*9). Edited by Hacked, F. C. ZCStt, Academic Frees, Raw lark, 56. Schroader, s. F., Collier, V. and Woodward, 0. S., Biochem. J., 1180 (.X939). 57. Conadea, S., Borden, A. H, wad Mertln, A. J. P., Biochem. J., la, 590 (191*7). 58. Block, B. J., Anal. Chew., 22, 1327 (1950). 59. Sanger, P. and Thompson, E. O. P., Bleakest. J., 353 (1953). 60. Sanger, P., Biochem. J,, lj9, 1*63 (1951), 61. MeParren, E. P., Anal. Che®., 2^, 168 (195l). 62. Feraud, K,, Bunn, M. S. wad Kaplan, J., J. Biel. Ohm., 112 . 323 (1935). 63. Porter, K, K., Methods of Radical Research, 2» 256 (1950). 6b. Blacidmra, S, and leather, 1. 0., Biochem. J., 1*8, 126 (1951). 65. Bleak, R. J., Burrnm, B. 1. and Zweig, 0., »A Manual of Taper Chromatography and Piper Electrophoresis,* p. Id, Academic Press, Hew Tork (1955) * 66. Oreeaberg, B. St., "Chemistry of the Amino Acids and Proteins,* 2nd ed. p. 557, Charles C. Strong, %>ringfisld HI. & Baltimore, Md. (I9bb). Edited by Schmidt, C. L, A. mrwJSBKmwmJhf 81 CM V\ *. V\ * * m • CM H 1A* 1A in in in in* in in in in CM CM CM CM i * o#- o#■ * -St f i in♦AA \a*1A CM * CM * .^* ..• .i o *• o I'l i 1A n o H * ©* in C*“ a a Continued next page 82 r*f "U\ lA* in* in o• W\m V\# CNjt \ in* in» in* o# in * t ? ? ? f SO* sO 'i o o tX A o* r-l 0# <3 A A I 4 § O nO *5“ rrf H ri H i a V °# o o © © o in m in in A o. c> <3 in A A n in m & CO * Q O CD «K £? r4 £5 H H o a H m ♦t 3 S 8, 8. 8, Sf la S"* S »; *S l r » 3 - t O 8, 8 O 3 3 4 8. 8. 8, © 6* K* 8 * & • S *a 4 N a °. n *" 74 i i p o , V <**» » t*4O m* A*»O* O HI % O H O# et\ o O* XA A I +i S'* S'* o. * w O H 1® *■? n *^9; rt -" Os » *i < < -I #HI H CM rt j lJ 9 9 $ o XA# o £3 • O* XA XA A 4*| ♦:I 4 H CMi £ (*>J n H (5*3| Eetest i * ° . © 1 -? ? CD* PA MO © © ♦ XA T XA CM XA « © rf o4 XA * © OHI© * od f c ©a r*f XA ♦1 ♦♦I © ? -=£ * m ' i *•%Q CM C J> T w-t if"4o V © ) also demonstrated the incorporation of the methyl groups of glycine betaine into the nico­ tine N-methyl group to an extent approximately equal to the incorporation of the methyl group of methionine. Byerrum and ling (6) showed that the methyl groups of choline can give rise to the nicotine methyl group to about the same extent as formate. Byerrum et al* (7) have also shown that th© alpha carbon of glycine can give rise to th© N-methyl carbon of 88 oicfttind a slightly greater extent than the methyl group of methionine, la this study (?) the carboxyl carbon of glycine did not give rlao to the aiootiio B~methyl carbon* Dewey (8) fed oa^eioi glycolate-2~C** to tobacco plants and observed a labeling of the nicotine in th# methyl group to about the same extent as observed in the earlier study (2) when methionine was administarsd to the tobacco plant#* Syerruai et al, <9) have dec demonstrated that can give riee to nicotine labeled in the 8*metfeyl group to about the name extent as that which re­ sult# from the methyl group of methionine, Various other investigator# have studied comparable metabolic re* actions in other plant#* Kirkwood et al. (10,11) sere unable to demon* strata the imerpcration of the methyl carbon ate®# of choline into the methyl carbon stem# of the alkaloids fcordentne of barley and ricinlne of caster beans , although methionine resulted in the labeling of both alkaloids, Srtbney and Kirkwood (12) m m able to demonstrate that glycine betaine east donate methyl group# to the alkaloid# K-methyl tryaroina and hordenlne of barley plants. The above studies, especially those in tobacco plants, indicate the occurrence of am over-all reductive metabolic reaction for the sym* thesis of methyl groups in addition to the reaction resulting in the formation of methylated compounds by transmmtfeylation, It was therefore of interest to study the imteiwediary metabolism of the reductive re­ action* fhe alpha carbon of glycine was not converted to the methyl carbon of nicotine to a m&ior extent via the carbon ate® of formate since the 89 latter 1# incorporated into nicotine to a meh leaser extest than the alpha carbon of glycine* ■Similarly the alpha carbon of glycine was sot eonTerted to the nicotine methyl group to a major extent eia toe methyl group# of choline, beiaine or raetliionine or via the alpha carbon of glycolato slums these ©amon atoms are Incorporated into the nicotine methyl group at an equal or lower rate than is the alpha carbon of glycine* &4 2n the animal serin©~>*C ■has been sltown to giro rise to the methyl group of methionine to the same or a soiaewhai greater extent than the carbon atom of formate (13) while the alpha carbon of glycine wee in­ corporated only to about ©ns-stxtb the extent of the beta carbon of serine* Steftel (Ih) demonstrated that the beta carbon of serine enters -the methyl gret^s of choline in greater amounts than does either formate or the alpha carbon of glycine * Fomaldahyde and formate appear to enter methyl groups to a similar extent in animal metabolism (15,16) * M animal metabolism it has been postulated that tbs alpha carbon of glycine may enter methyl groups through serine (13,17)# In tobacco plants, however, this does not appear to be the case since the beta carbon of BI*-serin© was incorporated into th® methyl gro^p of nicotine to an equal or somewhat lesser extent than was the alpha carbon of glycine. It was therefore considered possible that the beta carbon of serine and toe alpha carbon of glycine might give rise to a 1-carbon unit at the oxidation state of foimXdehyde. With this postulate in mind formaldehyde-C*4 was fed to tobacco plants and tbs extent of incorporation of C-Oi* into the nicotine methyl group determined. If the hypothesis 90 sere correct the carbon atm of formaldehyde would to expected to be incorporated Into th© nicotine netbyl carbon to a greater extent than either the beta carbon of serine or the alpha carbon of glycine, the present study tend* to indicate that s m h a hypothesis night be correct. 91 Previous studies i» this laboratory (2-9,27) have shone that tobacco plants can absorb various organic compounds through their root systems from a nutrient solution* The hydroponic procedure of admin— at4 laboring the forpaaldehyde^ was also used in this study sines it was desired to make a valid comparison with the earlier work* Preparation of Plants The tobacco plants used in these experiments were of a high nicotine Strain, fficotlana rustics 1*, w * hurailis* The seeds were planted in ■* flats containing vermiculite and transplanted after a two to three week period* The plants were watered twice weekly with a nutrient solution composed of 1 g* HgS04*7Ha0, 1 g* Ka8P04# 5.0 g* Ca(HOa)a*hKsO and I* 1. of tap water* On the remaining days of the week they were watered with ordinary tap water* The plants were grown in the flats in the greenhouse for about three months* Although the extent of growth varied with seasonal eon* ditoons, the plants were approximately six Inches in height after this period of growth* Sam budding and flowering was noted in the first experiment during the experimental growth period, which was (hiring the summer months. Sowever, budding and flowering were absent during the second experiment, conducted in the autumn, and during the third experi­ ment, conducted during the early winter months* * A commercial brand ef heat expanded mica* 92 Tojrepar© the plants for hydroponic administration of the form­ aldehyde they were £litt removed from the flats and the vermiculite carefully removed from ths root® as completely as possible. The roots vwr© t$j©a washed with water to free them of any retraining extraneous material. After washing tbs roots the plants sore Immersed in SO al, of an Inorganic nutrient solution contained in a 125 al. Brlenmeyer flask. Th® nutrient solution m s prepared by diluting a stock nutrient solution 1*3 (v/v) with distilled water, the coapositio® of th© stock nutrient solution 1® given In Table I. The various weight® of th® Inorganic salts shown in Table t refer to th® anhydrous compound, On® milliliter of an aeneous solution containing 0.5 mg . of m m m y o X n was also added to each flask to reduce the population of microorganisms. Htt I GmmmMm w tm mmx mmnm rnmmi ^Sater 1 Calcium nitrate 1 a. Potassium chloride Ferric chloride 1 . 250 m * Magnesium sulfate 250 mg. wHWlwilaJUWM* 25® mwiMUXae l i w Potassim dihydrogen phosphate 250 mg. 2 mg. &i ths actual feeding experiments the fomaldebyde-C X4 m e then added to the irlesneyer flask and a cottas plug placed in the neck of the flask around the stem to decrease the loss of formaldehyde through volatilisation• The plants wore allowed to grow for 7 days in a apodal 93 t & m hood to ovoid asi$r health h&aard that migftt arise due to the use of rmtfioaotAva material* Artificial lighting m m used during the ? day growth period* The source ®f light consisted of too 36 inch 30 watt fluorescent tubes and one 100 watt 1ncandescent bulk, The lights were placed shout lit inches shove the top of the plants and were found to produce a light intensity in the range of 20P-250 foot candles at the top of the leaves* The lights were left on for 12 hours each day* During the ? day period water 9m: added to the Erlemeyer flasks as necessary to maintain the volume of solution at approjdraately $0 ml. 4 standard formaldehyde solution was prepared by diluting about 1 ml. of a 3 6 per cent foraaldahyde solution to about 1 1* The result­ ing solution was standardised by the dimedon method as described by Toe and Held (IS) * Triplicate 25 ml* aliquots ©f tise formaldehyde solu­ tion were used, Th® formaldehyde solution was added to a solution con­ taining 100 ml, of the sodium acetate-by*h^ehX©ric acid buffer pH ii,6 aad 25 ml, of a saturated solution Of dimedon. After standing about 18 hours with occasional shaking the dimedon derivative was filtered off on a bared sintered glass crucible and dried at 60°C, The weight of the precipitate times ths factor G,X027 gave the weight of formaldehyde contained in the soAuticn used for the determination, AS The formaldehyde-C solution (purchased from the Isotopes Special­ ties C®., Inc., 0~360°C. and maintained at that temperature for h$ minutes. After the heating juried the apparatus m s allowed to ecd. The stream of nitrogen was continued during the eooling period. The delivery tube was then rinsed with ethanol into the receiver which was then stoppered, shaken and allowed te stand ewer*d.ghi*t m m ie%cratur©. The neat meriting i b major portion of the ethanol and excess triothylamim were enumerated ever an infrared lamp. The last ef the ethanol and triethylamine were remewed in a vacuum deaioo&tcr* The i^hyltrieth^Klaramoni^ iodide recovered was a shits crystalline compound, The quartern&ry compound was dissolved in a small of ethanol ami plated on tared aluminum counting discs* Th® ethanol was evaporated over an infrared leap and the disc reweighed to obtain the sample weight* The discs m m counted as described previously* The results are ex­ pressed as counts per minute per millimole (c.p.ra.) in Table XI. The observed radioactivity in the quariernary compound was divided by the factor fc*3f to correct for the- larger radioactivity used in experiment 2. 100 mmvrs Tjms ti IHGOHPORAfXOH OF FOHJUZiJEHTDB INTO THE M B E CKCW OF NICOTINE Experiment Number Maximum Specific Activity (counts per minute per millimole) Nicotine Kthyitrieti^yl*Diplcrate ammonium Iodide Per cent Radio&ctivity Recovered in Methyl Group 1 1.6k x 104 2 2.39 at ID4 2.31* x 10* 98 3 0.8? x 10* 0.80 x 10* 92 — — — These results show that the carbon atom of formaldehyde is incorporated into th© nicetine N~raethyl group* They also show that, within the limits of experimental error, non© of the formaldehyde was utilized for the bio-synthesis of the nicotine ring system under th© conditions of this experiment * 101 DISCUS3I0B Th* reason far tea variation in radioactivity of the nicotine iso­ lated in the terse experiments la unknown. Since the experiments Here conducted during different seasons of the year it ia considered possible that the variation is e^Utead by a seasonal variation in growth aad metabolism . A e«np»r*blo variation was also Observed ia the previous studies conducted la this laboratory. Comparing the results shown in Table XI to the previous studies conducted in this laboratory, formaldehyde appears to eater the nheetlae B-srnteyl group ia about 3-h times the quantity of the bet* carbon of BL-eerine. The alpha carbon of glycine wee Incorporated to about twice the extent of the methyl earhsm of methionine and only slightly greater than the beta carbon ef serine. Methionine was incorporated to about 'tee ease extent as glyeolate which mas about 10 times the extent of formate. f r m this cowparisen it is obvious teat forsaldehyds was incorporated to tee greatest extent of any of the precursors studied. It mould therefore appear that in the synthesis of tee nicotine H-methyl group neither formaldehyde nor tee alpha eaxtosa of glycine is ■etteeHsed to a major extent by may of the beta carbon of serine. Xa awtmal metabolism it has been postulated that tee alpha carbon of glycine eaa enter methyl grasps through serine (13,1?). Based sn studies la this laboratory, a more reasonable suggestion mould be that tee reverse reaction occurs with serins giving rise to 102 ptem a t o m t o m wit closely related to formaldehyde. The possibility of m m a reaction Is in lime with the Observation by si A * tfcfc* smsias Is sot oxidised to formate in the formation of methyl groups in the rat* The incorporation of the alpha carbon of glycine into the methyl group of nicotine to a lesser extent than formaldehyde mi$*i suggest at least three possible mechanisms. The glycine might be hydrolytically desalinated to 0 m glycolate which could then split to few tee 1carbon units* The l-earben wit arising from the carboxyl group might be expected to be at the oxidation state of caibom dioxide and the 1-carbcn unit arising from the alpha carbon at the oxidation state of formaldehyde. Such a pathway would be in line with the observation that the carboxyl carbon of glycine did not outer th® methyl group of nicotine (?) since there is no indication that carbon dioxide can be reduced to any one carbon unbound (15,25,26). Sheerer, if such a path­ way, from glycine to glycolate, does exist glycolate would be expected to be incorporated into the nicotine SWwthyl group to a greater extent than is the alpha carbon of glycine. 3$m e the reverse order of incorporation was observed, glycine is apparently not hydrolytically deaminated to produce glycolate in the metabolic pathway trm the alpha carbon of glycine to the methyl grow «f nicotine. A second possible pathway from glycine to the nicotine H-methyl ^pow might be through glyo^late. Such a reaction pathway would involve oxidative deamination of the glycine followed by cleavage into two l«earh0H units, one at the oxidation state of carbon dioxide and a 103 8609)9(1 slii tlift 03ddatl(m abate of formate (27) • Such a pfttbK%y would also m m to be unlikely sine© formate is incorporated into the methyl grm^ of nicotine to * much leaser extent than is the alpha carbon of glycine, the third possibility for the conversion of the alpha carbon of glycine to the nicotine B~m®thyl group might Involve reaction of the glycine as such, ®,g. tilth th® nitrogen and alpha carbon intact* Such a possibility has been suggested by toaill (28), If such a pathway does exist It would suggest that the incorporation of the alpha carbon of glycine and the carbon atom of formaldehyde take place by a different pathway. Such a possibility might be investigated by using glycine labeled with H-i# in th® amino group and G-llt in the alpha carbon. If the la-15 to G-Xlt ratio remains th® same in th© isolated nicotine the possibility of such a mechanism would seem to exist, In conclusion# If the present work is compared to the previous studies it would see© to indicate that th® beta carbon of serine, th® alpha carbon of glycine and th© alpha carbon of glycolate can give rise to a 1-carbon malt which would appear to be closely related to formaldehyde. Such m *&etiv© formaldeliyd©* lias been postulated by Berg (29) and also by Ilsliuk and Sakaoai <30>. Her© recently Klsliuk and Sakami (3l) have presented the results of an additional study which would sem b© Indicate the ©adstenc© of an "active formaldehyde” in win*"* metabolism which spears to result tram the ©<»abinaiioa of form­ aldehyde and tetrahydrofolic acid. If such a compound does exist in th® metabolism of higher plants it sight espls&n the failure of radioactive lot fe:na*Mehyde being detected after giving plants radioactive carbon di­ oxide (32) , since such an observation vonld tend to indicate that formaldehyde does not exist free, m such, in the plant* Obviously a final decision as to the nature of the "active formaldehyde*, if such a compound does exist, in tbbaece plants amst await the result of addi­ tional study* 105 amtxx 1. Fo*iaaldah$tie^ was attednislered to a highnicotim strain of tobacco, Kjcotiana rastioa var. hmniXis. Th© nicotine isolated from the tobacco plants was found to possess radioactivity. Bamethylation easperintents shoved that within the limits of experi­ mental error all of the activity located in the methyl group. 2. The results show tts&i the carbon atom of formaldehyde is incesrporated into the nicotine SM&eihyi group to th© greatest extent of any of the methyl grot^j precursors studied in this laboratory, 3. A ecmiparison and a dismission of the results in terms of the previous studies Is given. 106 nmzommix ^ **'* Chandler. 4. P», Ckfcon, M, and Broun, G. B., if. Bi^L, Olwaie, ^ ?87 2. Br©***, &» A, and Borrow, K. B., 4, Am, Chera. Soc#, 7b, 1523 (1952). 3. and M l , C, D., 4. Am. Ghem, Soc,, it. % 8 m » , R, G*,flftfestr*, 4* M., n®my, 1. 4. and Ball, C. 0., 4* Biol. Cham,,M l, 633 (195b) . 5. Sato, G. S., "Methyl Group Synthesis la Plant Metabolism,” Ph. B. Pfeooio, Michigan State Bniwreity, (1955)* 6. Byerrum, B, G. and Mag, R. 8,, 4* Biol. Ghem., 205. 63T (1953). ?. Byormaa, R„ B.. W B , ft* 1. and Ball, C. B., 4. Biol. Chera., sa* c*«u*. 8. Bewey, I». 4., "Studies on the Biosynthesis of Hicotine and Idgnin," Ph. B. Thesis., Michigan State bniwraity, (195b) . 9. Byerrum, B. G., BAngler, B. 1.. RMll, ft, 1. mad M l , G. B., 4. Biol. Chesft., J&6, 371 10, Kirkeood, S. and Marion, 1., Canad. 4. Ghost., 2£, 30 (1951). Matehett. T, 4., Marion, 1. and itrfewood, s., Canad. 4. Chan., b88 (1953). 11, Baboek, M. and mrkwood, S., 4. Biol. Gteni., Ig9, 3^7 (1952). 12, Sribney, M* and Kirkwood, S., Canad. 4. Chem., ^2, 918 (195b). 13# imsteln, K. R. If, and Beuberger, A., Blochem. 4,, Jg, 259 (1953). lb. Steteol, 4. A,, Weiss, S., Smith, P. and %iss, K., 4. Biol. Chea., 15. da Vigneaud, 9., Verly, W* C. and Vftlson, 4, E., 4, Am. Chexn. Soc„ 2£# 2819 (1950). 16, Mttoma, 0. and Greenberg, B. M., 4* Biol. Chem., 196, 599 (1952). 107 17* 18* A*» vl5^S®/« a* and Wulas, S., J. Biol, chem., 165. 271 8*ld» *>• 0.* IlJd. & Eng. Chem,, Anal. Ed., 1£, 238 19, TaneriWatt, M. and Bricker, 0. E., Anil. Chea., 22, 351* (1951). 2©. Craig, 1. C., Gregory, J. D. and Rwisjaan, a . a*ith, 0 , a., & a . & &>g. cb«#., Anal, Chea., 22, 11*62 251 (191*2), 22, Progl, F,, "Quantitative Organic Hleroanalyeis,* tptb Sag, Sd., HP. 156-160, Th® Blaidaton Co,, PMladelpIiia, Pa. (191*5). 23. Stasaonds, S., Cohn, M.« Chandler, 1. P. and du Tlgneaud, V., d. Biol. Cham,, 11*9. 519 . Elwyn, 22, 5509 (1951). A» and Sprlnoon, D. B., J, Aa. Chew. See., 25. Hathews, M. B. and Vennealand, B., J. Biol. Chent., 186. 667 (1950). 26* Siekavltss, P. and Gresnbsrg, B. K., 1. Biol, Cham., 180, 8lt5 (191*5). 27. Tolbert, K. S., Clagett, C. 0. and Burris, R. H., 3. Biol. Chem,, 181. 90S (19U9), 28. Hamill, K. The Bole of th® Alpha Carbon of Glycine in Motliylation Studies in Tobacco PlantsM. S, Thesis, Michigan State University, (1953). 29. Berg, P., 3, Biol. Chew., 205, 30. Kislink, B. 1. and Salami, V., J. Am. Ch*». Soo., J6, 11*56 3 1 . KLsliuk, B. 1. and Sakaai, w., J. Biol. Chem., 2|1*, 1*7 (1955). 32. Rvftwn, S., Batten, H. B. and Haasid, w. Z „ d. Am. Chem. Soo., 3W3 (19W». a*'??***:jot; The formula used in correcting the observed count to aero sample thickness was* A_ * 0o* M FTT where specific activity (counts/kiimte/millimole) G0 « observed count (counts^nimte) H ® molecular weight ®£ compound ¥ * weight of ©ample counted b * fraction of nasdMn activity at th® sample thickness used (T)-— obtained from self-absorption curve. Sample calculation* Ificoiine dlpicrate «*-* C0 • 11Q£ c.p .®* W ** ^2,S 3ngj#