The Isolation of 3-Indole Acetic Acid from Immature Corn Citation Dandliker, Walter Beach (1945) The Isolation of 3-Indole Acetic Acid from Immature Corn. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/kxfg-5c11. https://resolver.caltech.edu/CaltechTHESIS:03282025-223427071 Abstract No abstract. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Biology) Degree Grantor: California Institute of Technology Division: Biology Major Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Haagen-Smit, Arie Jan Thesis Committee: Unknown, Unknown Defense Date: 1 January 1945 Record Number: CaltechTHESIS:03282025-223427071 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:03282025-223427071 DOI: 10.7907/kxfg-5c11 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17109 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 31 Mar 2025 19:42 Last Modified: 31 Mar 2025 19:49 Thesis Files PDF - Final Version See Usage Policy. 19MB Repository Staff Only: item control page

The Isolati on of 3-Indole Acetic Acid from Immature Corn Thesis by Walter Beach Dandliker In Pa rtial Fulfilment of the Req uirements for the Degree of Doctor of Philosophy Californi& Institute of Technology Pasadena, California 1945 Acknowled,:;emen t The author wishes to express his ~ratitude to Profes so r A. J. Haa; en-Smit for hi s gu i dance a n d adv ice in this inve st i ~ati on, and also to thank Profes so r F' . W. Went and Professor James Bonner for their many kin d and helpful su~ ~es ti ons. The writer recalls many friendly ass o ciations with Mr . Ge o r g e Fei3 en, Mr . Co nrad Fon3 , Mr. Car l Hedema nn, Dr. Paul Saunders, Dr. rla lph Siu, and Dr. Sam Wildman. He wishes also to thank Dr. Gertrude Oppenheimer for the micro-analyses and Miss Priscilla Hoth for carryi ng out the Avena tests. Ta ble of Co ntents Introd u ct i on ............. . ............ pa g e 1 Exper ime n ta l Se c t i on. . . . . . . . . . . . . . . . . . 11 33 Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 50 Concl us i on..... . . . . . . . . . . . . . . . . . . . . . . . 11 54 Summe. r y·. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1, 59 I nt r oducti on Introductory facts concernin ~ aJxins J' he c o ncept that plants c ontain special substances which move a b o ut within the plant and serve to correlate and inte grate the functions of the organism is n o t new. The ideas bro ug ht forth by Sachs near the end of the l a st century accounted for the kn -.J wn phenomena o ccurrin ·~. h 1 pL,nts and even today a re not outdated. His basi c postul Rte was that differen ces bet we en plant orga~ s must be d ue to differences i n chemical compos i t ion, and that these differences must be present even before tje e ~ rlie st visible sta ~es of organ developm e nt. He pointed out that these differences mi g ht be extre ~ely minute and perhaps not detectable by ord ina r y micro-c h e mical methods. He al s o consi d - ered the possible i~ J ort an ces of optical isome rism of compounds takin ,; pRrt in reactions in yivo. de assumed th~t there were suostan ~es elabo r ated in the plan t which tr a vel i n definite directions and which brin3 abo ut special responses, fo r example, the formati on of flowers or roots. Darwin, about the sa 1i: e time, showed that the stimuli of g r av ity and li3 ht were perceived oy only the t i ps of seedling s an : concluded tha t s ome i ~ fluence w?s tra -sm i t te d fr om t he tip to the lo ~e r parts ( 1) of the plant, which then re?cted wi t h a ch2racteristic movement. The material nature of this influence was demonstra ted by early exp eriments of Boysen Jensen . He found that Avena seedling s depr ive d of their tips showe d no response to li 5 ht, but that by replacin 3 t h e cut-off tips on th0 wounds the sensitivity to lL•:, ht w2.s restored. I Paal repeated these experiments and f o und that the substance responsible for this behavior could pass throu:;h a layer of ~j elatin but not cocoa butter, mica , or platinum. Later ~V§_nt succeeded in removin:', the active substance from plants by e.llowin r, it to diffuse out of oat coleoptile ti p s i nt o ag ar blocks. This sub- stance , wh ich has been shown to be necess a ry for the process of cell elon 7 a tion in Avena, is a member of the class of phytohormones known as aux ins. The actions of aux ins in plants explain not only effects due to increase i n len g th, such as photo- and ~eotro p i sm , but Rlso a vari e t y of othe r phenomena , such as inhibition of l a t e ral buds by terminal ouds , root formati~n , and c a.mbial j rowth. A number of bioassays for auxins have been devis ed , an1 it is with the s e 8s tools that subsequent isola tion and chemical wo rk has been ma de pos s ible. (2) Probably the most widely used of these bioassays is the standard Avena test as described by Went (43). Briefly, the proced~re invol ~ed is to cut off the tip of an oat coleoptile and to place a small agar block containing an unknown amount of auxin on one side of the cut surface of the coleoptile. The auxin in the a~ar block diffuses out into the oat seedlin~ and stimulates ~r~wth on only the side on which the block is placed. This results in a curvature of the plant away from the a 3ar block, which curvature is propor- tional, within limits, to the auxin concentration. A modification o f the standard Avena test has been made by Skoos (33). The endosperm is removed from the test plant and the plant held in a cotton plu ; . This test is sensitive to much lower auxin concentrations than the standard Avena test since the seed acts as a source of auxin for the coleoptile and lessens its sensitivity to any added auxin. (3) Isolati on a nd chemical work on au x ins The Avena te s t f o rm ed t h e ba s is fo r t h e cl a ssical r e searc h es of Ko" g l, Haa g en-Su it, and £rxleben in isol 8 tin ~ a uxins a and b, and 3-indoleac e tic acid fr om natural sources (14,16,18). In their work the Avena Einheit (AE) was t ~k en as a unit of curvat J re- p ro d ucin g a cti v ity and wa s d efined a s the amount of a uxin n e c e ssary to ~ ive a ten de g ree curvature in the Avena te s t, usin ~ an a s a r block of 2mm3 in volume. After an examina ti :J n of the Vg,ri o us :)os s iole sources o f s ubs tances acti ve in th e Av ena test, huma n urine wa s concluded t o be the r a w m terial of cho ice and wa s 0 foun d to c o ntain about 10 8 AE per liter. Other sources inv e sti ; ated i n clude d the tips of ma ize a nd oat plants, cultures of Rhizo pu s, vari o u s yea sts, a n d dis t ill e ry sl cp s.(1 4 ) The acid fracti o n fr :Jm 200 1. o f p r e g nanc y urine was subjected to a, l 2 r ; e numb er of p '-J.rif ic a ti o n steps, the a ctivity of e a ch fracti on bein 3 follo wed by the Avena test. The proced ~res used includ ed f r a cti on- ati o n between solvents, extracti o n of inacti v e material with various solvents, precipitati on wit h le a d i o n a n d calcium ion, l Rct o nization of auxins a and b with acid methanol, an distillation of the a ctive SJ bstances in hi g h vacuum. The fracti on distillinG at 125-130 Co in ( 4) hi gh vacuum was found to be the most act i ve and crystallized on standing . Recrystallization g ave 6.9 mg . of a product which melted at 173 C. and the activity of which was 3ox10 6 AE per mg . This sub- stance proved to be the l a ctone of an a cid (a~xin a) which melted at 192 C. The activity of auxin a was found to be 50xl0 6 AE per mg. The isolation of auxin a and its lactone was repe a ted successfully on mixed urine and v a rious plant mat e rials. The first plant material subjected to the isolation procedure s was corn oil, Rn d here a new substance, auxin b, was obtained along with auxin a. Auxin b melts at 183 C. with decompos ition and has the same biological activity as auxin a. Malt, like corn oil, yielded both auxin a and b. In the c o urse of the search for rich sources of the auxins, 36 k inds of rna Lt were inve st i g ated. Most of them contained O to 540 gammas of a,uxin a plus auxin b per k g . One sample was found, however, whic h contai ned 10 to 13 m _,; . of .'?.UXin per k 2; . , but in samples subsequently in ~esti ; ated only much lower c on centra ti ons have been found. Two samples of malt examined by Bergren (7) were about 4 and 8% as pote nt respectively as the very rich source above. ( 5) The best sample of corn o i l located by K; g l c on ta ined 700 g ammas of auxin perk~ -, but this too was an unusually ric h sample. The reasons for the extraord inar il y h i 3 h aux in c o nte n ts of the mat e r i a ls ., u s ed by Ko ~ l are n ot known. Durin3 the co~rse of abo ut 3 years a t o t a l o f about 700 mg. of aux in a and b var i ous sources. amount of mater i a l W"l. s obtained from Wi t h only a part of this small the structure of bo th c ompo unds was elucidated i n a series of brilliant r esear c hes (17,21} a ltho un;h neither c ompoun d h as yet been synthesiz ed . A' rernarks.ble ch? ra c teri s t ic of both aux in a and b i s the ir spon t aneous inacti vat i on by i nter nal mo lecula r rearran g ement (15, 22 ). Th is react i o n i nvo l ves t h e shif t of a do uble bond and a h ydroxy l g r o up to ; ive the biolo g ic a lly inacti ve pseudoauxin. -_--,-cwa~~~~c"o~tMo~~oo~ M.G. OH The fact that the above rearra n g ement is practi c a ll y comp l ete in two months g re atly incr e ases the d i ff ieulty of isolating the a ~xins from even ve ry rich sources. ( 6) In workin s up further large amounts of human urine it was discovered ttmt an auxin was present which had different solubilities and c h emical pro c erties than either auxin a orb (18). The isolation pro- cedures were modified so mewhat and finally a crystalline substa ~ce was obtained which after several recrystallizations melted at 165 C. and had a biolo g ical activity C o f abo ut 2OxlO° AB perm~ . What was more surprisins still w,· s t at the elementa r y analysis shLJWed the p 1·esence of nitro en. 0 The c ompound was finally proven to be 3-indoleacetic acid, a substance known perhaps sixty years previously and the presence of wh~ch in human urine h11d aL ,eady been established . •• In 1934 Ko g l and Kostermans succeeded in isolating the same substance from yeast (~O), and in 1935 Thimann succeeded in obtainin s a small amo ~nt of 3-indoleacetic acid from cultures of 8.hizopus (40). The discovery that 3-indoleacetic acid occ J rs naturally and ha.s an activity of t h e sa me or d er of magnitude in the Avena test as t 0at of a uxin a presente t a di f ficult a n d critical proble ,.. or □ Is its biolo s ical activity a mere coincidenc e of no importB.nce 1·- n '" ture, or does it pe r h.9ps pl e. y t h e ro l e o:f' ,rowth ho r mon e P lan ; with auxin a and b? ( 7) The co m~lete answer to this qJestion eve n t o d2y is not known with certainty , but an immense amount of work h a s been d one and c o nsiderable evidence amassed on th is point. The hi s tory o ~ this problem goes back to the exp er i ments of We nt who, in 1928, measured the diff u sion con s tant and calculated the molecu ar wei ght for the auxin in oat plants and determined it to be 376. •• Ko , ;l and his co-workers repeated this work for the 1 tipa of young corn plants and obtained a value which indicated the presence of auxin a orb (19). It is of intere s t to n ote that their value for the molecular wei ~ ht of crystalline auxin a as determined by the diffusion method was 376, the same value as t hat obtained by Wen t approxima.tely six years before. Further charac- terization of th e aux in in youns corn plants wa s oot a ine d by d iffere r tial destruct i o n tests in acid and alkali, an d these 1 ikewi.s e in ( ice ted tba t auxin a ws,s predominant (19),* 'l'he followin- : tec cmi que is employed in the determinati on of the diffusi on constant. A small a Ga r block is prepared c onta inin g an amount of the aux in the diffus ion constant of whic h is des ired. Three other a g ar * The ~ i fferen tial acid-alkali destructi on test relies on th2 f a ct that Ruxin a i s stable in hot acid but labile in hot alkali; 3-indoleacetic ac i d i s stable in h o t alkali and l ab ile in hot a cid; aJxin bis destroyed by eit her medi 1;_m. ( 8) blocks the same size as the first are stacked on top of the first and diffusion allowed to taKe place for a measured time. Then all four ag2,r blocks are assayed .l'or their auxin contents. It is a m"'tter of greatest impor- tance to avoid air bubbles between the a r ~locks and also to maintain a constant temperature and a humidity hi ; h enoua-_h to prevent the a :;t'U' bloclrn from drying. The auxin contents obtained for the four agar blocks are · now multiplied by a f2.ctor calculated to make the sum o f the relative amounts equal 1000. Kno¼in s these four rela- tive concentrati~ns, the thickness of the a g ar blocks, and the time for which diffusian took place, the diffusion constant can be determined from the following diffusion .. tables, Stahler, (35), Arndt (1). X• h2 Number of Layer .4Dt 0.040 0.048 0.058 1 241 233 222 2 247 243 238 3 254 257 262 4 259 267 278 0.068 0.078 0.090 209 194 177 233 227 219 267 274 280 0.102 0.116 0.130 161 14L~ 128 212 205 197 0.144 0.160 0.194 114 100 76 0.230 0.270 0.314 56 41 30 - x- h2 Number of Layer 1 21 16 11 2 106 94 82 3 0.360 0. 410 o. 462 320 317 311 4 552 575 596 291 307 323 0.518 0,578 0.640 8 5 4 71 62 53 305 299 292 616 634 651 287 293 299 340 358 375 0.706 O. 7740.846 2 2 1 46 39 34 285 277 271 667 681 695 190 182 166 305 310 317 392 409 441 0.922 1.000 1.082 1 - 29 25 21 264 257 250 707 718 729 150 1-;:i:;_ _.I./ 120 322 324 323 L.J-72 500 527 1.166 1.254 l. 346 1. 440 - 18 15 13 11 243 236 230 739 748 757 765 (9) 4Dt - - 22L~ In the functid:m 2 X -_h_ - 4Dt h = the thickness of th~ a gar b locks in cm. t =. t b e time of d i ffusion i n day s D = t h e diffus i o n c o nstant based on t i me i n day s Dy interpolati o n each of the four rel a tive concentr2t i o n s g ive s a value of' X a.nd co nsequently of D. If p i s the d if fe r en ce between the two r e l at ive concentrat i o n valu e s in the table , between which in t erpol ~ti on was made , then the final va lue of Di s ca lculated as fo llows : D:------------------since the s i ~nifica nce of a va lue of D increases with incre as ing d i fferences between t he two concentrat i o n values. In the ca s e of aux ins, if f o ur qu it e di ff ere nt va lues of Dare obta in ed with a defin ite trend from o n e a g~ r block to the n ext , one of the fol l ow ing sit ua ti o ns i s in dicated : a. An inhi bjtor i s prese nt wh ich di ff u ses a t a r a te d i ffer ent from that of the a uxin. b. Mo re t h a n one aux i n i s present . c. Factor s of both a a nd bare at work. (10 ) The change of D with change in temperature is expressed by the followin ✓ equation: D2 D1 : 1 + a( T2-T1) The coefficient a itself is a function of D and can be evaluated approximately for aqueous solutions from the followin ,,, table, taken from Taylor, Treatise on Physical Chemistry. 2.0 1.6 1.2 0.8 - D a 2.4 1.8 1.4 1.1 0.7 0.018 0.020 0.022 0.02.:5 0.029 0.035 0.040 0.4 - 0.3 0.2 - 0.1 The followin 3 empirical relationship between the diffusion con,3tant and the m )le cular we i (;ht has been foun d to hold ; ood for a v~riety of compounds of molecular weL,;ht UD to a ran,~;e of about 5C: 0, with an ex- pected error of 10 - 20~ in the predicted molecular D fM ~ constant The value of the above constant was determined by 0holm (26) and for a variety of compounds was g iven an avera~ e value of 7.0 for aqueous solutions at 20 0. Since the diffusion constant usually increases almost linearly with dilution in the region of 1.0 to ( 11) o.l mola r, it i s n e c e s s 2 r y t o extrapolate t o i nf inite d iluti o n to obtain Di n the above equatio n. I t is an inte r·esting and perti nent fact that the d if f usi on c on s tant a t a g iven temper a ture i u about the sa me in mo st s els , such as a ~ar , as i n wa t er . t he derivati o n o f the d if fus i o n c onstant has been c arr i ed out by E ins te in (8) with only mea s re ass umpt i o ns. Cons i de r a cylind e r Z f ille d with a solution a nd divided int o c~mpartments A and B by a movable semi-permeable memb rane K, the concentrati on of solute in A bein g grePter than i n B . z B A K Now in order to prevent K from movin ~, a for ce must be applied i n the d irec ti o n o f A, or i f' n o for ce is applied then K will move in the d irec t i on of B until the c onc entra t i ~ns in A an d Bar e equal. lh i s simple con- sideratio n illu str a tes the i den tit y o f the for ces ca u s in3 osmotic pressure and those re s po nsibl e for diffusion. Consider now a cylinder of 1 cm 2 cross secti ~n with no membrane, but filled with a solut i on in which (12) diffusion is takin g place because of a concentrati on g rad ie nt . p p' - E" E I At the plane E call the osmotic pressure P, and at E', a distan ce dx from E , the press ure P' (P)P'). Then the force K acting on the solu t e mo lecules in dx cc. of sol uti on can be ex pressed: - K - P - P' dx - -dP o_x ( 1) But the osm o tic pressure due t o n moles of s o lute per dx cc. is (2) P=nRT ( 3) and combining (1) and ( 2 ). Now if a force K due to osmotic pressure a cts on one mol e c ule then v, t he vel o cit y , is v• -Fl ( 4) where Fis t h e fric t i o n functi 0 n, depe n ding on t he size (1 3 ) and shape of the solute molecule and the viscosity of the medium. If in dx cc. there are n moles of solute, there are Nn molecules where N is Avogadro's number, and if the above force K is divided up oetween Nn molecules, the velocity of the molecules is K F' V-::-1- Nn (5) or combinin 7 (3) and (5) vn - -R T - - dn_ dx NF ( 6) The diffusion constant Dis defined by Fick's first law of diffusion, assuming a cross secti~n of 1 cm 2 : I dN --=..:--: -D dt dn d~( ( 7) where N' is the number of moles of solute and t i s the time, but dN' ___ ___ dt - so that D - .,,~- -- vn ( 8) (9) RT N F Stokes law Gives the friction function for molecules lar3 e in cbmparison to those of the solvent and spherical in shape. In this CRse F = 6i\'\r where~ is th e viscosity of the medium and r the radius of th e diffusing molecule. ( 14) Since m, the mass of a spherical molecule, is m= ~) 1i r3 d whe re d is t h e apparent density in solution, we would expect t he followin3 to hold true for the d iffusi on of spherical molecules : 3 D The fa.ct that D ~ = constant fM constant holds better than the above relationship for a variety of substa nces investi g ated me a ns simply that as a rule their molecules are not approximate s~heres i n solution. No doubt a statistical treatment of chain-like and dis c-like molecules wo uld result in fricti :m functions which could satisfactorily describe their diffus ion behavior. The d i ffus i o n techni q ue was further employed by Heyn i n 1935 (13) who in vest i ~ated auxins from s e vera l sources . He found that the a u x i n in Phycomyces cul- tures ha d a molecular we i ~ht clo s e to that of 3-indoleace t ic aci d , while the aux in from Aven a coleoptiles, Vicia faba ~oat s , a nd r e3enerated Avena tips gave values close to t ha t of auxin a. Th i mann in 1935 (40) publ i shed hi s results on the i solat io n of the aux in found in cultures of rlhizopus suinus. This c ompoun d has t he same ac id- base stab ility as 3-indoleacetic acid and both compounds d i sti ll in (15) hi g h v~cuum ( molecul ar still) at about 100 C. A sma ll amoun t of the crysta lline substance was obtained and mel t i n :::; point detern1 i n2.tLms were used t o establish i dentity with 3-indoleacetic a cid. 1 933 fh i mann n oted in (38 ) tha t the a uxin produ ction of Rhizo pus cul- tures wa s corre l :-:1ted with the con st i tuents of' the medium. Ce~ta in peptones resulted in very l ar3 e auxin product i on , while othe r s yielded almost none at a ll. I t has b e en shown that 3-indoleacetic acid aris in g i n cultu res of micro- or·,;anisms is produced by ~he oxida tive deam i nat i a n o f tryp t ophane so that t h e effects o bserved by fh i mann are due si~~ ly to the tryptophan e c anGent of the ue u tone u ~ed . I n 1 936 an i m~ ortant p henom eno n was d i s cover ed by van Overbeek (27). He found that i f crystall ine auxin a ( obtained fro m K~ ::; l) i s e.pplied to Avena pl2.nts i n the dark , a \ re a~ er curvature i s obtaine d than i f the pl ants are i n the li s ht . fh i s effect is not shown, ho ¼ever, by 3-indoleacet ic ac id. Moreover , a d i st ill ed water extract of cor~ me a l yields an auxin solut i on which be haves lik e aux i n a i n thi s re 2~ ect and n o t l ike 3 -indole a cetic acid . auxin a fhis charact e ristic lis ht sensitivity of trr v ivo e.fi'ords an a dditLmal t:)ol f'or the cha r~cteriz~ti on o f unkno wn aux i ns but as yet has not been s enerally appl i ed . ( 16) Van Ove rbeek and Bonner (30) usin~ the ac id- alka li destruct i J n test in vest i : ated the auxin from pea r oots and c onc luded that aux in a was probably present. An examination of t he aux ins i n the algae Ma c ro cystis an d Br yops i s and in ~ lode a , a hi g her p l ant , e mp l oy in; both d i ff usi ~n an d acid - alkali tests (van Ov er beek 3 1) s h ow ed that the aux in uresent has the cha racteristics of 3-indo leac et ic acid. I Lefevre ( 25) applied the ferric c h l o ride and color reacti ons for in do le deriv~tives to vari ,Ju s p l an t extracts and made subse que n t absorpt i on spectra measur e me nt s . He conclude d that 3-indoleac et ic ac id, or a ne ar relative, is present in numerous hi g her plants. It must be reco ~nized, h owe ve r· , that co l or reactions c a rried out on mixtures of unkno wn comp os i t i o n and c o rnple x i ty , such as the expressed jui ce of a plant, can easily l ead to erroneous conclusi ~ns. Neve rtheles s , the spectrop ho tometr ic a nalysis of the p i 3men ts prod uc e d i n color r ea ctio ns s h ould afford a po ;. erful t o o l c ompo un d s . i n the i de ntificati o n o f unknow n o r new The fo ll ow i n 3 , for example, i s a p lo t of the absorpti o n of a mix t ure of 10 cc. of 0.001 molar 3-indo l ea cetic acid and 0.1 cc. of 0.1 mo l a r ferric chloride i n 1 mo l ar hydroc h loric ac i d about on e half hour after mi x i ~s - ( The absorot i ~n of this re ac ti ~n mi xture i s not constant wi th t i ~e .) (17) l,a Lo Gr 'I.c, 0,5 -I 'ioao 5000 a,oaa O 1000 WP\\JELEN(;TH A Absorpt i o n s p e ctr um of indo le ac e ti c a ci d plu s ferr ic chloride . I t i s thus c once iva bl e t hat i nf o rmat.,Lm of this s o rt c on c ern i n 3 the abs o rp t i a n max i ma anf ~ i n i ma of t h e products of co l or reac t i ons mi J ht ive i nformatio n about t h e st ructura l deta il s o f the reac tants . Goodwin in 1939 (9) emp l oyed t he d iffusion method on c orn rnea.l extra.c t s . he concluded tha t certain i n - hi b i tors of a ux i n were present , wh i ch wh en removed 3 a ve diffusi on const an t s near those of aux i n a a n d b. In 1 941, H2a e;e n- Smi t an , co -workers ( 11) reported t h e first i s o l at i ~n of 3 -indo leacetic a ci d fr om a hi J her ( 18 ) plant source. Corn meal was treated with di i ute sodium hydroxide solution and hydrolysis allowed to take place. The extract was fracti~nated an d one of the potent concentrates was crystallized. 3-indoleacetic acid was the chief produ ct but also a small amount of pseudoa uxin a was obtained. It was concluded that the latter substance arose from the rearran :;ement of a,uxin a during the isol ~ti on procedures. Wildi'T11=i.n and .: ;ordon ( t'.1 4) in their \·.1orlc on the enzymatic hydrolysis of spinach proteins concluded that an auxin is li be rated which has a lower molecular wei s ht than that of 3-indo leacetic acid, the liberated auxin bein,,;; char-'?'.cterized by the diffusi ::m tec0.,nique. The same autho rs (10) studied the acti on of alkali on tryptophane, ation. casain, and ~e latin with res uect to auxin liberUnder the mo:Jt favorable conditi -J ns found for the liberati on of 8.uxin from tryptophane approximately 0.008% of the tryptophane appenred as aux in e fter hy- drolysis. Casein 2.nd '.\ elc1.tin were round also to li bera te small amou nts of an auxin on treatment with alkali. Avery in 1941 (3) published his re ~ults of treating dormant maize endosperm with vari ous solve r ts and oorat e buffers at pH 9-10 qn d 100 C. Extraction with several solvents successively or with water alone removes only (19) about one tenth o f the a v a ilable aux in (2). stitutes the so-called free auxin. This con- The remainder exists in a bound form li berated by a lkaline hy d rolysis, a nd in do rmant c o rn amounts to the equivalent of 7xlo-5 gm. of 3-ind oleaceti c ~cid per gram of corn. This is essen- tially c o nfirmat i o n of the results obtained p r eviously by Haap;en- Sm it (11). Avery found that, the auxin in corn h as the a cid-alkali stab ility of' 3-indo leace t ic acid. In a subseq uent paper (4) Av ery applie d the same tre a tment to wheat g rains wi th a most int erest in g result. The tot a.l auxin frorn dry whe at was eq uivalent to of 3-indoleacetic acid per g ram of wheat. On hea t i n g this auxin with l•ON. s o d ium hy drox ide, marked destru c t i on take s pla ce, an ~ he c onc l udes that 70% or more of the aux in present is a compound or c ompounds differen t from 3-indoleacetic acid. In cont inuin g work on the auxin i n dormant corn, Berg er ( 6) purifi ed a n II • aux in p re c urso r 11 which on al- kaline h ydro lysis g i v es as o ne of the products about 3. 7% of 3-indo le a cetic acid. The lB.t ter was purif,ied f u rther and cry sta llized t u t was concluded to c o ntain 10 to 30% of an adcli t i ma l as yet unidentified su bstan ce. Bot h Avery (5) and .•,• it twer (45) have stud ied the chang es in a u x in con J entr aL i o n in immat u re c orn kern e ls (20) during the peciod followin 3 fertilization. that for II Avery found Country ;J-entleman" corn the peak in total au x in co nte n t occu r red about ten days after pol i inat i o n. At this stag e the total is equivalent to about ~-3 to 0.5 mg. of 3-indoleacetic acid per g ram of dry wei g ht, and ofthis total only about 6% is free, that is, ex~ractable by water or org anic solvents. At this stage the ker- nels are about 35 to 40% dry wei s ht, so that on a fresh weight basis one g ra ~ of kernels c ~ntains the eq~ivalent of 1.2x10- 4 to 2.0xlo-4 g m. of 3-indoleacetic acid of which only 7.2x10-6 to l.2xlo-5 gm. is present in an unbound form. The results of Wittwer indicate a similar peak in the free auxin concentrati :m about 10 to 15 days after pollinati o n. Moreover, his results bring out the inter- es t in g fact that unfertilized ovules do not contain any demonstrable amount of auxin even at a time when the fertilized ones contain comparatively lar~e concentrati·ms. As the development of the kernels proceeds, both the total aux in and free auxin content decrease rapidly to the value for dormant corn, that is, a total auxin content equivalent to about 7.4xl0- 5 gm. of 3-indoleacetic acid per ; ram of corn. (21) Of th i s total roughly 5% is in an uncombined form readily extractable by water. Bergren (7) found that ether extraction of wh<ille corn yielded the equivalent of 2 to 7xlo- 7 gm. of 3indoleacetic acid per gm. of corn. This is in contrast to the case of rye studied by Hatcher (12). Up to the attainment of maximum a,uin c oncentration, all the auxin exists in a free form. Thereafter the free auxin increases in amount. about dis a ipears an d the bound form In the dormant stag e, as in corn, 5% exists still in the free form. (22) Summary of Information about Characterization of Auxins Source Auxin found Avena tips Pregnancy urine Corn Oil Malt Human urine Yeast Corn tips Auxin of MW , 376 Corn tips Phycomyces Avena coleoptiles Auxin a Method of Ref. Characterization Diffusion 43 Isolation 14 Auxin a and b II 16 II II II Auxin a and IAA IAA II II Auxin a Diffusion Acid-alkali Diffusion II IAA 18 20 19 19 13 Auxin a II II Vicia faba roots - 1, ·11 II i1e E;;enerated Avena tips II II II Rhizopus Corn mea_1 IAA Isolation Li ,: .: ht Inactival.,ion Diffusion a,nd acid-alkali Acid-alkali Auxin a ', Ivlacrocyst is IAA Pea roots Auxin a Corn meal Auxin a or b Bryops is IAA Elodea Corn meal Corn meal II Pseudo auxin a and I r, p,_ I AA Wheat c;ni,ins 70% not IAA Spinach proteins Auxin of lower MW than IAA Corn ke t·nel s IAA (23) Diffusion Diffusion and acid-alkali Diffusion and ac id-allG::i.l i 40 27 31 30 9 31 II Isolation 11 Aci d-a.lkali 3 II 4 Diffusi ,.:m 44 Isola Lion 6 Evaluation of th~ methods used to characterize auxins Of all the methods employed to establish the i dentity of an auxin the method of isola t ion is by far the safest and most likely to lead to uneq uivocal conclusions. Althous h there exists the possibility of alter- ation of substances during isolation, this is certainly a minor objection when compared with the disadvantag es of the other methods employed. The determinati ,, n of ~iffusi on constan Ls is theoretically a good metho d but subject to the approximati ons already noted. Inasmuc h as inhibitors are probably not uncommon in pla nt ~xtracts, (24,36) co g nizance must be ma de o f this factor in interpreting diffusi on data. The determina tion of li ~ht sensitivity, it seems, should af f'ord ~n excellent me t h o d to distin guigh between auxin a and 3-indoleace t ic acid, but because it has not been extensively applied its advanta,: ;es an d disadvanta g es are not kn o wn. The differential acid-alkali destruction test has be e n widely used and althou s h there is no specific eviden c e to sh aw that its results on crude extra cts mi ~ht be unreliable, they should be interpreted with c~uti o n. ( 24) Comparison of the biolo ,{ical effects of 3-indoleacetic acid and other ~ i n s (1) Effect on cell elongation. The action of crystalline auxin a and of 3-indoleacetic acid on cell elongat ion in Avena in investi gat ions thus far undertaken has p roved to be qualitatively the same. The ~c tivi t ies in the Avena test as determined ~ by Kogl and hi s co-workers are for auxin a, 50xl0 6 AE per rn ':. ( 14), and for 3-indoleacetic acid, about 20x10 AE per m,;; . (18). 6 'l'he velocity of tra.nsport in the Avena coleoptile is sli ghtly different for auxin a and li n doleacetic acid, auxin a bein J trans po rted in the cut coleoptile at a r ate of 10 to 12 mm. per hour, or about 10% faster than 3-indoleacetic acid (Went 43, p. 90). (2) Effect on roots. The action of cry sta lline aux in a and 3-indole acetic ac i d on the roots of Avena was studied by Ko g l ard co-workers (19). Concentrati o ns of auxin a down to 10-7 gm. per liter showed an i n hi b itory effect on the growth of roots. The sa ~e effect is sho wn by 3-indoleacetic acid a lt houg h at very low co n ce ntrati ons , that is about 10- 9 mol2, r , a stimulatLm of root 6 rowth is observed. Thimann and Koepfl i ( 41) h ave de :;1ons tra Ged tha t pure 3-indoleace t ic acid is fully a ctive (25) 1 ·,1 the stimu- la tion of root ini tia tL:;n an ( g ro wth. However, a differe n ce in the acti o n of auxin a and 3-indoleacet ic acid has be e n observed on root formation, (Went 43, p. 195). In these experiments pea cuttings were treated with the two auxins separately and to gether. If very small concentrati o ns of ~uiin a are ~dded ~tQ o~timal concentrati ons of 3-indoleacetic acid, the number of roots formed is far g re a ter than the number formed by 3-indoleacetic acid alone. Ho ½ever, the addition of small amounts of 3-indoleacetic acid to o p timal concentrations of auxin a does not show this eff e ct. (3) Effect on Dud development. It is a v; idely kn own f a ct that the l a teral buds of plants a r e inhi b ite d by t h e te r min a l bud. If the termin a l bud be removed, the laters l buds be g in to develop a lmo st imme d iately. This, of c our s e, is t he theo- retical basis o f o run ing trees &nd shrubs. The s e effects we r e inve s ti gated b y fh i mann and Skoo g (39) using young pl an ts of Vicia faba. The y found that young termina l buds produced larg e quantities of auxin and tha t t h is prod u c t L , n f a lls of f in ol d er bud s. Moreover, normal late ra l buds produce almost no auxin, but if the terminal bud of a shoot is r emove d , the latera ls be ~ in to develo p a n d ; ive o f f a uxi n which acts then (26) to inhibit the other lateral buds. The in h ibitory ef feet of the n o rmal terrnin 2,l bud ca.n be reproduced by p utti ng in its pl ce an a g a r bl ock cont a in in s 30 indoleacetic acid. As i n the case of roots, lo w c oncentrati ons of 3-indoleacetic acid exert a stimulating effect upon the g rowth of buds. These relationships are illustrated i n the fpllowing plot ta '.: en from Thimann (42), the bud and stem curves being partly hypot hetica l. + 0 - I\ 10 't I '7 ' - Loa M, oF ~U'1.\N I;' &f 3 1.. t ONt. E.~ Tft f'"T\O N Inhibi tLm and :::;rowth promoti o n of different organ s a s a functi on of aux in c oncentrat i o n. The abscissae for the b ud and stem curves ar e only approximate . (27) In subse ~uen t work Skoo g and Thimann (32) compared the action o f crystalline auxin b with that of 3-indoleacetic acid on bud inhibiti on. The auxin b was obtained from Professor F. Ko g l. Solutions of the . auxin to be tested were pl~ ced in small wax cups molded around the stem of a d ec ap itat ed seedling and the effects observed after a number of days . Thes e workers c onclude d that in res p ect to bu d inhibition the a cti o n of auxin band 3-indoleacetic acid was indisti ngu ishable in kind. (4) Effect on cambial g rowth. Snow (34) studied the action of c rystalline auxin a (presente d by K~ g l) and 3-indoleacetic acid on c ambial g r owt h and f ound that t he ir effects on th±t tissue ~ere identical. While the lateral buds of a s tem are inhibi- ted by the auxin libe rated by a te rminal bud, the activity o f the cambium layer is stimul t e d. 0 (5) Effect on phototropism. 'rhe physiolo :-; ical effects of auxin a and 3-indoleacetic acid thus far discussed a re different in only very minor res pe cts. The physiolog±cal differences are much g reater, at least in Avena plants, when the effects of li ght are consi d ered. The exp eriments of van Overbeek (27.28.29) first detected this imp ortant physiolo g ical ( 28) distincti on between the two aux ins. He showed that Avena curvatures produced by auxin a were up to two ti me s as g reat in the dark as in t he li 3 ht , whi le thos e ~f 3-indoleacetic a cid were the sa me in e i ther case. Th e same prob l em was st udied by Koning sberg er a nd Verkaa i k ( 23) u s in~ the chemical results of K~g l as their starting point . the l a tter ( 22 ) foun 6 that , in mak i n g ultra-vi ole t a bsorpt i on me as ur ements on auxin b solut i ~ns, an eno l fo rm coul d be detected by the ferr ic chlor i de test after a ti me , but auxin b i tself gave no col or re ? ct i Jn with this r ea~ ,.., ent . Moreover , aol uti ons of auxin a l acto ne ra p i dly lo st their biolo g ic a l activity on ex pos ure to ul t r a - vio l et r ad i at i on . the se cha n ~es were formulated as follows : JI. .., ~ Au~, N ~ ( A\'."T' '" E ) 0 ,-o - S1'AGLl:. f" Et L\l~\ ~\lY.\M 0... L~c."f0"'~ (1t-l~"'-"1"'iE) ( 29 ) •• Ko g l (22) mentions the possibility tha.t the above photochemical inactivation of auxin a and b might conceivably be of im o ortance in the normal responses of plants to lig ht. Konin~ sberg er and Verkaaik first re peated the work of van Overbeek and obtained fully similar results usin::; a hic; hly purified preparation supplied ·• 5 l. by Ko They, how~ver, added a very important and enli g htening exp erime n t. First, deseeded and decapi- tated Avena plants wer e pre pared; these have been shown to be practically free of auxin and mor e over do not form more auxin during the test period. Now on one set of such plants were placed agar blocks containing auxin a and on another 3-indoleacetic acid. Diffusion was allow e d to take place, anc5 then the plants illuminated on one side o nly. After a peri od the test plan ts were photographed. The result obtained was that expected from theory. Seedlin g s g iven auxin a sho ~ed a curvature (7 to 8 degrees), b ut those g iven 3-indoleacetic acid showe d no sig nificant curvature (less than 1 degree ). It was postulated that the observed phototropic curvature was due t o the i.D_vl_vo .. inactivation of auxin a lactone . It was l a ter found in KO [!; l Is la bora.tory that solutions of auxin a lactone are inactivated by visible waveleng ths in the presence of carotene. (30) Results were obtained by Stewart and Went (37) which apparently contradicts~ those of Koning sberger. They found first that exposure of Avena coleoptiles to li g ht decreased the amount of auxin which could be extracted by ether. This is in harmony with the findings of van Overbeek and Koningsberger. How- ever, they were unable to find any destruction of an auxin orepared from corn meal on allowing it to be transported throu::,;h illuminated coleoptile sections. Stewart and Went conclude that this is due to light sensitivity of auxin when it is bound to some constituent of the cell an :: to 1 i ,;ht stability of freemoving auxin. It is perhaps possible that this auxin which was prepared from corn meal contained 3-indoleacetic acid al thou·;h the available information indicated that the 11 free 11 auxin of corn meal is cw.xin a or b ( 9, 27). To summarize in general what has been said about auxins and their comparative physiological properties, we see that the differences, for example, bet½een auxin a and 3-indoleacetic acid are not merely quantitative in nature. This is an inescapable result of their dif- ferences in structure and, as investi3ation proceeds and more information accumul 0 tes in this field, it is to be expected that further differences in biolog ical activity will be discovered. (31) The foregoin g review of the litera ture brino; s us to the re r l purpose of this problem. The •11 bound 1I or combined auxin of dormant corn kernels h as been conclusively identified by isolation b y both Haa g en-Smit (11) and Berger 11 (6). However, the free 11 portion (that rea.dily available by water extra c- tion) has not been isolated for either immature or dormant kernels. It is with this fraction in imma ture ker- nels that the present work is concerned. This II free 11 portion is of special in te rest and signific ~nce for the followin 3 reasons: (1) It i s present in a rapidly growing tis s ue. (2) It ca n be e x tracted und er very mild conditions, i.e., wit h water or org anic solvents. (3) There is evidently s om e connection between auxin production a nd the chan5es which occur subsequent to fertilization. Whether or not this relationship is causal is at present not known. ( 32) Experimental Section Bioassay The auxin concentrations throug hout this work were determine d by the standa rd Avena test, as described by Went (43). The interva l between application of the agar blocks (2 mm3 in v6lume) and photographing of the test pla nts was 110 minutes in all c as es. The follow- ing procedure was adopted in evaluating the experimental dat a . The an3 les of cu r v a ture were measured a n d those between O a nd 2 de g rees discounted unles s the mean curva ture was under 5 degrees. 'rhis is justified on the basis tha t errors in techni que can result in approximately a O degree curvature even thou g h a larg e concentra t ion of e.uxin is present. remainin ::,; curvatures was c 0 The aritb..metic mean of the .culated and those means within the proportionality range taken as proporti onal to the a ~xin concentrati ~n. In each test, the Avena seedlings were calibrate d by applying blocks soake ~ in solutions of known c o ncentrations of 3-indoleacetic acid. One mg . of ~-indole- acetic acid, Merck, diluted uo to 3 1. (1:3x10 6 ) usually gav e curvatures of about 15 de g rees. Dilutions were made ~ 1th a medium compose d o f distilled water containing 0.005 moles of potassium chloride Pnd 0.003 moles of acetic acid per liter. (33) Description of Startin 0 :Materia l The crude extract used in th is isolation of 3-indoleacetic acid was prepared by Dr. S. H. Wittwer at the Uni v ersity of Misso uri . Approximately 100 k g . of fresh corn kernels i n the milk s tag e, that is about 15 days after fertilizati o n , wa ~ e xtracted by placin ~ it in 95% ethanol. About 80 k g . of kernels was extracted f or t wo weeks and t h e remainder about 24 hours . The alcoholic soluti 0n was concentrated under reduced pressure to 3700 cc. and the syrupy extract shipped to The California In st itute of Te c h n o logy via Air Express. Abo ~t two days were spent in tr ansit but on arriva l ther e was no noticeable e v i dence of fermentati Jn or spoilage . The total activity of this crude extract was equivalent to 1.48 5m . of 3-indoleacetic acid (Appendix Sept. 8.). The residual corn now once extracted was sent to us by Railway Express and s to red at 4 degrees C. The li qu id still held in this residue by capilla.rity was abo.:t 50 to 60% ethanol. Chemical fractionat i on (1) Preparat i on o f the crude acid fract i on . The c r ude syrupy extr2 ct was acidified to a pH of 3.0 to 3.5 by ~dd in 7 230 cc. of concentrated HC l. The resulting mixture was d ivided into two e q ual p a rts to f a cilitate handlin g a nd e ~c h part extracted in s epa ratory funnel wit h on 2 liter po r tion and two 1 liter portions of Et 2 o. All ether used was p urified by d i stillation over a mixture of FeS04, excess Ca(OH) 2 , a nd H20. The ether soluti on was dried over anhydrous Na 2 S04 and concentr s ted by evaporation under reduced pressure. It contai n ed 26.2 gm . of solids, equivalent in ~uxin activity to 0.186 g m. of IAA (A ppen d ix Sept. 8.). The concentrated ether soluti a n (735 cc.) was extracted in a s epar a tory with three 370 cc. porti J ns of sa t ura ted NaHC0 3 in water. The bicarbonate layer was acidified with about 110 cc. of concentrated HCl and extracted with three 1 liter portions of Et 2 0. On eva p ora t i on of t he l a tter ether soluti on und e r reduced pressure, 4.81 gm. of solid remained. This fraction was eq i ivalent to about 0.48 sm . of IAA and is composed chiefly of the free organic acids (Appen d ix Sept.11.). This sudden rise in a ctivity from 6.1 36 BID· to 0.48 s m. of I AA indicates that the bicarbonate fractionation removes an inhibit o r - or inhibitors. Probably then too the ori J inal m2 terial contained more than 1. 48 s ::1. of IAA. Since the ori ~inal crud e extract still contained a l~rg e amount of auxin activity (Appendix Sept. 8.), it was further extracted with Et 2 o. A total of 6 liters in three portions was used, and the aqueous phase was then almost free of aux in (A ppend ix Sept. 13.) . As before, the ether solutio n was concentrated under reduced pressure and extracted with saturated solution. A total of 1350 cc. of bicarbonate 3 ( seven porti :ms) was used; the b icarbonate was acidi- NaHco fied and the solutLm extra.cted with f ou r 500 cc. and one 250 cc. portions of Et 2 o. The ether was evaporated under reduced pressure and the residue c ombined with the pr~vious porti on of or3 a~ic acids . The resulting fraction wei 5hed 10.52 3m. and contained an amount of auxin e q "' ivalent in activity to 1.09 ~;rn. IAA (A ppendix Sept. 18) . Th is is equa l to about 7 4 jo of t h e act ivi ty in the original extract . This fraction which forms the basis for the succeedi ns steps of the isol ~tion will here inafter be referred to as the crude acid fractiJn . (2) Tentat ive identificat i on of the auxin present. At this juncture it was decided to determine how much auxin remained in the extracted c orn kernels and to attempt t o chara cterize the auxin or auxins in the extracted kernels and in the crude ac id fracti o n. A r e presentative sample of the extra cted kernels was ground up in a mortar and extracted with water at (36) room temperature (1 g m. of moist kernels per 15 cc. of water). The bioassay sho ~ ed that the 100 k g . of ke :c ne ls c ontained an pmount of aux in equi va1e n t to 1.2 gm . of IAA (App end ix Sept. 11). Thus the concen- tration of auxin in the fresh kernels as determined by extracti~n with ethanol plus wa ter was e quivalent to 2.7x10-5 gm . IAA per ?;m. of fresh corn. Cha racterizati o n of t he auxin present was accomplis hed by the use of th e differenti a l acid-alkali destruction test, as described in the intro ductory section. When this test wa s app lie d it was found that the auxin in the c r ude ac i d fracti on and in the residual corn kernels behaved like IAA. The following experiment will illustrate the techni q ue used and the results obtained. 53 mg . of the crude a cid fraction was d iss o lved in 0.48 cc. of 96% EtOH ana four 0.1 c c . portions of the solutiJn withdrawn. These port i ons were treated vari o usly, as follows: (a) 1.0 cc of 0.1 N. KOH was added an ~ the mixture he;:i_ted on a boilin_:; water bath for 3. 5 hours. (b) 1.0 cc. of 5% HCl was added and heated as above. (37) (c) 1.0 c c. of pH 7 phosphate buffer was added and heated as in (a). ( d) 1. 0 cc . of water wQs added and the mi x ture put i n the cold room as a c ontro l. After the he a t i ng period the mixtures were neutra lized an d d i lut i ons made . Th e following curva t ure s were obtained f r om a n Aven a test of the products . Tr ea t ment Diluti ons ( based on .-.eight of crude ac i d frac ti (m) 1: ~.s i: ,. '2 s 1:1,"2..S I: ~-S ~,~s X. ,0 -Lf "ll10S X. \0 s ""'-\o-" 12 0 > 12 '~ s ,~,.~s ~,o"' KOH ·--• HC l _ pH 7 buffer ---- Contro l 14 0 0 - > 10 10 0- >10 10 0- Th is shows that approximately 86% of the e.cti vi t_y was lost by tre2.t :nent wt t h h o t acid and n o ne by treatment with hot pH 7 buffer or 0.1 N. KOH Trea tment with 1.0 N. KOH likewise resulted in no d estruction (Appendix c• t '.)Q 0ep. _ )• Synthet i c IAA was likewise subjected to the abo ve tests, an( i t was fou.nd tha t ho t 5?; HC l dest r oys 40 to 50% of the a cti v i ty in } t hours at 98 C. whereas 1 N. KOH g ives n o measur eable destructi o n un de r the sa me conditi ons (Appendix Sept . 20). ( 38) An active extract ma d e from a representative sample of the residual corn kernels was found lik e wise to be stable toward bqse b u t labil e in acid ( Append i x Sept. 27). The r ea son for the differences observed in t he percenta 5 e destruction of pure IAA and t he aux in from c o rn is n ot known. The ac id-alkali test s on the crude a cid fracti o n wer e comp let e d 13 days after the alcoholic ex Lr act was received from the Unive rs it y of Mis souri, tho se on the residual corn kernels 7 days later. The abo ve r es ults ind ica t e t hat the chief component o f th e aux in in immature corn kern e ls behaves toward acid and alkali like I AA. From this informati o n it was evi- dent that the steps of the isol at i on procedure s hou ld be patterned after t hose for IAA from urine r ather than tho se for the i so l at i on of e i ther a uxin a orb. The small decreases in activity of the crude aci d fraction with time are difficult t o i nterpre t. Thus, on Se a t. 18, it was found to contain the eq u ivalent of 10.4% IAA, on October 2, 9,7%, and on October 6, 9,0%. These d iffer en ces a r e quite pr obably within the range o f experimental error. (3) Fra ctiona ti ~n of the crud e a cid fracti o n Approx imate ly h a lf ( 5. 02 e;m . ) o f t h e cr ude acid fraction containing the e qu ival ent of about 10.4% IAA was dissolved in 100 cc. of 60% EtOH a nd the resulting (39) soluti on extracted in a sepa r 2 tory funnel with twelve 50 cc. portions of purified benze ne . The benzene layer was then extracted with six 50 cc. oorti J ns of 50% MeOH and with six 50 cc. portions of wate r. The water and methanol extracts were c omb i ned and the methanol evaporated under reduced pressure. The remaining aqueous solu- tion was extracted with purified Et 2 o, two 100 cc. portions, and three 50 cc. portions, a nd the r es ultin g ether solution dr ie d over anhydrous Na 2 S04 and evaporated un der reduced pressure. The residue wei g hed 1.25 gm. and contai ned the equivalent of 0.26 gm. IAA (Appehdix October 13). This fraction will be referred to as the benzene-metha nol fracti.:)n. The activity here is only about fift y per ce n t of t hat present in the 5.02 g m. of crude acid fr a cti on , but the concentration of aux in is ne a rly twice as gre at . At this point an a ttempt was made to crystallize the benzene-metha nol fr e cti on. O. 87 i::;m. was d issolved in 20 cc of hot CHCl3: t h e solution wai filtered and allowed to cool. A few cryst a ls appear e d but they were obvi ously no t ~ell formed an d quite i mpure . Inas muc h as no more raw ma teria l could be obtained it was essential to get as hi g h yields as possible in th e crysta llization procedures and thu s it was neces sary t o further fractionate the benzene-metha nol fractLm by other means. ( 40) The above 0.87 gm. of acti v e material, after removal of the chloroform, was extracted on a water bath with five 5 cc. porticms of petroleum et her ( 40 to 60 de g rees) at its boiling point. The residue was then further extracted with five 5 cc. portions of hi bh boiling petroleum ether at its boiling point (100 to 130 degrees). A crude crystalline material deposited out of the hi ~h-boiling petroleum ether on cooling to room temperature. This de 1osit was filtered off, redissolved in xylene, and the solution crystallized in a few hours in the cold room. This product amounted to only a couple of mg . but was recrystallized later along with other hi s hly purified frRctions. The main bulk of material havini_s been extracted with petroleum ether was treated with five 15 cc. portions of hot xylene. A residue (83 rn5.) remained which proved to be ina,ctive in the Avena test (Appendix Nov. 8). The xylene s8lution was evaporated under reduced pressure an~ gave a residue of 505 m; . This residue was extrc1,cted on the water-bath with twelve 5 cc. Jor- tions, three 10 cc. portions, and two 25 cc. porti~ns of boiling cyclohexane. mained undissolved. About 317 mg . of material re- The cyclohexane extracts deposi- ted a crude crystalline material on st2nding in the ( 41) cold room, and t h is proved to c ont a in the equivalent of about 30% IAA (Appendix No v. 8). This material was recrystPllized from xylene and chloroform and l a t e r c o~b i n ed wi th other fractions for the final recryst a lliza t i ons. The mater i c:1. l rema in i ng after the cycloheatane treatment \vas now r e -ext r a c t ed with five 10 cc. port i ons of hot xylene. Only 13 m~ . of residue r emain ed , and as t h e xylene insoluble material was p r e vi o usly show n to be ina cti v e in the Avena test t h is residue was disc ard ed. The xylene soluti on was evaporated a n d t he resi- due (328 ms .) assayed an d foun d to c o nt a in the eq ~ival en t of a b out 2 1% IAA (A ppen d ix Nov. 8); thus it s t ota l c onten t was abo ut 69 m; . of the pure subst fm ce. This fr ~ cti on solidified partly in the cold but the crys t als were still contaminated by larg e amounts of o the r subst a nces. The fo l low i ng step des i gned to remove v ar i ou s c ontaminat ing i mpur i t i e s c onsists of a prec i p itati on with le ad ion in dilute a c id an der which c onditi ons IAA remc ins in so luti on . The 328 m,,; . of IAA was d i ss ol- ved in 10 cc. of 96% EtOH, and l cc. of s a tura t e d n eutra l l ead acet a te was a d de d slowly with st i rrin g , wh ereupon a dense p r e cipi t a te formed. ( 42) The mixture was filtered , the precipitate washed with a small volume of EtOH and the filtrate and was hing s c ~mb ined. The moist pre ci p itat e was immed i a tely d issolved in a mixture of 1 c c . of 1 N. HCl and 20 cc. o f 1 N. AcOH. The res ultin 3 soluti on was ex tra c te d with f ou r 10 cc. portions of Et 2 o an d the ether phase evaporated under reduced p res sure. t he res : due (1 23 mj .) pro v ed t o c o n- t B. in the e qu ivale nt of 38% I.AA (Append ix Nov . 1 3 ). The f iltrate fr om the lea d pre cipit at i o n wa s c on centra t ed in vacuo to r emo ve the EtOH, a nd a mixture of 10 cc. of water and 1 cc. of 1 N. AcOH added to the resi d u e . The solu t i 0 n wa s e x tra ct e d with five 10 cco portions of Et 2 o 2n d the ether l ayer evapo r a ted as be fore . The resultin g fraction (11 8 m; .) containin g the e quiva lent o f 33% I A.A (A ppen d ix No v. 13) Wern d i s s o l v ed in a small amo unt of CHC1 , the mixture filtered and 3 p l a ced in the c o ld roo m. Crystallization occurred in a s h o rt tiwe a n d resJlted i n 24 m~. of c rud e crystalline mster i a l. Th is was r e c rys t Rl li zed o nce fr~m CHC1 3 and the pro d uct reserved for the fi na. l pooling of all the a c t ive cry sta lli ne fracti ons . Inasmuch as IAA it self do es n ot yield a .f)l'ec i p itate with l end 2,cet : 1 te in wee.kly ? Ci d medium, the precipitatio n was repe ~te d on the fr ~ct i h ~ h ich was insoluble in the first tre atment. ( 43) This fr act i on (1 23 m~ .) was diss ~lved in EtOH and saturated neutral lead acet ate a dded. The p r e cipitate was d is solve d in d ilute AcOH and , a l ong with the filtrate, ext ra cted as before, This time the insolub le fraction, after remo val of the lead ion, wei g hed 84 mg. The filtrate afforded 27 m0o· . of a crystalline material which had the solubility behavior of IAA and gave a brilliant red c o lor with a mixture of Fec1 3 and HCl. This fraction was recrysta~lized f rom CHC1 3 and c ombin ed with the other crystalline fracti J ns. Evidently the mater i a l which wa s soluble in the second precipitati on was merely adsorbed on the precipitate dur i n g the f irst trea t ment. The cryst Alline ma t eri a l obtained from the petroleum e t her and c y clohexane e x tractions a nd from the two lead acetate precip ita.tions was c omb n ed and dissolved in CHC1 . 3 Th i s crude cryst2lline material amounted to e. b ou t 70 mg . from which 49 m;5. of th in fla.t pl a tes was obtained on recr ys t al li za ti J n . This subst ance sinte r ed on t he hot st age a t 16 3 C. and melte d t o a reddish liquid at 1 6 5 to 169 .5 C. Re crysta lli zed IAA, Merck , s i n tere d at 167 C. and melted at 168 to 170 .5 C. under th e same c ond i t i J ns. The 49 ms . of crysta ls was r e cr ys t a lliz ed from CHC1 3 and dried over ni '.·,ht ove r P 20 5 in vacuo . l 'he re- s ultin g crys t a lline compound (43 mr.; . ) sintered at 164 c. ( 44) and melted from 165 to 169.4 C. on the hot stag e, whereas the recrystallized synthetic product simter ed a t 164 C. and melted from 167 to 170 C. An intima te mixture of the two sintered at 164 C. and me lt ed at 166 to 170 C. (hot s ta c,;e) . Capill ~ry melting points we re made simultaneousl y on the material from corn an d on recrystallize d synthetic IAA. The former sintered at 165 C. and melted at 165.7 C. to 166.5 C. The synthetic compound sinter- ed a t 165 and melted at 16 5 .5 to 166.5 C. uncorrected). . (all m.p. 's An intimate mixture of the two crystalline rmteri a ls sintered at 1 6 4.5 a n d melted at 1 65 . 5 C. while a s imultaneously observed ca p illary of recrystallized synthetic IAA sin tered at 16 4.5 a.nd melted at 165 C. Ca rbon, hydr o ge n, and nitrog en micro- a n a lyses of the cryst .s lline m;:iteria.l gave the follow n g results: Cale. for Cl0H902N Found C2,rbon 68 . 541;; 68. 99Jb Hydro g en 5.18% 5.17% Ni tro ,., ,i en 8. OOJ~ 8.06% As an additional check on the identity of I AA and the subst a nce obtained from c orn, the two were assayed simultaneously in the Avena te s t. ing curva t ures wer e obtained: ( 45) The follow- Diluti on l; 1; l; 1: Crystals from corn Synthetic IAA Me rck ~ 6 25 de ,;•rees 3xl0 3xl0,,_ 9xl0° 27xl0 6 '~ ) 23 II 11 II 3 11 19 degrees II 14 7 4 11 11 This discrepancy in biological activity may be due to the fact that the synthetic IAA us ed in the Avena test was not recryst all ized but used directly from the Merck fontainer. All diluti ons for the above test were made on the same day . Thus the inform Pt i on afforded by rneltin 3 po int, mixed meltin; point, qua nt itative ana l ysis , and bio a ssay shows that the auxin isola ted from e a rn kernels is 3 -indoleacetic ac id. The overal l y i e ld of IAA making a llowance for the fa c t that only a p a rt of each active fraction was used for further purification is a,bout 9% . ( 46 ) (4) Summary of the fracti onati on scheme. The following will serve to summarize the isola- tion procedure which has been described in detail abbve. 100 kg. fresh corn kernels (2.7 ,;m. IAA) I gs% Et0H Residual~Alcoholic extract kernels ( 1. 2 gm . IAA ) l Concentrate in vacue 3700 cc. of extract (1.48 g m. IAA) Acidify Ether extract Aqueous phase (Ina ctive) Evaporate Water phase Ether phase Acidify Et 2 0 Water phase l Ether phase Eva.porate Crude acid fraction ( 1.09 3rn . IAA) 10. 52 gm. ( 47) 5.02 gm. Crude acid fracti on (0.52 gni. IAA) 60% EtOH Benzene Benzene layer water layer 50% MeOH Methanol layer Benzene layer \ Combine f ~Water layer ~ ~ Benzene layer Evaporate methanol ~ Aqueous solution ~o Water phase Ether phase ~ Evaporate 1.25 gm. Benzene-methanol fraction (0.26 gm. IAA) Pet. Ether Pet. Ether (40-60) Residue Pet Ether (100-130) ( 48) Pet Ether Residu.e j Cool Preci pi te..te iqo t Xylene /,.~Solution Xylene ~.e§idue ( 83 mg . inactive) Crude IAA Crystals r{esidue 505 (small amount) ! j Cyclohexane ! Residue cool P :'ecipi tate I Xylene plus chloroform Crudt;;r cry ·s··. t ,ls of IAA , 2 0 rw : . 1; t Hot Xylene 0oluti::m 't{2s idue \13 mg . l Ine,ct i ve) Residue 378 mg. ( 6 9 mE; . IAA ) 96% EtOH Pb ( OAc) Precipitate 2 Filtrate Acidify Evaporate Dilute AcOH Water layer Ether(l23 m,7 .) .b;t O 2 ( 47 mg . IAA)' Solution 96% EtOH Pb ( OAc) Precipit.c::te -----.... 2 Filtr,-: , te 1 Acidify Et 2 o 27 mg . crude IAA ( 49) l hesidue 118 mg . (39 ms . IAA) ! CHC1 3 24 m6 . crude IAA A;,pe ndix Appendix , Date and Materia l , Being Testea. Dilution Mean Curvature ' Degrees ;Fercentage IAA ,sept. 8, 1944 IAA Standard l:9xl0 6 1:27x10 6 Orie; inal Crude BxtrAct (370 0 c ,:: .) 1: 100 1: 500 1: 2500 <.20 22 10 Et 2 0 soluble fracti on of crud~ e•tract ( 26 .2 gm) 1:10 4 4 l:5xl0 4 1: 25x10 14 9 Tra-c e _Gri g inal crude extract after ex~raction with Et20 1:10 1:50 1: 250 Sept. 11, 1944 7 0 O. 04% 8 8 0. 0067'~ 16 IAA Stan ciard 1:9x10 6 1: 27x10 6 Orig ina l crude ex t ract ( a hso tested Sept 8 4 0, tak en to be 0.0 ;o IAA 1: 50 0 1: 2 500 1: 12,500 0 o· 14 Trace 0 ) 10 7 Crude acid fracti on (first pnrt ~n ual 4. 81 gm.) 0.04% 10% 0 Residua l co rn kernels ( about 100kg .) ,f:3-ept 13, 1944 ~AA StandP.rd 1:15 1:75 1: 22 5 14 l:9xl0 6 1: 27~no 6 3 D _0.0012% 0 b Residual crude ex- 1:50 0 1 : 2500 tract after 2 ext~s 1: 12500 with Et 2 o 0 0 Ina c tive ll ' ( 50) li:)ate and Mater i al 32eing Tested Sept 18, 19Lt4 IAA Standard Diluti on f>ilean Curvatu re Degrees 6 l:3xl0 0 l:9xl0 <s 8 10. 4% Crude acid fractLm ( 10. 52 ,s m) $ept. 20, 191+4 IAA StAndard 1:3x10 6 1:9x10 6 0 Crude acid fracti on he ated with i N. KOH 4L l: 2~xl0 t4 1: 1· 5xl0 14 10 0 Crude acid frac iion control (un tre r,1 ted) 1:5x10 4 4 l:25xl0 1: 125xl0 4 12 8 0 Synthetic IAA he F.1.ted with 5% HCl 1: 25xlo 4 l:125xl01 l:625xl0 t 10 tra ·.: e Synthetic IAA he e ted with 1 N. KOH 1: 50xlo 4 ,, 1: 250xl0 ., 4 1: 1250xl0 12 1:3x10S 1:9x10° 14 5 1: 5xl0 - 8 9 8 0 100% pept 27, 191;.4 ,IAA Standard Active extract from ~esi dua l corn ~.Le ~nels \1 gm. - 1060 ~m ker• ne 1 s -dontro l (Untre ~ted ) Same extra.ct as above he ~ted wlth 5% HCl Same extra. ct as i bove hes ted with N. KOH + 8 4. 4% 0 0 4 1: 25xl0 4 1: 125xl0 4 l: 62 5xl0 l:25xlo 4 4 1: 125xl0 4 l:625xl0 ( 51) 0 0 0 Inactive 10 0 0 5-5% Diluti on Mean Curvature De gre es IAA Standard 1:3 x109 l:9 xl0° 0 Crude ac id fr a ction 1: 10 4 4. l:5 xl 0 4 1: 25xl0 Date and Materia l Be ine; Tested Oct 2 , 1944 6 Oct 6, 19 44 22 20 7 9-7% 13 13 IAA Stande rd 8 10 ca 10 ca 10 14 trace Crude ac id fr 2 cti )n 9 .Oju Oct 13, 1944 IAA .S tandard 1: 3x10§ 1:3x10 l: 9x l0 13 14 4 Benzene-m e thanol fra.cti :- n (1.2 5 ,')m.) 1:10 6 c 1: 3x10° t race IAA Stne.d,'?.rd 1:3x1og l:9xl0 19 11 Xy l e ne insoluble fr a cti-:J n ( 83ms . ) l: 5x10 4 1: 25:xlO Lj. l:lOb 0 0 0 Cryst a lline m2.t er i al from cycloheae2.ne extrRcts 1: 5x10 4 . 4 l: 25xr:: l 0 1:10° Xylen e soluble fracti::m ( 328 ffi <">' ) 1: SOllO 1:10 C 1: 3x10° 6 20.5% 8 No v 8, 1944 4 ( 52) Ina ct ive Max . an; l e 11 Ma.x . 19 33;0 arn::~le 17 7 2lfo Date a nd Material Being Tested Dilution Mean Curvature De g rees %-age found , _) Nov 13, 1944 l:3xl0~ l:9xl0 6 l:27xl0 26 Ppt. from first Pb pptn (123 m(7) . ) 1:10 6 , -, 1 0 b 1 :)x JL;9xl0 6 )10 8 0 F' '. ltra te from first Pb pptn. (118 mg.; 1:10° r l:3 x l 0~ l:9xl0 ° IAA Standard 7 4 38;o ,,- (53) 14 7 tra ce 337° Conclusion Actually there were two prime objectives in view in undertakin g thls present work. The most immediete of these wa s to conclusively identify a n a ~xin which occurs in a readily watersoluble, biolo 3 ically active f o rm in immature corn ke r nels. This has been acc ~mplished by t b e iso- lation ~nd identification just described. total available dat a concernin; Thus the the distributi on and identity of auxin in the corn pla nt is as follows: Auxin Present Method of Characterization Tip of youn,_:; plant auxin a Diffusion Acid-alka li Imma ture kernels (free portion) IAA Isolation Part of !Blant Corn 1vieal (free portion) auxin a auxin a orb Li g ht ina ctivation Diffusion Dormant kernels or corn meal (bound portion) IAA and Auxin a Isol B. tion Corn oil Auxin a and b IAA Isolation and Acid-alkali Isolation From this information the re2.der may wonder why IAA did II I n ot appe a r in the course of Ko ; l s wo rk on corn oil or why the isola ti o ns fr om mat~ re c o rn or corn meal did n o t y i eld a uxin a and b. In the l,g,tter work already discussed evidence was found for the existence of auxin a in corn meal by the ( 54) isolati o n of a small amount of a substance which had t he correct meltin g point for pseudo-auxin a . . In the work o f Ko 3 l one o f the obvious poin ts in the isolati on procedures to examine for clues for the presence o f IAA is the lactonization step. This was car- rie d out in hot 1-5% HCl in MeOH and here i t mi ~ht be expected that any IA.A ptesent woul d be lar gely destroyed al thowi; h the stability of IAA under the s e donditi ons is n o t known with certainty. In the first isola tion from corn oil, f' or example, the active fr R.ction before l a ctonization contained 55 mg . o f aux in and afterwards appa rently 28 mg . from wh i ch 7.4 mg . o f crysta lline auxin b was fin a lly obta i ned . This app rent decrease 0 in activity during l a ctoniza ti on does not oc cur with the concentrates from urine which did not yield auxin b.and can be satisfactorily explained by the form a tion of auxin b d imethyl aceta l l a ctone wh l ch is much less active than ~uxin b. In a se cond isolati on from c orn oil containing presumably 70 mg . of auxin, a uxin a an d 15 ms . of crystalline 15 mg . of auxin b were obtained. This is a very excellent yield eve n Lif all the auxin in the starting material were aux in a and auxin b. In the puri f ic a ti :J n of auxin from malt the activity practically di§ap~eared on l acton ization but reap- ( 55) peared almost entirely on d istill at i on . The purification ste p s employed in al l o f K; 3 l 1 s isola tions were such that probably a n appreciable amo unt of !AA would be c a rried alon~ with auxin a 2nd bat least to the lactonization st a~e if it had been present in the starting rnateria,l. These considerations are complic a ted by ~he p ossible pres~nce of inhibitory substan ces. .. In the wo rk of Ko g l there was evide n ce f o r these only i n the case of corn o il. An isolati o n was begun with 16 k g . of corn oil containing 700 gammas o f auxin per k g . Th is was ex- tracted with H20 and the water extr '"' cted with Et 2 ()). The resulting fract i on contained no m~ . of aux in, an appa r ent 10 fol d increase in auxin c o ntent. Simil ar evidence of an inhib itor in imma ture c orn kernels has alre 0 dy been pointed out; it is evidently n 8n-acidic since it was not present in the crude acid fraction. It is inte resti r s to no t e that both the ma lt and ti the corn oil used by Ko 6 1 were probably rat he r fresh samples or else the a uxin would have already di sappeared by rearrangement. Moreover, the auxin in both these ma t er ials was probably present in a free form since it was extracted by simp le water extr 0 cti J n. ( 56) The pos s ibility tha t t h e imma ture c o rn used in the present i s olatio n c ontained a uxin a and/orb is not conclusively ruled out b y the fore g o in;; data, but the evidence indics,tes t hat if pre s ent at a ll, probably they ar e pre se n t in only low concentrati o ns. Abo u t 70 k s . o f t he corn used in this isola tion was harve s ted on Aug . 15, 1944, a n d a bo u t 30 k g . on Sep t . 1, 1944. The crude acid f raction was first c hara c- t e rized b y the a cid-alkali t est on Sep t. 18, presumably before any a u x in a orb c ould hav e c o mp l e tely rearranged to t h e ina ctive pseudo-a ux in. The fact tha t no destruction occu r r e d i n alkali ind icates o ne o f the followin g situati o ns: (1) Auxin a and/or b a r e pres e n t but a re st ab ili z ed b y some o t her comp on e nt of t he mixture. (2) Auxin a and/orb are pre s ent along wit h an inhibi t or, which itsel f is an acid a nd wh ich is destrc y ed by alk a li in a n a mount biolo ~ ically equivalent to t he a u x in d e s t r oy e d . ( 3 ) Auxin a and/orb ~re n o t presen t in a ppreciable c o ncent ra tions. To ~he a utho r t h e l as t is by f a r the most like ly c oncl u sio n. ( 57) The se cond objective of this work was to attempt to apply the inf orma tion 3alned experimentally to shed additional li s ht on bhe identity of the native plant g rowth hormone. As we have just seen in the case of corn, this problem i s very complex. About the most we can say iB a General way is that if aux in a orb or 3-indoleacetic acid are found in a locality of a p la nt where the correspond in g c om ound has been proven to exert a s~ ecific effect, then the auxin found is probab ly responsible for this effect in the intact plqnt. In the ca J e of the corn plant , 3-indoleaceticc a cid h~s been found in immature kernels, a r ap idly me taboli z in g tissue. Inasmuc h as the role, if any, of the g rowth hormone in maturing seeds has not been investi gated, the si 3nifica n ce of 3-indoleacetic a~ id in corn kernels is at present n ot known, and we must await further physiological findin s s to be able to correctl y interpre t these data. (58) Summa ry Summary '.l1he followirn:; surnma,rizes the main results obtai ned in the f a regoin s wo rk. (1) An auxin occurrins in a water-soluble and biologically active form in the kernels of immature corn h as been pro ven t o be 3-indoleacetic a cid by isol a ti on and compar is on with a n authentic sample of 3-indoleacetic acid. (2) The c 8 n centrat i J n of aux in in these corn kernels was fo un d to be e qu ivalen t to 2.7xl0-S gm. 3-indo leacetic acid per g ra m of fresh corn. (3 ) l' he va,1 i d ity of t h e acid-alkali test h as been :onfirmed for a mixture contai n ins about 10% 3in doleace tic ac id. (4) An isolat i on pr ocedur e for 3-indoleacetic acid (based on the results of previ o us workers) has been de vis ed . This proc e du r e possibly c a n be applied to a va r 1e t y of plant materials with only minor a lt erat i ~ns. ( 59) Biblio g ra 9hy Bi blio ~·. raphy (1) Arndt, K., Handbuch der physikalisch-chernischen Technik. p. 614. (2) Avery, G. S. Jr., Crei g hton, H. B., and Shalucha,B. Extractio n Metho ds in rlelation to Hormone Content of Maize Endosperrns. Am. J. Bot. ~7: 289-300 (1940). (3) Avery, 1}e o. S. Jr., Berger, Julius, and Shalucha, B. The Total Extraction of Free Auxin and AuxinPrecursor from Plant Tissue. Am. J. Bot. 28: 596-607 (1941). -- (4) Avery, Geo. S. Jr., Berger, Julius, and Shalucha, B. Total Auxin Extracti ~n from Wheat. Am. J. Bot. g~: 612-616 (1942). (5) Avery, }eo. S. Jr., Berger, Julius, and Shalucha, B. Auxin Content of Maize Kernels During Ontogeny, from Plants of Varying Heterotic Vi gor. Am. J. Bot. ~2: 765-772 (1942). ( 6) · Berger, J. , and Avery, Geo. S. Jr. Isolation of an Auxin Precursor and an Auxin (Indole Acetic Acid) from Maize. Am. J. Bo t. 21: 199-203 (1944). (7) Be rgren, W. R. Dissertation. of Te chnolo s y (1941). California Institute (8) Einstein, A. Elementare Theorie der Brownschen Bewec;ung . Z. Elektrochem. 14: 235-239 (1908). (9) Goodwin, R.H. Evidence for the Presence in Certa in Ether Extracts of Subst 0 nces P~rtially Maskins the • Activity of Auxi n . Am. J. Bo t. 26: 130-135 (1939). (10) Gordon, Solon, A., 2,nd Wildman, Sam G. The Conversion of fryptophane to a Plant Growth Subst Rnce by Concfil:tions of Mild Alk2 lini ty. J our. Biol. Chem. 147: 3 39...:3,93 ( 1943) . (11) Haa gen-Smit, A. J., Leech, W. D., nd Bergren, W.R. Estimation, Isolation, an d Identificati o n of Auxins in e lant M2 te riEi,l. Science 93: 624-625 ( 1941) . 0 (12) Hatcher, E. S. H. 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Ko ,;l, F., Haage n-Smit, A. J., and Erxleben, H. Ueber ein neues Aux in ( 11 Het eroaux in 11 ) aus Harn. Z. physiol. Che m. 228 : 90 -102 (19 34) . •• Ko g l, F ., Haagen- Sm i t , A. J., and van ' H~lsijen , C. J. Ueb er den ~influss der Auxine a uf das Wurzelwa ch stum and ue be r die chemisc h e Na tur des Aux ins der }raskoleopt i len . Z. ~hysiol. Chem . 228 : 104-112 ( 1934) . (20 ) Ko• •g l, F ., an d Ko ste r mans , D. G. F . rl . He t ero auxin als Stoffwec h selp r odukt nie derer pflsnzIicher Jrganisrnen. I so lierun5 au s Hefe. Z. phys iol. Chem . 228: 113-121 (1934) . 0 ( 21) Kcg1l._,, B'·•.. ,. a.nd Erxleben, H . . ',Synthese der 11 AuxinII g lutarsau re _ u. n d ein i ; er I somerer . . z. phystol. C.hem .. _g3 2 : 1_81-200. ~ 1_93 5) . (22) Ko ~l, F., Konin~ sberger , C., and ~rxl ebe n, H. Ue ber d i e bel ~st inRctiv ie r un ; der Aux i ne a und b . Z. physiol. Chem . 244 : 266 - 278 (1936). '. (23) Koningsberger, V. J., and Verkaaik, B. 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