BIOCHEMISTRY.THE past year has seen notable advances in our knowledge of thcchemical constitution of the sex hormones and of vitamin B,.The more purely chemical aspects of these subjects are dealt with inthe Report on organic chemistry. The identity of vitamin C withascorbic acid has been fully established, and some progress has beenmade in determining the nature of vitamin B,. The mode ofbreakdown of carbohydrate by the enzyme systems of muscle andyeast has been studied further, and evidence has been supplied ingeneral support of the Embden-Meyerhof schemes reported lastyear, as well as in defining more closely certain of the stages involved.Attempts have also been made to link the formation of lactic acidwith other reactions which also take place in muscle.I n amino-acid metabolism a number of somewhat isolated observations havebeen made, and attention is directed in this Report to a renewedinterest in the possibility of carbohydrate formation from fattyacids-a problem which still seems to elude a definite solution.Much valuable work has been done on the nature of the proteolyticenzymes and their substrates, but further clarification is necessarybefore a short review of the situation is possible. During the year,however, the synthesis has been carried out of substituted diketo-piperazines which are claimed to undergo hydrolysis by enzymes-an event of sufficient importance to warrant a special note.Considerable space has been devoted this year to the question ofplant growth-regulating substances, investigations of which aredistributed over a number of years.The establishment of auxinas a definite chemical entity has focused the attention of chemistson these growth substances, and some account of the more biologicalaspects of the subject seems called for. Since the matter has notpreviously been dealt with in this section of the Report, the cus-tomary practice of extending the period under review has beenadopted. The practical agricultural importance of legume inocula-tion also demands reference to the organisms concerned. Interestin the varied and complex problems of the mineral nutrition ofplants and the related subject of metabolism continues to increase.The current year’s work represents a steady general advancementwithout isolated spectacular achievementSTEWART AND POLLARD.323ANIMAL BIOCHEMISTRY.The Secondary Sex Hormones.Interest in the secondary sex hormones continues unabated, andthe elucidation of their chemical structure has made further greatprogress during the year. Improvements in the methods ofseparating the different hormones, and the artificial preparation oftwo of them, justify the hope that the advances in the more purelychemical study of these substances will soon result in similaradvances in our knowledge of their biological effects. The chemicalconstitution of the -secondary sex hormones is considered in detailelsewhere in this volume,l and it will therefore be sufficient heremerely to indicate the suggestions as to structure which are accepteda t present.Testicular Hormone (Androsterone) .-The structure (I) suggestedfor the hormone isolated from male urine and from testes2 hasbeen confirmed by its preparation from epidihydroch~lesterol.~Like cestrone, it is a hydroxy-ketone, but it differs from the ovarianhormone in being saturated, and in retaining the methyl group ofthe sterols at Clo.A noteworthy difference between androsteroneand cestrone is the apparent specificity of the former. As is wellknownY4 a large number of substances, all containing the phen-anthrene ring system but otherwise differing widely from cestrone,possess the power, sometimes in high degree, of inducing cestruswhen injected into immature animals. The formula for andro-sterone allows the theoretical existence of 128 isomerides, of whichRuzicka and his colleagues prepared four.Two of these, derivedfrom dihydrocholesterol and epidihydrocholesterol, differed in thespatial position of the hydroxy-group but agreed in possessing thetrans-configuration of rings A and B ; the other two, from coprosteroland epicoprosterol, had cis-configurations of rings A and B butdiffered similarly with respect to the hydroxy-group. The twocis-compounds were without noticeable effect on comb growthin the capon even in doses 15 times as great as those of natur91androsterone which sufficed to produce a 20% increase in comb area,and only the substance obtained from epidihydrocholesterol had anactivity equal to that of the natural hormone. The specificity,however, is not absolute, and K.Tschernig has actually found athreefold increase in activity to accompany reduction of the keto-group to a secondary alcohol. Moreover, a substance exhibiting3 L. Ruzicka, M. W. Goldberg, J. Meyer, H. Brungger, and E. Eichen-4 Ann. Reports, 1933, 30, 340.P. 206. Ann. Reporte, 1932, 29, 241.berger, Helv. Chim. Acta, 1934, 17, 1395; A., 1221.ti Wien. klin. Woch., 1934, No. 20324 BIOCHEMISTRY.androsterone activity (1 capon or mouse unit in 25-100 x g.)is reported as being obtained by hydrogenation of crude or crystallinef ollicular hormone (oestrone) .Two forms of the hormone are suggested by T. F. Gallagher andF. C. Koch,' one occurring in the testis itself and the other inurine. They find that the hormone obtained from human maleurine is not affected by boiling 3.3% potassium hydroxide solution,whereas hormone prepared from bull testis loses activity in thesecircumstances, and that the loss is not prevented by urine or hormoneprepared from urine.A. A. Adler 8 finds further that in malehuman urine the hormone is present in an inactive form, extractableby butyl alcohol, and activated by boiling with trichloroacetic acid.The Follicular Hormone ((E'drone) .-The accepted structure ofcestrone (11) shows the hormone as differing from androsteronein that ring A is fully aromatic (and has therefore lost the methylgroup at C,,), the 3-hydroxy-group being therefore phenolic insteadof alcoholic.The excretion of estrogenic hormone in male urine has beenconfirmed by B.Zondek? and the isolation and identification of thissubstance with a-folliculin (cestrone) have been accomplished byE. P. Haussler, 10 and by V. Deulofeu and J. Ferrari.11 Zondek 99 l2finds that stallion's urine contains an average of 42,000 mouse unitsof folliculin per litre, but the non-pregnant mare excretes less than500 units per litre. Man and other male animals also excreteceatrogenic hormone, though in much smaller amounts-less than200 units per litre. These findings, in conjunction with the furtherfacts that horse testis yields 54,000 units of follicular hormone perkg., and that implantation of 50 to 100 mg. of testis into spayedinice induces estrus, are taken t o indicate that the testes areconcerned in the formation of oestrone by males.Zondek 12suggests, indeed, that the metabolism of the sex hormones is, in themain, the same for both sexes, and that the male hormone is firstproduced and then converted into the female hormone.6 W. Dirscherl and H. E. Voss, Naturwiss., 1934, 22, 315; A., 815.7 J . Biol. Chenb., 1934, 104, 611; A,, 568.* Nature, 1934, 133, 798; A., 815.xi Helv. Chim. Acta, 1934, 17, 531; A., 702.11 2. physiol. Chem., 1934, 226, 192; A., 1269.L3 Nature, 1934, 133, 494; A., 567.Ibid., p. 209; A , , 332STEWART AND POLLARD. 325The great range of synthetic oestrogenic substances (all containingthe phenanthrene ring system) was discussed last year.4 Theresults of Cook and his collaborators have since been published indetail,13 and another active phenanthrene derivative has beenadded to the list by J.C. Bardhan,l* who distilled 2-carboxy-3 : 4-dihydrophenanthrene-l-propionic acid with acetic anhydrideand obtained an active substance, Cl,HI,O (probably I11 or anisomeride) .CH,A(111.) (IV.) l-Keto-1 : 2 : 3 : 4-tetrahydrophenanthrene.(V.) 9 : 10-Dihydroxy-9 : 10-di-n-butyl-9 : 10-dihydro-1 : 2 : 5 : 6-di-benzanthracene.J. W. Cook, E. C. Dodds, and A. W. Greenwood l5 find that two oftheir synthetic estrogenic substances (IV and V) are devoid ofandrosterone activity, causing no acceleration in comb growthwhen injected into brown Leghorn capons. They bring about analteration in the plumage, however, with development of femalecharacteristics.The increase in the activity of androsterone aftgr reduction of theketo-group is paralleled in the case of cestrone.E. Schwenk andF. Hildebrandt l6 find that reduced cestrone has an activity of30 x lo6 mouse units per g. in the Allen-Doisy test (about threetimes as great as cestrone), and K. David 1' states that the reducedsubstance, cestradiol, has about twice the activity of cestrone.S. L. Cohen and G. F. Marrian l8 report in detail a method forthe separation and colorimetric determination of cestrone andestriol in urine, using a modified Kober reaction l9 (a red colourwithout green fluorescence on boiling with sulphuric and phenol-sulphonic acids, and dilution with water), with analysis of thecolour in a Lovibond tintometer. They state that, although theseparation is not strictly quantitative, the method is sufficiently13 J.W. Cook, E. C. Dodds, C. L. Hewett, and W. Lawson, Proc. Roy. SOC.,14 Nature, 1934, 134, 217; A., 1102.1 5 Proc. Roy. Soc., 1934, By 114, 286; A., 457.16 Naturwiss., 1933, 21, 177; A., 1933, 540.1 7 Actcc Brev. Ne'erl., 1933, 3, 160.18 Biochem. J . , 1934, 28, 1603; A., 1269.1934, B, 114, 272; A., 457.Is Biochem. Z., 1931, 239, 209326 BIOCHEMISTRY.accurate to allow the detection of abnormal amounts of eithersubstance in pregnancy urine.The International Standard of estrogenic activity is definedas that contained in lo-' g. of ketohydroxycestrin (aestrone).The Corpus Luteum Hormone (Luteosterone).-Early in the yearunder review, the pure corpus luteum hormone was isolated,practically simultaneously, by three groups of workers.21, 22y *3, 24There was some difference of opinion a t first as to the exact chemicalcomposition of the hormone, and as to its physical properties, buti t is now agreed that the main active substance is an unsaturateddiketone, C21H3002, melting a t 128".A second active substance,also an unsaturated diketone, melting at 120" and converted intothe first by heat, was considered by Slotta and his collaborators tobe a stereoisomeride of the first. Butenandt at first thought it tobe C21H3202, but now 25 considers the two substances to be chemicallyidentical and to differ only in crystalline form and melting point.Luteosterone thus resembles oestrone in existing in polymorphousmodifications. The close relationship of the corpus luteum hormoneto oestrone, androsterone, and pregnandiol has been shown byA.Butenandt, U. Westphal, andH. Cobler,26 who succeeded in de-~ l - - c ~ o c ~ 3 grading stigmasterol to a substanceH& I I I (probably VI) with physiologicalactivity only slightly inferior to that(VI.) of the natural hormone. The sub-stance originally obtained melted at129-135", but later work 27 gave a pure diketone of melting point129", identical in physical and chemical properties with the naturalhormone, and with the same physiological activity. The pure sub-stance has also been obtained from stigmasterol by E. Fernholz,28and from pregnandiol by A. Butenandt and J. S~hmidt.2~Pure luteosterone has not been known long enough for completeinformation as to its specificity to be obtained, but already anumber of interesting observations have been made, tending to theCH,Ap/VI A//v20 C.Lormand, Bull. SOC. Chim. b i d , 1933, 15, 1566; A., 1934, 457.21 A. Butenandt and U. Westphal, Ber., 1934, 67, [B], 1440; A., 1039.22 A. Butenandt, U. Wostphal, and W. Hohlweg, 2. physiol. Chem., 1934,23 K. H. Slotta, H. Ruschig, and E. Fels, Ber., 1934, 6'7, [B], 1270, 1624;24 M. Hartmann and A. Wettstein, Helv. Chim. Acta, 1934, 17, 878, 1365;25 Ber., 1934, 67, [B], 2055.26 Ibid., p. 1611; A., 1265.28 Ibicl., p. 2027.227, 84; A., 1268.A., 931, 1268.A., 1039.27 Ibid., pp. 1903, 2085.29 Ibid., p. 1901STEWART AND POLLARD. 327conclusion that it is at least much more specific than oestrone.Acurious fact is that reduction of the keto-group attached to thepentamethylene ring reduces the activity very markedly %-aneffect the opposite of that found with cestrone and androsterone.Equally, reduction a t C, to the 3-hydroxy-ketone (actually thesubstance from which the synthetic hormone is obtained by dehydro-genation) gives an inactive 28 and cholestane,28though it resembles luteosterone spectroscopically, is withoutphysiological activity. The dihydroxy-compound correspondingto luteosterone is, of course, pregnandiol, which is physiologicallyinactive. Luteosterone thus differs markedly from the other knownsex hormones in that the keto-groups seem t o be essential to itsactivity.Origin of the Sex Hormones.-The fact that in all three hormones,the configuration of rings B, C, and I, is that of cholesterol and thebile acids is significant evidence that in the animal body they ariseby degradation of cholesterol.The relationship between preg-nandiol and luteosterone suggests that the former (which could bederived from cholesterol through lithocholic acid) may be theprecursor of the corpus luteum hormone in vivo. Pregnandiolcannot, however, be the natural precursor of androsterone, for it is amember of the coprostane series, whereas the testicular hormoneis derived from epidihydrocholesterol. Ruzicka has suggestedthat androsterone arises in nature by a process similar to t,hat bywhich it is obtained in vitro-epimerisation of the hydroxy-groupof dihydrocholesterol, followed by oxidation of the side chain.J.W. however, points out that an unsatdrated hydroxy-ketone recently isolated from urine by A. Butenandt 32 may, if thesuggested position of the double bond is correct, be the trueprecursor. The substance can, in vitro, be hydrogenated to andro-sterone .Little can yet be said of the origin of oestrone, since thestereochemical relations of its ring system are not known, butreference has already been made to Zondek’s suggestion that it isderived from androsterone by a, process of dehydrogenation anddemet hylation.Vitamin B,.Although there is not yet absolute agreement as t o the identityof the various crystalline vitamin B, preparations obtained bydifferent workers, it seems probable that they all contain the samesubstance but differ in purity.A. G. Van Veen 33 reports a crystal-30 A. Butenandt arid J. Schmidt, Ber., 1934,67, [B], 2092.31 Nature, 1934, 134, 758.33 Nature, 1934, 133, 137; A., 333.Wien. klin. TVoch., 1934, 47, 936328 BIOCHElldISTRY .line preparation of oryzanin (from rice polishings) which has acurvative action on polyneuritic rice- birds corresponding to 500international standard units per mg., an activity which he claimsto be greater t,han that of torulin from bakers' yeast. He describesthe substance as containing sulphur, with C 40.7% and H 5.5%.On the other hand, H. W. Kinnersley, J. R. O'Brien, and R. A.Peters point out that an equal activity has been attained in someof their preparations, but that torulin crystals contain 42.2% C,a difference which they regard as significant.As they state thatmost vitamin B, crystals contain inactive material, it follows thatsmall differences in activity by no means necessarily point to completenon-identity of different preparations. Indeed, crystallographicmeasurements by J. D. Bernal and (Miss) D. Crowfoot 35 suggestthat the different groups of workers are actually dealing with thesame substance, for they find that crystals from several differentsources are substantially identical in form and X-ray pattern.They consider that their measurements suggest a conjugated ringstructure, with the heavy atoms, sulphur and chlorine, not both atthe ends of the molecule.A similar identity of the various crystalline preparations ofvitamin B, is suggested by the most recent work of F.F. Heyrothand J. R. Loofbourow,36 who find very similar absorption spectrawith a number of samples obtained from different laboratories.They state that there is marked correlation between vitaminactivity and absorption a t or near 2600 8. The absorption curves,with maxima a t 2650 and 2350 pi., resemble those of cytosine, andit is therefore suggested that the vitamin molecule contains apyrimidine of the cytosine type. The discrepancy between theseresults and the earlier ones of R. A. Peters and J. St. L. P h i l p ~ t , ~ 'which gave the characteristic absorption maximum at 2450 8., isexplained as probably due to the different solvents used.by oxidising vitaminB, with nitric acid, have obtained two substances, each containingfive carbon atoms.The first of these was isolated as an ethyl esternitrate, C,H,,05N3, the free acid being, therefore, C5H,0aN,. Itwas not identical with any of the four possible glyoxaline acids ofthis formula, and it was suggested that it might be a dioxymethyl-pyrimidine. The second substance, C,H,O,NS, gives a methylester, loses ammonia and hydrogen sulphide when warmed withA. Windaus, T. Tschesche, and R.34 Nature, 1934, 133, 177; A., 333.Ibid., 1933, 131, 911; A., 1933, 768.38 Ibid., 1934, 134, 461; A,, 1270.37 Proc. Roy. SOC., 1933, B, 113, 48; A,, 1933, 645.3* 2. physiol. Chem., 1934, 228, 27STEWART AND POLLARD. 329alliali, and gives the zinc dust-pine splinter reaction which is heldto indicate the presence of a pyrrole ring.The formula (VII) wastentatively suggested for this substance, but the suggestion hassince been disproved by K. N i e ~ s e r , ~ ~ who synthesised a substance offormula (VII). He suggests that the compound isolated by Windausmay be represented by one or other of the formulae (VIII) and (IX),of which the latter is the more probable. The complete identifi-cation of these two substances, which account for ten of the twelvecarbon atoms of vitamin B,, will form an important step in deter-mining the constitution of the vitamin.H 2 v - p HO,C$-RH H$-G* C0,HS:Cvc'Co2H NH Hs*cxcH HSmcvCH NH(IXJ (VII.) (VIII.)Vitamin B,.Last year it was reported that the flavins, yellow water-solubledyes extracted from both 'animal and vegetable sources, possessedintense vitamin B, activity,and that lactoflavin from wheywas claimed by Kuhn et al.40 HTw'\f'ycHsthe vitamin. The elucidation ofCO N CHto be probably identical withthe constitution of the flavinshas proceeded so rapidlythat, asis described in more detail else-where,41 they are now known H8 9 - v y C H 2 * O HOH OHto be derivatives of alloxazine, and a substance of constitutionrepresented by formula (X) has been synthesised and stated to beidentical with la~toflavin.~~Flavins have been obtained, though not always in pure crystallineform, from a number of new sources. K. G. Stern has describedthe isolation of hepatoflavin from horse liver, and has shown thaton irradiation it gives products identical in absorption spectra withthose from other flavins, and P.Karrer et aLM give a detailedaccount of their preparation of hepaflavin, which is identical withlactoflavin in elementary composition, melting point, crystallineform, and absorption spectra. W. Koschara 45 has described39 Ber., 1934, 67, [ B ] , 2080.40 R. Kuhn, H. Rudy, and T. Wagner-Jauregg, Ber., 1933, 66, [B], 1950;A , , 1934, 227.4 1 P. 263.43 Nature, 1933, 132, 784; A., 1934, 97.44 Helv. Chim. Actu, 1934, 17, 419; A., 538.4a R. Kuhn and F. Weygand, Ber., 1934, 67, [B], 2084.46 Ber., 1934, 67, [BJ, 761.L 330 BIOCHEMISTRY.uroflavin, from urine, as differing from lactoflavin in elementarycomposition, containing CH,O extra, and in melting point (thoughit does not depress the melting point of lactoflavin), but indis-tinguishable from it by chromatographic examination.B. C. Guhaand H. G. Biswas46 have obtained renoflavin from ox kidney.F. Plant and K. Bossert 47 have found a chloroform-soluble pigmentresembling lumiflavin in the serum and cerebro-spinal fluid of manand rabbit.The identity or, alternatively, the exact differences, of thesevarious flavins are still undecided. P. Karrer and K. Schopp4*find the flavins of malt, milk, egg, liver, and dandelion to be identicalin crystalline form and melting point, whereas Koschara hasdescribed uroflavin as non-identical with laotoflavin. It has beensuggested 4g that the results of elementary analysis a t presentindicate non-identity, but the existence of a group of closely relatedsubstances, alloxazines substituted in the 9-position.This questionof the identity of the flavins bears importantly upon the furtherquestion of the nature of vitamin B2-whether the vitamin is asingle naturally occurring flavin, whether, as in the estrogenichormone, the activity associated with the vitamin is a property ofmany substances possessing a common grouping, or whether, asin the case of carotene, the substances isolated are vitamin pre-cursors. This problem cannot yet be regarded as definitely solved,though there is no doubt as to the relationship of the pigments tovitamin B, activity. The flavin content of various tissues parallelsthe vitamin B, activity ; 50 lactoflavin retains its activity afterrepeated and varied purifications ; 61 (a) and tetra-acetyl lactoflavinalso shows activity 52 and retains this activity on regeneration ofthe original flavin by hydr~lysis.~l ( b ) But even more impressive isthe fact that the synthetic lactoflavin, in doses of 15 mg. per day,produces a growth of 9-10 grams per week in &-gram rats fed on adiet deficient in vitamin B,.53 One may reasonably incline to the4 6 Current Sci., 1934, 2, 474; A,, 1041.47 Klin.Woch., 1934, 13, 450; A., 1022.48 Helv. Chim. Acta, 1934, 17, 1013; A., 1233.40 K. G. Stern and E. R. Holiday, Ber., 1934, 67, [B], 1442; A., 1041.5O E. Adler and H. von Euler, Svensk Kem. Tidskr., 1933, 45, 276; A.,1934, 226; H.von Euler and E. Adler, 2. physiol. Chem., 1933, 223, 105;A., 1934, 544.5 1 (a) R. Kuhn, H. Rudy, and T. Wagner-Jauregg, Zoc. cit.(b) R. Kuhn and T. Wagner-Jauregg, Ber., 1933, 66, [B], 1577; A.,82 P. Gyorgy, R. Kuhn, and T. Wagner-Jauregg, 2. physiol. Chem., 1934,53 R. Kuhn and F. Weygand, bc. Cit.1933, 1320.223, 241 ; A., 706STEWART AND POLLARD. 331view that lactoflavin (or a lactoflavin-protein compound) is itselfvitamin B, on account of the specificity shown. Both the aromaticmethyl groups and the side chain attached to the 9-position of thealloxazine ring are necessary for activity, but as yet completeinformation as to the degree of specificity in the side chain is lacking.A compound with n-amyl in place of the E-arabinose of lactoflavinhas been synthesised, but no report of its biological activity has yetcome to the Reporter’s notice.54 Lactoflavin combines with protein,and according to P.Gyorgy, R. Kuhn, and T. Wagner-Ja~regg,~~it exists largely in that form in liver and yeast. The proteincompound retains vitamin B, activity.56The yellow oxidation enzyme of 0. Warburg and W. Christian 57consists of flavin combined with protein. The reduced leuco-formof the enzyme gives the coloured form and hydrogen peroxide whenshaken with atmospheric oxygen ; anaerobically it reduces methyl-ene-blue and re-forms the yellow enzyme.58 H. Theorell 59 findsthat the crystalline enzyme, dialysed against dilute hydrochloricacid, is split into two inactive components, pigment and protein.By mixing electrolyte-free solutions of these components, heobtains restoration of enzyme activity.Kuhn ,53 indeed, believesthat the biological activity of lactoflavin consists in its assumptionof enzymic properties by combination with protein.One of the biological tests for vitamin B, depends on its power ofpreventing pellagra or pellagra-like dermatitis, and this power isnot-possessed by flavins. This naturally raises the question of theidentity of vitamin B, in spite of the close correspondence betweenthe vitamin and flavin by the growth test. C. A. Elvehjem andC. J. Koehn 60 simply deny the identity of the flavins with vitaminB,, on the ground that colourless liver preparations, after removalof flaviiis, were highly active in preventing pellagra.They wish,in fact, to retain the name B, for the still unknown anti-pellagrafactor and to find another term for the activity shown by theflavins. P. GyorgyY6l on the other hand, considers that the oldvitamin B,, the anti-dermatitis factor, consists of the real B, (flavin),and a factor, B,, responsible for the prevention of the pellagra-likedermatitis of rats. He finds that B, is not identical with any ofthe water-soluble vitamins hitherto described. The difference54 R. Kuhn and F. Weygand, Ber., 1934, 67, [B], 1939.5 5 2. phyaiol. Chem., 1933, 223, 241; A., 1934, 706.56 R. Kuhn and G. Moruzzi, Ber., 1934, 67, [ B ] , 1220; A., 932.57 Ann. RepoTt8, 1933, 30, 159.58 0. Warburg and W. Christian, Biochem. Z., 1933, 266, 377; A,, 1933,59 Ibid., 1934, 272, 155; A., 1136.60 Nature, 1934, 134, 1007.979.61 Ibid., 1934, 133, 498; A., 560332 BIOCHEMISTRY.between the two workers is obviously one of nomenclature alone,but, the existence of a separate factor having been independentlyobserved, only confusion can result from the multiplication ofnames.It seems reasonable, in view of the general acceptanceaccorded to the identification of lactoflavin with vitamin B,, t oadopt the suggestion of Gyorgy, and to apply the term B, to theassociated anti-pellagric substance until it, in its turn, has beenidentified chemically.It has been suggested on several occasions that vitamin B, is anactive factor in the liver preparations used for the treatment ofpernicious anaemia, and there is no doubt that such preparationsare rich in vitamin B, or that, conversely, vitamin B concentratesmay possess definite curative value in pernicious anaemia.It isworth recording, therefore, that yeast extracts have recently beenprepared which-possess high curative value but little or no vitaminB, .61aVitamin C.The year 1933 produced very strong evidence that vitamin Cis identical with ascorbic acid, but complete acceptance had toawait the biological investigation of the synthetic material whichhad just been obtained by Haworth and his collaborators.62 Com-parative tests on guinea pigs of synthetic Z-ascorbic acid andhighly purified natural material have now demonstrated the com-plete identity of the substances in physiological as well as in physicaland chemical proper tie^.^^ Further accounts of the synthesis ofascorbic acid have been published by the Birminghamand other syntheses by T.Reichstein and A. Grussner 65 and byF. Micheel, K. Kraft, and W. Lohmann 66 confirm the claim thatthe synthetic substance is physiologically identical with the naturalvitamin C.It thus appears that 2-ascorbic acid is, without doubt, vitaminC ; but other closely related compounds, which, however, havenot yet been found to occur in nature, seem to possess someantiscorbutic activity along with a resemblance to ascorbic acidin readily undergoing reversible oxidation. For instance, K.Maurer and B. Schiedta' obtained a substance which theyC. C. Ungley and G. V. James, Quart. J .Med., 1934, 8 (new series), 523.62 Ann. Reports, 1933, 30, 336.1x3 W. N. Haworth, E. L. Hirst, and S. S. Zilva, J., 1934, 1155; A., 1091;V. Demole, Biochem. J . , 1934, 28, 770; A., 934.64 J . , 1934, 62, 1192; A., 279, 1091.65 Helv. Chim. Acta, 1934, 17, 331; A , , 510.86 2. physiol. Chem.., 1934, 225, 13; A., 869.6 7 Rer., 1933, 66, [B], 1054; A.. 1933, 936STEWART AND POLLARD. 333later6* identified as d-arabo-ascorbic acid and showed to haveabout &th of the antiscorbutic activity of the vitamin itself.The occurrence of toxic symptoms following the administrationof excessive amounts of vitamin D has naturally directed attentionto the possibility of hypervitaminosis in other cases. G. Gothlin 69finds that a t least 1-33 mg. of crystalline ascorbic acid are requireddaily to protect guinea pigs completely against pre-scorbutic changesin the molar teeth, and calculates the minimum daily dose for adultmen of 60 kg.at 19-27 mg. (3-16-4.5 mg. per kg.). These figuresare rather higher than those previously reported for the protectionof guinea pigs 7O (06---1*0 mg. per day), but the test used is morerigorous. On this basis the doses which E. KramQr 71 found to bewell tolerated by infants (15-50 mg.) cannot be considered excessive,but V. Demole 72 found no ill effects to follow the administrationto guinea pigs of amounts up to 2.5 g. per kg. of body weight forsix days.The mode of action of vitamin C is still by no means clear, thoughthe availability of the pure substance will undoubtedly facilitatethe attack on this problem.The tendency is naturally to examineascorbic acid with reference to various enzyme systems andespecially oxidising systems in view of the ease with which it under-goes reversible oxidation. J. H. Quastel and A. H. M. Wheatley 73find that slices of rat liver or of scorbutic guinea pig liver (but notalways of normal guinea pig liver), suspended in glycerophosphate-Locke solution containing sodium butyrate or crotonate, give anincreased production of acetoacetic acid and an increased oxygenusage when ascorbic acid is added. Further, they find that ascorbicacid prolongs the steady uptake of oxygen by liver slices. Theoxidation of fatty acids by liver slices is largely inhibited by iodo-acetic acid, and this inhibition is partly neutralised by ascorbic acid,although there is no evidence of a chemical reaction between thevitamin and iodoacetic acid analogous to that between the lattcrand glutathione.Quastel and Wheatley suggest that ascorbic acidplays some part in maintaining the general respiratory metabolism,on the integrity of which the oxidation of fatty acids (and, presum-ably, of other substrates) must ultimately depend. Ascorbic acidhas also been described as having an activating effect on proteases of68 Ber., 1934, 67, [B], 1239; 0. Dalmer and T. Moll, 2. physiol. Chem.,69 Nature, 1934, 134, 569; A., 1271.7 1 Deut. med. Woch., 1933, 59, 1428; A., 1934, 1271.72 Biochem. J . , 1934, 28, 770; A , , 934.73 Ibid., p. 1014; A,, 934; cf.D. C. Harrison, ibid., 1933, 27, 1601; A.,1933, 222, 116; A., 1934, 227.'O Ann. Reports, 1932, 29, 253.1933, 1340334 BIOCHEMISTRY.the cathepsin type 74 (from which the natural activators had beenremoved by acetone), increased by Fe", Fe"', and Cat.*, as well as onarginase 75 in presence of Fe" and Fe"' (dehydroascorbic acid actssimilarly on arginase). On the other hand, wheat amylase andcatalase are said to be inhibited by ascorbic as is tyrosinasewith respect to tyrosine, 3 : 4-dihydroxyphenylalanine, and dl-adrenaline. 76The Breakdown of Carbohydrate to Lactic Acid in Muscle.The important Embden-Meyerhof theory of the chemical changesinvolved in the conversion of carbohydrate into lactic acid bymuscle or into ethyl alcohol by yeast was reviewed in some detaillast year.77 Since that time, a considerable volume of evidence hasappeared in support of the theory, although there are indicationsthat the series of reactions proposed for the formation of lacticacid in muscle may not represent all the changes taking place.The crux of the matter is, of course, the position of methylglyoxal,which Meyerhof ignores as an unimportant by-product (or possibly,78when it is isolated, an artefact) from triose-phosphoric acid.Thisview, however, is difficult to reconcile with a number of experi-mental observations, and a dual or multiple route has been suggestedby several workers.79$ 85The isolation of methylglyoxal from muscle pulp has frequentlybeen reported, and, especially when the glyoxalase system has beeninhibited (by anti-glyoxalase, or by iodoacetic acid), the amountproduced represents a considerable fraction of the added substrate(hexose-diphosphoric acid or glycogen).Claims of this naturehave recently been renewed by N. Arayama,80 who reviews the earlierpapers on the subject. Dealing with lactic acid fermentation byyeast, E. Auhagen and T. Auhagenal confirm the production ofmethylglyoxal and state that it is a primary product of the reactionand is not an artefact due to the addition of " fixing " reagent(2 : 4-dinitrophenylhydrazine). Also using yeast, M. Kobe1 andH. Collatz 82 find that, under conditions shown to be optimal forthe production of met hylglyoxal from hexose-diphosphoric acid,7 4 H.von Euler, P. Karrer, and F. Zehender, Helv. China. Acta, 1934,7 5 P. Karrer and F. Zehender, ibid., p. 737; A., 1034.7 6 E . Abderhalden, Perrnentforsch., 1934,14, 367; A,, 1138.7 7 Ann. Reports, 1933, 30, 327.7 8 Ann. Inst. Pasteur, 1934, 53, 221; A., 1137.7 9 M . Jowett and J. H. Quastel, Biochem. J., 1934, 28, 162.80 J . Biochem. Japan, 1934, 20, 371; see also, E. Aubel and E. Simon,81 Biochem. Z., 1934, 268, 247 ; A., 662.17, 157; A., 461 (cf. A., 1933, 873).Compt. rend. Soc. Biol., 1933, 114, 905; A., 1934, 807.82 Ibid., p. 202 ; A., 449STEWART AND POLLARD. 335only lactic acid is formed from added glyceraldehyde-phosphoricacid. T+ey conclude that methylglyoxal is formed from sugars,but not from this particular triose-phosphoric acid-a conclusiondefinitely suggestive of a dual route.One of the strongest arguments in favour of the non-participationof methylglyoxal in lactic acid formation is the finding that dialysedmuscle extracts, which contain no glutathione (co-enzyme ofglyoxalase), still produce lactic acid on addition of a magnesiumsalt and adenylic acid pyrophosphate.This argument still holds,at least as showing that methylglyoxal is not an obligate inter-mediate, The fact that glutathione is destroyed in viko by iodo-acetic acid, which also inhibits lactic acid production, meets theclaim that iodoacetic acid acts in other ways 83 than by preventingglyoxalase activity. This claim is supported by certain facts to bereported later, and by the observation that glycolysis in shed bloodis inhibited by iodoethyl alcohol, which, however, does not formhydriodic acid with glutathione in vitro.84 Nor is it overthrown bythe fact that added glutathione can prevent iodoacetate poisoningin the isolated frog’s ventricle and can even cause recovery from thepoisoning when the ventricle has almost ceased to respond tostimulation.85R. Caddie and C. P. Stewart 85 have advanced evidence whichsupports the Embden-Meyerhof scheme of lactic acid production,but which again is suggestive of a dual route. Using the isolatedfrog’s ventricle, exhausted of available carbohydrate in an atmo-sphere of nitrogen, they found (confirming N. Freund and W.Konig,86 and their own earlier work with A. J. Clark 87) that thepower of contracting on stimulation was entirely restored by glucose.Incidentally, it was also restored by mannose, but not by galactose,fructose, or by any pentose or disaccharide.It was a reasonableassumption that intermediates in the conversion of glucose intolactic acid should also restore the power of responding to stimu-lation, and actually a partial restoration was obtained by additionto the perfusion fluid of sodium pyruvate with sodium glycero-phosphate, though neither substance was effective alone. On theother hand, a partial, but rather better restoration was given bymethylglyoxal : this at least shows that, however methylglyoxalis formed, the energy of its conversion into lactic acid is utilisableby the contractile processes. I n both cases the partial recovery83 Ann.Reports, 1933, 30, 330.84 D. M. Mowat and C. P. Stewart, Biochem. J., 1934, 28, 774; A., 912.85 R. Gaddie and C. P. Stewart, J. Physiol., 1934, 80, 457.88 Arch. exp. Path. Pharm., 1927, 129, 193.87 J . Physiol., 1932, 75, 321336 BIOCHEMISTRY.became full recovery on further addition of glucose. While, then,no evidence suggests that the chain of reactions required by theEmbden-Meyerhof scheme does not take place, there is definiteevidence that other reactions, also leading to lactic acid production,may occur simultaneously-or perhaps under slightly differentconditions.The first stage in the conversion- of fructose-diphosphoric acidinto lactic acid is the splitting of the six-carbon chain with theproduction of two molecules of triose-phosphoric acid.It hasalready been shown 88 that one component of the synthetic (racemic)glyceraldehyde-phosphoric acid is capable of conversion by muscleextracts into phosphoglyceric and glycerophosphoric acids-thesecond stage in lactic acid formation-or, under different conditions,into pyruvic and glycerophosphoric acids-the third stage. Earlythis year, 0. Meyerhof and K. Lohmanns9 found that hexose-diphosphate, added in low concentration to co-enzyme-free musclejuice or yeast maceration juice, was rapidly converted into a triose-phosphoric acid (in yields up to 60%) which closely resembled, butwas not identical with, synthetic glyceraldehyde-phosphoric acid.They suggested that it was slightly impure dihydroxyacetone-phosphoric acid, and later found that it was indeed identical withthe synthetic substance, which was prepared by W.Kiessling 91and found by him to be fermentable. This ester is readily hydrolysedby 0*5N-sodium hydroxide to lactic and phosphoric acids, and byacid to methylglyoxal and phosphoric acid. Meyerhof and Lohmannfound that, whether they used hexose-diphosphoric acid or thesynthetic dihydroxyacetone-phosphoric acid as substrate, therewas, in the presence of enzyme, the same ultimate equilibriumbetween the two esters, and it was possible to prepare hexose-diphosphoric acid from the triose ester. The effect of temperatureon the equilibrium was the same whether the initial substrate wasnatural or synthetic dihydroxyacetone-phosphoric acid or hexose-phosphoric acid.The equilibrium was disturbed by potassiumcyanide and by sodium bisulphite, the presence of the latter allowingthe isolation of dihydroxyacetone-phosphoric acid (from hexose-diphosphoric acid) in 90% yield. The enzyme concerned in thisreaction (zymohexase) is water-soluble, moderately thermo-stable,and is unaffected by iodoacetate, fluoride, or o ~ a l a t e . ~ ~ A point ofconsiderable interest is that the conversion of hexose-diphosphoricacid into two molecules of dihydroxyacetone-phosphoric acid is88 Ann. Reports, 1933, 30, 329.89 Naturwiss., 1934, 22, 134; A., 660. Ibid., p. 220; A., 807.B2 0. Meyerhof and K. Lohmann, Biochem. Z., 1934, 871, 89; A., 927.Ber., 1934, 67, [B], 868; A., 764STEWART AND POLLARD.337an endothermic reaction, the measured heat of reaction being givenas - 33.5 g.-cals. per gram,93 or - 6000 g.-cals. per gram-molecule,Q4of the hexose-diphosphoric acid.It has thus been shown that both the triose-phosphoric acids tobe expected from hexose-diphosphoric acid are capable of yieldinglactic acid in the presence of muscle extracts, and of doing so by wayof the intermediates suggested by Embden and his colleagues. Thework of Meyerhof and Lohmann suggests that dihydroxyacetone-phosphoric acid is the main triose ester to be formed in vivo, and inits case its production from and conversion into hexose-diphosphoricacid have also been demonstrated. This does not yet appear tohave been done for glyceraldehyde-phosphoric acid, though it hasbeen shown that glyceraldehyde is capable of yielding glycogen inthe animal body.95The breakdown of phosphoglyceric acid to pyruvic acid has beenfurther studied.The enzymic breakdown occurs only in thepresence of adenylic acid pyrophosphate and a, magnesium salt,96and in the absence of these a phosphopyruvic acid is obtained whichexists in enzymic equilibrium with phosphoglyceric acid, and whichis hydrolysed by muscle extract containing co-enzyme to pyruvicand phosphoric acids. The degradation of Z-phosphoglyceric acidto pyruvic acid by fresh bottom yeast has been confirmed byW. Schuchardt and A. Vercell~ni,~~ and C. Neuberg and M. Kobe1 98have observed a similar reaction in the presence of pulped germinatedpeas and beans.A. E. Braunstein has observed it also in thepresence of haemolysed (but not intact) erythrocytes from rabbitsand pigeons. It is evident, therefore, that the responsible enzymeis widely distributed, a fact which lends further support to thesupposition that the conversion of phosphoglyceric acid intopyruvic acid is a general step in carbohydrate breakdown.The simple view that the enzymic hydrolysis of phosphagen tocreatine and phosphoric acid provides the energy for muscularcontraction, and that the resynthesis of phosphagen is a recoveryprocess at the expense of carbohydrate breakdown, is becoming lesssatisfactory as a result of recent work. E. Lundsgaard 99 finds thatin muscle poisoned by iodoacetic acid the energy utilisation isgreater than can be accounted for by the phosphagen breakdown,and a similar conclusion can be reached from figures given for93 0.Meyerhof and K. Lohmann, Naturwiss., 1934, 22, 462 ; A., 1034.D4 Idem, Biochem. Z., 1934, 273, 73; A., 1261.95 R. Stoher, 2. physiol. Chem., 1934, 224, 229; A., 919.96 K. Lohmann and 0. Meyerhof, Biochem. Z . , 1934,273, 60; A., 1261.9 7 Ibid., 1934, 272, 434; A., 1260. Ibid., p. 457; A., 1260.99 Ibid., 1934, 269, 308; A., 685338 BIOCHEMISTRY.anaerobic cardiac muscle by A. J. Clark, M. G. Eggleton, andP. Egg1eton.l Lundsgaard considers that the extra energy cannotbe accounted for by utilisation of adenylic acid pyrophosphate,since this only becomes perceptible a t a late stage after considerablefatigue, and he failed to find anaerobic synthesis of phosphagen orhydrolysis of adenylic acid pyrophosphate.He considers, in fact,that phosphagen breakdown is a recovery process.One may note here that hitherto no chemical reaction has beendiscovered which can a t present be regarded as initiating muscularcontraction ; first, lactic acid formation was shown to be a recoveryprocess, and now phosphagen hydrolysis is placed in the samecategory. It seems that more attention might well be given to theviews of Ritchie,2 who suggests that the contractile processes ofmuscle are physical (he regards the contracted as the true restingstate of muscle, and the relaxed as the condition in which tension isbeing maintained, thus reversing the usual notion), and that all theknown chemical reactions are recovery processes.A similar conception of phosphagen hydrolysis as a secondaryreaction is instinct in the suggestion of K.L~hmann,~ who considersthat the production of creatine and phosphoric acid from phosphagenis not due to a specific phosphatase, but is the resultant of tworeactions :Adenylic acid pyrophosphate = adenylic acid + 2H,P04.Adenylic acid + 2 phosphagen = adenylic acid pyrophosphate +creatine.In accordance with this view he finds 4 that, though adenylic acidand phosphagen separately are inactive as co-enzyme of lactic acidformation, a mixture of the two exhibits the activity of adenylicacid pyrophosphate. J. K. Parnas and P. Ostern accept Loh-mann’s view of the connexion between adenylic acid pyrophosphateand phosphagen breakdown, but go further, considering the break-down of carbohydrate also to be coupled with these reactions and tobring about the resynthesis of phosphagen :(1) Adenylic acid pyrophosphate + glycogen + water = adenylicacid + fructose diphosphoric acid.(2) Adenylic acid + phosphocreatine = adenylic acid pyrophos-phate + creatine.(3) Creatine + fructose diphosphoric acid = phosphocreatine +lactic acid.Recently, the same authors with T.Mann have adduced evidence1 J. Physiol., 1932, 75, 332.3 Biochern. Z., 1934, 271, 264; A., 1034. Ibid., p. 278; A., 1033.Nature, 1934, 134, 627.Ibid., 1933, 78, 322.Ibid., p. 1007STEWART AND POLLARD. 339that the resynthesis of phosphagen is particularly associated withthe conversion of phosphoglyceric acid into pyruvic acid.Phospho-glyceric acid, added to muscle pulp poisoned with iodoacetic acid,prevents ammonia formation which occurs in presence of freeadenylic acid but not of the pyrophosphate, and also allows theformation of phosphagen. Resynthesis of phosphagen in thesecircumstances is not permitted by addition of free phosphate or ofany of the other intermediates in the Embden-Meyerhof series.Moreover, addition of pyruvic acid and phosphate together tofluoride-poisoned muscle pulp stops ammonia formation andallows synthesis of adenylic acid pyrophosphate (and therefore,according to Lohmann, of phosphagen). It is suggested, therefore,that the real intermediary phosphate carrier is phosphopyruvicacid, and that the phosphate from this substance is transferred tocreatine and thence to adenylic acid.Reaction (3) above thusincludes, as one of its stages, the reaction :Phosphoglyceric acid + creatine (= phosphopyruvic acid +creatine + H,O) = pyruvic acid + phosphocreatine + H,O.Parnas and his colleagues point out that their experimentsindicate an inhibitory effect of iodoacetic acid on the earlier stagesof carbohydrate breakdown, i.e., on some reaction prior to theformation of phosphoglyceric acid. Meyerhof and Lohmann 92have found that iodoacetic acid does not interfere with the formationof triosephosphoric acid from hexose-diphosphoric acid. It followsthat iodoacetic acid inhibits either the formation of hexose-di-phosphoric acid or the conversion of dihydroxyacetone-phosphoricacid into glycerophosphoric and phosphoglyceric acids.SinceParnas and Ostern (Zoc. cit.) have found that in the iodoacetic acidpoisoned heart adenylic acid accumulates with formation, not offree phosphate, but of carbohydrate phosphoric esters, it seems thatiodoacetic acid must act in the latter of these reactions.There are, of course, difficulties in the complete acceptance ofthe suggestions of Parnas, though possibly they may be removedwhen a more detailed exposition appears. In particular it is noteasy to reconcile Parnas's series of reactions with the long con-tinuance of muscular contraction after poisoning with iodoaceticacid. Aerobically, cardiac muscle, poisoned with iodoacetate, cancontinue to contract for some hours without lactic acid formationand with maintenance of the usual phosphagen concentration.'It would appear, therefore, that phosphagen can be resynthesised a tthe expense of ot her-presumably exot hermic-reactions thanlactic acid formation, and that, although the phosphate trans-7 A.J. Clark et aZ., Zoc. cit340 BIOCHEMISTRY.ference postulated by Parnas may occur normally, it is not essentialto the process of contraction. This verdict is probably justified inspite of the admitted differences between cardiac and skeletal muscle(though it is doubtful whether any of them are really fundamental).To the number of these differences, one more has recently beenadded. F. Beattie, T. H. Milroy, and R. W. M. Strain8 haveobtained, from the heart of the rabbit, cat, dog, ox, and horse, asubstance which, on the basis of chemical composition and behaviourtowards acid, they describe as different from the adenylic acidpyrophoshate of skeletal muscle, and as resembling a dinucleotidecomposed of one molecule of adenosine-diphosphoric acid and onemolecule of adenosine-triphosphoric acid.Only about 60% of itstotal phosphorus is labile (as compared with 67% in the case of thesubstance from skeletal muscle), and it is stated to be more powerfulthan the ordinary adenylic acid pyrophosphate in reactivatinginactive extracts of both voluntary and cardiac muscle. The samesubstance has apparently been obtained from fresh heart by P.O ~ t e r n , ~ who described the isolation of a diadenosine-pentaphosphoricacid, but failed to obtain it from pig heart, which yielded adenosine-5-phosphoric acid instead.The Metabolism of Amino-acids.Cystine and Methionine.-H. S.Loring, D. Dorfmann, and V. duVigneaud 10 have confirmed the observation that d-cystine does notpromote growth, but find that mesocystine can be used as the Bolesource of cystine. They suggest that this is due t o reduction withliberation of Z-cysteine. Corresponding to this difference in growth-promoting power, the cystine isomerides show differences in the easewith which they are oxidised in the animal body.ll When thelaevo-acid is fed to rabbits, about 80% of the extra urinary sulphuris in the form of sulphate, but with the dextro-acid only about 45%.Racemic and mesocystine give intermediate results.Similarresults have recently been reported by J. A. Stekol,12 who findsthat dogs excrete as sulphate 40% of the sulphur from dZ-cystinebut only 12% of that from Z-cystine. V. du Vigneaud, H. S. Loring,and H. A. Craft l3 find that homocystine, dl-methionine, and S-methylcystine are all readily oxidised by rats, the percentage of theextra urinary sulphur appearing as sulphate being practically the8 Biochem. J., 1934, 28, 84, 90; A., 431.9 Biochem. Z., 1934, 270, 1 ; A., 677.10 J . Biol. Chern., 1933, 103, 399; A., 1934, 212.11 V. du Vigneaud, H. A. Craft, and H. S. Loring, ibid., 1934, 104, 81 ; A.,12 Ibid., 1934, 107, 225.322.l3 Ibid., 1934, 106, 481; A., 921STEWART AND POLLARD.341same for the first two. 8-Methylcystine, however, does notstimulate growth.The ready oxidation of dl-methionine has been confirmed 14, l6and the fact that a-benzoylmethionine is not attacked by the animalbody is reported by R. W. Virtue and H. B. Lewis,15 who suggestthat methionine is demethylated to homocysteine. These authorsalso find that the urinary substance (after methionine feeding)giving the cyanide-nitroprusside test 16 does not respond to theSullivan test for cystine.N. W.'Pirie 17 has begun a study of the intermediate stages in theoxidation of sulphur-containing amino-acids, having succeeded indemonstrating their oxidation to sulphate by slices of rat liver andkidney. He finds that cystine is oxidised only after reduction tocysteine ; glutathione only after hydrolysis ; methionine is oxidisedrather slowly, and during the oxidation the fluid contains a substancewhich gives the nitroprusside reaction ; ethylcysteine is oxidisedslowly and, curiously, ergothionine not a t all.Pirie suggests,therefore, that in all cases oxidation starts from a sulphydryl com-pound the formation of which by reduction or dealkylation is thefirst stage. He suggests, tentatively, the following series of reactions,of which the third has already been shown to proceed spontaneouslyat body p H :R*S*S*R +- 2R*SH -+ 2R*S*OH -+ R*S02H -+ H2S03 -+ H2S04or \2R*S*CH3 L k R~SHV. du Vigneaud et al.18 have reported the synthesis of pento-cystine, [ HO,C*CH ( NH2)*( CH,),*S-], , and homomet hionine,CH,*S*(CH,),*CH(NH,)*CO,H, but so far no report of their meta-bolic effects has appeared.Tryptophan.-It is interesting tn view of the specific growtheffect of I-cystine, that the stereoisomeric forms of tryptophanare equally effective in promoting the growth of rats.l9 Acetylation,however, introduces specificity, since acetyl-d-tryptophan is notutilised as a growth promoter (possibly because it is not easily hydro-lysed in the body).Neither d-tryptophan nor its acetyl derivativeleads to kynurenic acid excretion, and acetylation considerablyl* J. A. Stekol and C. L. A. Schmidt, Univ. Calijornia Pub. Physiol., 1933,8, 31 ; A., 1934, 440.l5 J . Biol. Chern., 1934, 104, 59; A., 322.l6 Ann. Reports, 1933, 30, 333. *Biochem. J . , 1934, 28, 305; A., 440.l8 V.du Vigneaud, H. M. Dyer, C. B. Jones, and W. J. Patterson, J . Biol.lQ C. P. Berg, ibid., 1934, 104, 373; A., 440.Chern., 1934, 106, 401; A., 1094342 BIOCHEMISTRY.reduces the amount of kynurenic acid formed from the lzevo-isomeride. L. C. Bauguess and C. P. Berg20 found further thatp-3-indolylacrylic acid and a-oximino-~-3-indolylpropionic acidwere both without effect on growth and were not converted intokynurenic acid by the rabbit, Racemic p-3-indolyl-lactic acid,however, both supported growth and led to kynurenic acid excretion.Part of the amount fed was recovered from the urine (more of theE-compound than of the d-, which suggests a greater efficiency of the&-acid). p-3-Indolylpyruvic acid was also converted into kynurenicacid, and was not recoverable from the urine.The same authors 21found that amides of Z-tryptophan support growth and yieldkynurenic acid. These results suggest, of course, that, whatevermay be the mechanism of the growth-promoting action of trypto-phan, it involves the formation of an optically inactive compound,presumably the corresponding ket o- acid.R, S. A1cock,22 indeed, points out that kynurenic acid formationis probably a side or " shunt '' reaction (possibly a detoxicatingmechanism) dealing with excess of tryptophan by way of indole-pyruvic acid ; the ease with which various tryptophan derivativesyield kynurenic acid is no more than a measure of the ease withwhich they are converted into the keto-acid. He finds that rats ona tryptophan-deficient diet do not grow when tryptophan is injected,but do grow when given small daily doses of the anterior pituitarygrowth hormone (0.1-0.15 ml.of the extract per day, injected).He suggests that the animal can synthesise tissue proteins withoutreceiving tryptophan in the diet, but that it requires tryptophan forthe synthesis, in the liver, of some substance essential to life.Histidine.-S. Edlbacher and M. Neber,23 in a further study of thebreakdown of histidine by histidase, consider that the reactioninvolves the opening of the glyoxaline ring with addition of 2H,Oand formation of ammonia and o-formylglutamine. The product ofenzyme action is hydrolysed by sodium hydroxide to ammonia,formic acid, and glutaminic acid, and on oxidation with hydrogenperoxide it gives succinic acid semialdehyde, as does glutaminicacid.Amino-mid Anhydrides.-It is perhaps invidious t o single outany one item from the mass of interesting work on peptides and theirhydrolysis by enzymes, but the subject 8s a whole seems hardlyready for review. A point of particular interest, however, is thepossibility of the occurrence of djketopiperazines in the proteinmolecule.The great obstacle to the acceptance of this possibility2o J . BioE. Chem., 1934,104, 675, 691; A., 554.21 Ibid., 1934, 106, 615; A., 1252.z3 2. physiol. Chern., 1934, 224, 261; A., 920.22 Biochern. J., 1034, 28, 1721STEWART AND POLLARD. 343has been the failure of proteolytic enzymes to hydrolyse diketo-piperazines.Recently, however, K. Shibata 24 has obtained foursubstituted diketopiperazines which underwent enzymic hydrolysis,and two of these contained only naturally occurring amino-acids.CHR-CO Glycyldiaminopropionic anhydride, NH<c-,CHR,>NH (R = H,R’ = CH2-NH2), and diaminopropionic anhydride (R = R’ =CH,*NH2) were hydrolysed by pepsin ; and glycylglutaminicanhydride (R = H, R’ = CH,*CH,*CO,H) and asparagine anhydride(R = R‘ = CH,-C0,H) were hydrolysed by trypsin, cathepsin, andpapain.Indirect Calorimetry.Last year, E. P. Poulton 25 amplified a previous note 26 on certainerrors in the measurement of heat production by calculation fromthe gaseous exchange using the ZuntzSchumburg factors. Hefound that, though the heat produced was proportional to the carbondioxide, the correlation between heat production and oxygen usagewas bad.There is, he Bays, considerable variation in the oxygenconsumption-the heat and the carbon dioxide production remainingconstant-but on the whole the amount of oxygen used is unex-pectedly small a t R.Q. above about 0.8, and unexpectedly highbelow that level. He suggests that under basal conditions there isactually a constant combustion ratio of carbohydrate to fat, andthat the errors in calculating the heat production from the oxygenconsumption are to be explained by the use of oxygen for partialoxidations or its liberation in reductions, whereas the carbon dioxideproduction represents complete oxidations.One of the most obvious of incomplete oxidations is, of course,the formation of carbohydrate from fat, and the vexed question ofwhether this does or does not occur in the animal body still receivesattention.The great criticism which is directed against all attemptst o prove this conversion is that they depend upon measurements ofthe R.Q., and that even if the accuracy of the measurements begranted, alternative explanations may still be possible. In adiposesubjects undergoing dietetic weight reduction, where conditions forsugar formation from fat ought t o be ideal, V. Forbech and F.Leegaard 27 found that the carbohydrate metabolised frequentlyexceeded the intake, and that the low R.Q. supported the idea thatfat was converted into carbohydrate. In both man and the pig,24 Actu Phytochirn., 1934, 8, 173; A., 1260.25 Proc.Roy. SOC. Med., 1933, 20, 1591.26 T. W. Adame and E. P. Poulton, J . Physiol., 1932, 77, (Proc.) 1.27 Acta, Med. Xcand., 1934, 81, 351; A., 1025344 BIOCHEMISTRY.following high fat diets, Hawley et aLZ8 found values of the R.Q.too low to be accounted for by ketosis or by carbohydrate formationfrom protein or glycerol.find that, contrary to the usual view, only 30% or less of glycerolis converted into glucose.) They found considerable individualvariation, the occurrence of a low R.Q. depending less on theamount of fat in the diet or the carbohydrate/fatty acid ratio thanon the tolerance of the subjects. They point out that otherexplanations than that of glucose formation from fat are conceivable,and direct attention t o the effect of cold in producing a low R.Q.andto the specific dynamic action of butter fat.J. M. Peterson30 has compared the R.Q. in decerebrate and indecerebrate eviscerate cats, with corrections for displaced carbondioxide. He finds that the true oxidative R.Q. in decerebrateeviscerate cats is about 0.825, and calculates the R.Q. of the removedviscera to be 0.69, a value which he considers to be associated withliver gluconeogenesis .The subject of carbohydrate production from fat has been critic-ally reviewed by H. H. Mitchell,31 but the work reported during thepast twelve months has not forced acceptance of such a productionupon the sceptical. As J. Needham 32 points out in dealing withoxygen consumption without carbon dioxide production in acidifiedhen or crab eggs, such a phenomenon may be due to a variety ofcauses other than the production of sugar or keto-acids from fats;e.g., oxidation of sulphur to sulphate, of lactic acid to pyruvic acid,of glucose to glycuronic acid. It is, however, doubtful whetherthese, in man, are of sufficient quantitative importance to accountfor the low values of the R.Q.which have been recorded.(F. H. Lashmet and L. H. NewburghHeavy Water.The biological behaviour of D,O is receiving attention, though,naturally, not a great deal of progress has yet been made. Never-theless, a number of short papers have appeared which combine togive the impression that living cells are by no means indifferent tothe proportions of H,O and D,O in the surrounding media, and thata normal concentration of heavy water may be essential to the pro-cesses of life.The fact that pure D,O is definitely toxic and kills certain small38 E. E.Hawley, C. W. Johnston, and J. R. Murlin, Amer. J . Physiol.1933, 105, (Proc.) 46; A., 1934, 441; J . Nutrition, 1933, 6, 523; A . , 1934,320.29 J . Clin. Invest., 1933, 12, 968.80 J. Phyfliol., 1933, 79, 508; A., 1934, 683.a 1 J . Nutrition, 1933, 6, 473.32 Proc. Soc. Exp. Biol. Med., 1932, 30, 182; A., 1933, 1324STEWART AND POLLARD. 345organisms a t a rate generally proportional to their degree of com-plexity was reported last year.33 G. N. Lewis,% however, statesthat the toxicity is not very great to such varied organisms astobacco seeds, yeast, flatworms, and mice; and lower organismstolerate it in high concentrations.He finds that the rate of thevital processes is roughly inversely proportional to the concentrationof D,O, and that in pure D,O growth is extremely slow if it takesplace at all.Even low concentrations of heavy water have a definite effect oncellular metabolism and on enzyme systems. T. C. Barnes35found that in water of d 1.000061, filaments of Spirogyra nitidawere characterised by lack of movement, absence of abscission orcell disjunction, and greater longevity. Later,36 he reported that inheavy water (d 1.000061) a filament of 31 cells of Xpirogyra nitiduhad increased to 43 cells (3 dead) after six days, whereas in ordinarywater under,&nilar conditions no cell division had taken place, inice-water there were 15 abnormal cells out of 50 after five days, andin freshly condensed water all the cells died.Similarly he 37 foundcells of E. gracilis to multiply more rapidly in water containing alow percentage of D,O than in ordinary distilled water, and astimulating effect of D,O (in small concentrations) on the vegetativegrowth and development of Aspergillus sp. has been reported byS. L. M e ~ e r . ~ ~Dealing with enzyme systems, Barnes and Larson found thatheavy water (d 1.000061) decreased the rate of starch hydrolysisby pancreatic amylase, and the amount of carbon dioxide evolvedduring zymase fermentation. OQ the other hand, oxidation ofguaiaconic acid by a peroxidase-oxygenase system was increased bymaking the solution in ice-water and allowing it to warm to roomtemperature.H.Erlenmeyer and H. Gartner 39 find that, within experimentalerror, the animal body does not change the D,O content of water,since 8 litres of pure water yielded 20 C.C. of heavy water of d 1.00087,and cow’s milk similarly treated gave water of d 1.00083. This is,of course, an important observation for those workers who use heavywater as an indicator of water movement in the animal body, Thishas been done in the case of fish (goldfish), in which it appears thatnearly all the water leaves the body and is replaced by fresh withinAnn. Reports, 1933, 80, 34.34 Science, 1934, 79, 153; A., 557.35 J . Amer. Chem. SOC., 1933, 55, 4332; A., 1933, 1329.36 T.C. Barnes and E. J. Larson, {bid., p. 5059; A., 1934, 217.Science, 1934, 79, 370; A,, 692.38 Ibid., p. 210; A., 562. 89 H e b . Chirn. Acta;, 1934, 17, 334; A., 540346 BIOCHEMISTRY.a few hours,40 and in man, where the exchange takes many days.In man, G. von Hevesy and E. Hofer 4 1 found that half the ingestedheavy water was excreted in 9 (-+ 1) days, and calculated the average" life " of a water molecule within the body to be 13 (& 1-5) days.Their calculations involved the assumption that the ingested waterwas completely mixed with the water present in the body, and thisassumption has been verified by H. Erlenmeyer et ~ 1 . ~ ~ Theseworkers found that injection of isotonic heavy water (1.64%)solutions of xylose into jejunal loops of rats led to reduction of theheavy water content to 0*05--0-07% without diminution of the totalvolume.Calculation showed that this meant the distribution of thoheavy water over 127-135 c.c., the total water content of the bodybeing 132 C.C. The absorption was very rapid, the figures quotedbeing obtained in one hour.PLANT BIOCHEMISTRY.Fixation of Nitrogen by Nodule Bacteria.Investigations of the symbiotic relationships between leguminousplants and Rhixobia, culminating in recent years in the developmentof practical methods of field inoculation, have brought in their traina much wider knowledge of the nutrition and metabolism of thesebacteria. Contrary to earlier views, it is now firmly established thatthe nodule bacteria are unable to fix nitrogen in the absence of thehost plant.112, 3 Moreover, the organism, previously supposed to bestrictly aerobic, has been shown to maintain its activity underanaerobic conditions in the presence of a hydrogen-acceptor.Itsgrowth in nodules in which the oxygen supply is, to say the least,much restricted, now receives adequate explanation.The general association of rapid vegetative growth in legumes withextensive nodule formation in the roots has led to many investig-ations of the balance between the ability of the plant to supply car-bonaceous energy material to the nodule bacteria and that of thebacteria to provide assimilable nitrogen for the plant. In thisconnexion, P. W. Wilson and colleagues*, 5, 6 have examined the40 G.von Hevesy and E. Hofer, Nature, 1934, 133, 495; A., 557.4 1 Ibid., 1934, 134, 879.42 H. Erlenmeyer, H. Gartner, E. J. MacDougall, and F. VerzBr, ibid.,1 P. W. Wilson, E. W. Hopkins, and E. B. Fred, Arch. Mikrobiol., 1932,p. 1006.3, 322; A., 1932, 549.F. E. Allison, J. Agric. Res., 1929, 39, 893; B., 1930, 258.3 M. P. Lohnis, Soil Sci., 1930, 29, 37.4 P. W. Wilson, E. B. Fred, and M. R. Salmon, &ad., 1933, 35, 145; B.,P. W. Wilson, P. Wenck, and W. H. Peterson, i b d , p. 123 ; B., 1933,323.P. W. Wilson and E. B. Fred, J . Bact., 1033, 25, 64; A., 1933, 647.1933, 323STEWART AND POLLARD. 347frequently observed beneficial effect of an artificially increasedproportion of atmospheric carbon dioxide on the growth andnodulation of legumes.With Rh. trifoliurn it is shown that in anormal atmosphere the rate of carbon assimilation by red clover,rather than that of nitrogen fixation by the bacteria, is the limitingfactor in this symbiotic system. With a carbon dioxide content ofapproximately 0.3 yo, carbon assimilation reaches an intensitysufficient to meet the energy requirement of the nodule organismsfor maximum fkation of nitrogen. The critical carbon dioxidecontent for balanced conditions depends on external conditions,notably those controlling the difference between the intake andrespiration of carbon dioxide, and the rate and extent to whichcarbon dioxide liberated by the bacteria can diffuse through thegrowth medium and again become utilisable by the leaves. In-creased assimilation results not only in an increasing growth andnitrogen content of the plant and a greater number of nodules, butin enlargement of the nodules and a tendency for their normal dis-tribution round the crown of the roots to be extended to lateralroots.By increasing the pressure of carbon dioxide around sand-culturedplants, C. E.Georgi, 3'. S. Orcutt, and P. W. Wilson find that allthe above effects are accentuated even if the total amount of carbondioxide to which the plant has access remains unchanged. Theproduction of similar effects by the addition of sucrose or glucose toculture media is attributable to the utilisation of the sugars by thebacteria and to increased carbon assimilation of the plant followingthe production of carbon dioxide by the organisms.Mamito1produces no such effects and even in moderate concentrationsdepresses nitrogen fixation. In an examination of soya bean organ-isms, F. Allam 8 calculates that approximately 15 g. of dry matterfrom the plant are consumed during tbe fixation of 1 g. of nitrogen.More recently E. B. Fred and P. W. Wilson9 confirm the directrelationship between the carbohydrate content of clover and thenumber of nodules formed, and further show that excessive amountsof sugars in the early stages of growth may cause a temporarilydelayed fixation of nitrogen.A reduced supply of carbohydrate, e.g., when plants are kept indarkness, checks fixation by the bacteria, which then attack thecellulosic matter of the plant tissues.10 Sugar metabolism inRhixobia takes the form of a butyric fermentation with the7 Soil Sci., 1933, 36, 375; B., 1934, 114.2.PJEanz. Dung., 1931,20, A , 270; A., 1931, 876.Proc. Nat. Acccd. Sci., 1934, 20, 403; A., 1273.1" H. G. Thornton, PYOC. Roy. SOC., 1930, B, 106, 110348 BIOCHEMISTRY.production of the customary proportions of carbon dioxide andhydrogen and small amounts of alcohol and acetic acid.llBy providing an alternative source of nitrogen to clover,E. W. Hopkins and E. B. Fred l2 find that the size of root nodules isdecreased to extents which vary with the nature of the materialused, but, in general, vary inversely with concentration of thenitrogen source (potassium nitrate, ammonium sulphate, urea,asparagine, and yeast extract are examined).Under these condi-tions nodules are found principally on lateral roots. In the pre-sence of mannitol and either nitrogenous substance nodulationis largely confined to tap roots.The high specificity of the various species of Rhizobium in respectof the host plant, sufficiently accentuated to permit the use ofagglutination and complement -fixation tests for classification pur-poses,13 is reflected, to some extent, in their response to differentforms of fixed nitrogen. Thus R. H. Walker and D. A. Anderson l5record that the oxygen consumption of cultures of four differentspecies is uniformly low in nitrogen-free media, but is increased, byadditions of yeast extract, in proportion to the amount of nitrogenthus added. The effect is maintained until the supply of nitrogen isexhausted.If, however, nitrogen is added to cultures of Rh.leguminosarum, Rh. trifolii, or Rh. phaseoli in the form of sodiumnitrate or alanine, there is only a small upward trend in oxygenconsumption. With ammonium chloride or urea, small concen-trations produced little or no effect and larger amounts had adepressive action. The growth and activity of Rh. meliloti weredefinitely increased by all forms of nitrogen examined, the actionof urea being intermediate between that of yeast (high) and that ofthe remaining three simpler compounds, which produced generallysimilar effects. Characteristic physiological differences betweenRh. meliloti and other species are shown by A. W. Hofer andI. L. Baldwin16 to develop in media rich in nitrogen.The decomposi-tion of various nitrogenous compounds by different nodule organismsis also examined by G. G. Pohlman ; l7 Rh. meliloti produces ammoniafrom glycine, dl-amino-n-butyric acid, dl-alanine, asparagine, andurea, whereas Rh. japonicum acts only on the last three substancesl1 A. I. Virtanen, M. Nordlund, and E. Hollo, Biochern. J., 1934, a, 796;A,, 928; Suomen Kern., 1933, 8, B, 62; A., 1933, 752.la Plant Physiol., 1933, 8, 141; A., 1933, 647.l3 W. R. Carroll, Soil Sci., 1934, 37, 117, 227; A., 453; B., 468.l4 J . Bact., 1933, 25, 53; A., 1933, 638.l5 With P. E. Brown, Soil Sci., 1934, 37, 387; A., 811; ibid., 1934, 38,l6 J . Bact., 1932, 23, 65; A., 1932, 1066.l7 Soil Sci., 1931, 31, 385; A., 1931, 876.207; A., 1265STEWART AND POLLARD.349named. Differences in the utilisation, without ammonia formationof the amino-groups in glycine, 1-tyrosine, dl-amino-n-butyric acid,and urea are also shown by these two species. It is not yet clear,however, to what extent these differences are characteristic ofspecies. In some cases, various strains of the same species appearto vary in their action on nitrogen compounds.The diffusion of bacterial nitrogen from nodules into the surround-ing soil and its ultimate utilisation by other plant species arematters of obvious practical importance. The implied breakdownof the bacterial cell is variously ascribed to the action of plantenzymes or to the presence of a specific bacteriophage within theroot.According to A. I. Virtanen and S. von Hausen,ls thediffused nitrogen is almost entirely in the form of amino-acids,which are effective sources of nitrogen for Grumineue. In mixedcrops of peas and oats, the latter obtained additional nitrogen if theproportion peas : oats was between 1 : 1 and 1 : 2. With smallerproportions of peas, the growth of both crops was depressed.19H. G. Thornton and H. Nicol20 show a similar utilisation of nodule-nitrogen to occur in rye grass when grown simultaneously withlucerne.Biochemistry of the Higher Plants.Nitrogen Nutrition and Metabolism.-The great practical andacademic interest in the nitrogen relationships of plants is reflectedagain this year in a heavy output of research work on this subject.The relative efficiency of nitrates and ammonium salts in plantnutrition continues to form the basis of many investigations inwhich the varying aspects of this intricate problem are being steadilyelucidated.Continuing earlier work,21 Shive and his colleagues 22 show thatammonia intake by the tomato, as by other plants, is most rapidfrom neutral or alkaline media, and that of nitrate from acid media.In older plants, however, the absorption of nitrates becomes lessaffected by pH, and, in the range pH 4.0-7.0, usually exceeds that ofammonia at any given pH.Further, the rate of absorption ofammonia per gram of dry matter in the plant decreases, whereasthat of nitrate increases with advancing plant growth. Inammonium nitrate-fed plants the proportion of ammonia in roots2. Pjlanz.Dung., 1931,21, A , 57; A., 1931, 1101; Szcomen Kern., 1933,6, 23, 55; B., 1933, 243; (with H. Karstrijm), Biochem. Z., 1933, 258, 106;A., 1933, 437.19 U. Wartiovaara, Z. Pjlanz. Dung., 1933, 81, A , 353; A., 1933, 1092.2o J . Agric. Sci., 1934, 24, 269; B., 550.2 1 Ann. Reports, 1933, 30, 320.22 H. E. Clark and J. W. Shive, Soil Sci., 1934, 37, 203, 459; B., 468, 776350 BIOCBEMISTRY.increases with the p= of the medium, and the simultaneous appear-ance of large amounts of basic-free amino-nitrogen indicates rapidtransition of ammonium- to amino-nitrogen within the root system.This is confirmed by the relatively small effects of changes in therate of ammonia absorption on the ammonia contents of leaves.I n these experiments, also, the nitrate content of roots, stems, andleaves was but little changed as a result of variations of and ofnitrate intake.It would appear, therefore, that conditions favour-ing the intake of ammonia or nitrate are also those favouring theassimilation of the respective nitrogen sources. The high proportionof nitrate in stems as compared with roots or leaves of tomato istypical also of a number of other plants.23 I n a somewhat similarinvestigation of sand-cultured peach trees 0. W. Davidson andJ. W. Shive 24 show that optimum utilisation of ammonium saltsoccurs with media of pH 6.0, whereas that of nitrate shows twooptima, at pE 4.0 and 8.0. Here again the early stages of ammoniaassimilation occur in the roots, in which also the reduction of nitrateis practically completed.V. A. T i e d j e n ~ , ~ ~ working with tomatoand with apple, codrms many of the observations of Shive and alsoshows that ammonia-fed plants contain more soluble organic nitro-gen than those receiving equivalent proportions of nitrate. Highproportions of soluble carbohydrates apparently favour ammonium-assimilation.A comparison of the relative effects of different nitrogen sourceson soil cultures of a number of farm crops is recorded by K. Nehring.26With acid-sensitive plants, e.g., mustard, ammonium sulphateproduces better growth than nitrates at all ranges of pH examined.A slight advantage is shown by ammonia in alkaline and by nitratein acid conditions for the development of barley, wheat, and maize.At neutrality no differences are apparent.The nitrogen intakefrom the various sources, by acid-tolerant plants (oats, rye), is notgreatly affected by pH, and although differences in the growth ofammonia-fed and nitrate-fed plants are small, the latter containrelatively larger proportions of amides as compared with proteins.An interesting paper by J. P. Conrad2' records changes in thereaction of media as a result of the growth in them of maize andsorghum plants. The residual (titratable) acidity of culturescontaining ammonium sulphate, after absorption of the nitrogen by23 E. Parisi and G. de Voto, Atti R. Accad. Lincei, 1932, 16, 270; A., 1933,197.24 Soil Sci., 1934, 37, 357; A,, 821.25 Phnt Physiol., 1934, 9, 31; A., 821.26 Landw.Jahrb., 1934, 79, 481; B., 728.27 J . Amer. Soc. Agon., 1934, 26, 364; A., 1048STEWAXT AND POUARD. 351plants, is greater than that in corresponding media containingsulphuric acid, and the alkalinity of sodium nitrate cultures is lessthan that of sodium bicarbonate cultures. The residual values fornitric acid, ammonium nitrate, and ammonium carbonate media areall practically the same as when water alone is used. These resultsare attributed to the necessity of the plant to utilise hydroxyl ionsduring the assimilation of ammonia, and hydrogen ions for assimil-ating nitrates. It is suggested that the energy utilised in the absorp-tion of the hydroxyl or hydrogen ions is a factor controlling the effectof a on the proportional absorption of nitrate and ammoniumsalts.D. Prianischnikov 275 shows that ammonium nitrate innutrient media may exert a physiologically acid or alkaline reactionin accordance with the age of the plants and the concentration of thenutrient. Under conditions of excessive alkalinity, absorbedammonia may be re-excreted into the medium. The physiologicalreaction of ammonium nitrate is also influenced by temperature,since nitrate absorption increases with rising temperature, whereasthat of ammonium is unaffected.28Temperature effects on the nitrogen nutrition of apple areexamined by G. T. Nightingale.f9 Simple protein synthesis occursin the fibrous roots of trees grown in darkness, in sand cultures a t9", nitrogen being supplied as ammonium sulphate at pE 6.0 orsodium nitrate at pE 4.5.Formation of amino-acids and aspara-gine and the utilisation of carbohydrate proceed more rapidly in theammonia-fed plants. Protein formed at this temperature, however,remains in the roots, which develop rapidly. By increasing thetemperature to 21", translocation of amino-acids to buds beginsand these organs develop rapidly. The reaction of the internalroot tissues is unaffected by that of the nutrient media or the natureof the source of nitrogen. I n several leguminous plants examinedby L. Burkhart 30 the absorption of ammonium salts appears to beinfluenced to a considerable extent by the nature of the non-nitro-genous plant reserves. Ammonia injury in these plants resultsfrom interference with the normal process of utilisation of proteinand non-protein reserve substances, and is associated with lowproportions of reducing sugars. Amide detoxication is onlymoderately effective in reducing injury.The ammonia nutrition ofrice varies with the nature of the associated anion, absorptiondecreasing in the order sulphate, phosphate, nitrate, chloride.The differences persist throughout growth except in the case of the270 2. PJEanz. Dung., 1934, A, 33, 134; B., 417.28 P. Strebeyko, Polish Agric. Forestal Ann., 1932, 28, 357; A., 1934, 821.29 Bot. Gaz., 1934, 95, 437; A., 1044.30 Plant Physiol., 1934, 9, 351; A., 1273352 BIOCHEMISTRY.nitrate, the ammonia of which is relatively better assimilated inolder plants.31 Sugar cane plants supplied with ammonium saltsare observed by G.B. Ulvin 33 to produce less chlorophyll than thosereceiving nitrate.Carbohydrates in Plants.-Starch formation in sugar cane leaveshas been further examined by W. M. Coelingh and V. J. Konings-berger.33 Normally, starch accumulates only in the bundle sheaths.The process continues in darkness if excised leaves are placed insolutions of maltose, sucrose, glucose, or fructose, but starch pro-duction is localised in the palisade cells of the parenchyma. Lightis required for the translocation of starch to the bundle sheaths.The extent to which these processes occur differs somewhat with thevariety of cane examined. During the growth of the cane seasonalvariations in total solids and non-reducing sugars in the sap are of asimilar character, whereas reducing sugars vary in an inversedirection. I n cooler periods sucrose accumulates in stems and maybe utilised during the sprouting of stem cuttings, during tassellingand side-shoot formation, or in the renewal of denuded leaves,During such periods the proportion of reducing sugar in saps under-goes little change.34In an interesting investigation of the distribution of carbohydratesand other constituents of the cotton plant, T.G. Mason andcolleagues 35, 36 examine the concentration gradients of sugars invarious organs under different growth conditions. I n addition tosucrose, glucose, and fructose a water-soluble polyglucoside occursin leaves. Translocation of carbohydrate to the bundles occurs inthe form of sucrose.Stored polysaccharides are located chiefly inthe bark.I n the examination of carbohydrate changes in plants the practiceis frequently adopted of comparing analytical values on a total-dry-weight basis. F. E. Denny37 indicates that this may sometimeslead to quantitative or even qualitative error and advises the calcu-lation of data in terms of “ residual dry weight,” i.e., total dry weightrniizus carbohydrates.The significance of sugar accumulation in vines is examined byL. Rloreau and E. V i r ~ e t , ~ ~ who show that the rapidly increasingB., 1934, 33.31 R. H. Dastur and T. J. Mnlkani, Indian J. Agric. Sci., 1933, 3, 157;a3 Arch. Suikerind. Ned.-Indie, 1932, 1325 ; A., 1934, 464.34 S. Komatsu, S. Ozawa, and Y.Makino, Mem. Coll. Sci. Kyoto, 1933,36 E. Phillis and T. G. Mason, Ann. Bot., 1933, 47, 585; A., 1933, 988.36 T. G. Mason and E. J. Maskell, ibid., 1934, 48, 119; A., 335.37 Contr. Boyce Thompson Inst., 1933, 5, 181 ; A., 1933, 873.32 Plant Physiol., 1934, 9, 59; A., 818.A , 16, I ; A., 1933, 873.Ann. agron., 1932, 3, 363; A . , 1033, 102STEWART AND POLLARD. 353amount of sugar appearing in the fruit during ripening is obtainedvery largely from the carbohydrate reserves of the main stem.Moreover, sugar acdumulation in the sap is a controlling factor inthe development of new fruit buds. Variation in the carbohydratecontent of roots is discussed by A. C. from the practicalviewpoint of the importance of timing cutting operations in weederadication.The effect of disease on carbohydrate variations in plants formsthe subject of numerous investigations.Leaf-roll in potato disturbsthe normal translocation of sucrose as a result of damaged phloem,and although there is some passage of hexose through the paren-chyma, starch steadily accumulates in leaf lamin~e.40 Tobacco virusbrings it decline in leaf ~arbohydrate,~~ differences from normalbeing especially marked in periods of active photosynthesis. Duringthe storage of healthy leaves there is a, steady decrease in insolublecarbohydrates, whereas in diseased leaves the loss falls principallyon the disaccharide fraction.42Rust in wheat causes a reduction in the percentage of sucrose andan increase in that of starch in the grain, although the total starchper grain is subnormal.&Other observations of this character include the occurrence of ageneral increase in reducing sugars, sucrose, starch, and poly-saccharides in the leaves of lucerne and clover following injury bythe leaf hopper;44 a partial replacement of carbohydrate bypentosans and galactan in rutabagas exhibiting “ dark centre ” ;and the association of ‘‘ blind wood ” in roses with abnormallyhigh accumulation of insoluble carbohydrates in stems and leaves,as compared with the flowering shoots which contain large pro-portions of reducing sugars.GZucosides.-Seasonable variations in cyanogenetic-glucoside con-tent of Xorghum vulgare are recorded by C.N. A ~ h a r y a . ~ ~ Maximumyields of hydrocyanic acid were obtained from very young plantsor young, actively growing side shoots of older stocks.The pro-portion declined with advancing age to a minimum a t the flowering3% Minnesota Agric. Exp. Sta. Tech. Bull., 1932, No. 84; A., 1933, 328.40 E. Barton-Wright and A. McBain, Trans. Roy. SOC. Edin., 1932, 57,4 1 J. Caldwell, Ann. Appl. Biol., 1934, 21, 191, 206; A., 1030.42 H. Cordingley, J. Grainger, W. H. Pearsall, and A. Wright, ibid., p. 78;A., 466.43 R. M. Caldwell, H. R. Kraybill, J. T. Sullivan, and L. E. Compton,J. Agric. Res., 1934, 48, 1049; B., 978.44 H. W. Johnson, Phytopath., 1933, 23, 19; B., 1933, 565; E. B. Hollandand C. P. Jones, J. &Tic. Res., 1934, 48, 377; A., 822.45 Indian J . Agric. Sci., 1934, 3, 851; A., 710.309; A., 1933, 546.REP.-VOL.XXXI. 354 BIOCHEMISTRY.stage. Cyanogenetic material accumulates chiefly in leaves andthe yield is the same whether seedlings are grown in darkness orin daylight. Daily variations show minima in early morning andevening and a maximum soon after mid-day. In the preparationof material for analysis, treatment with chloroform or toluenedoes not check the enzymic liberation of hydrocyanic acid. Thepossibility of the presence in Xorghum of cyano-compounds otherthan glucosides is indicated. A daily periodicity in the glucosidecontent of Aesculus leaves is reported G. Kerstan,46 who makesthe interesting observation that ssculin in the bark of twigs is noteasily translocated and does not function as a carbohydrate reserve.In leaves, however, zesculin is mobile.An unusual practical applic-ation of glucoside 'analysis is indicated by an examination of NewZealand clovers. It is shown that the amount of hydrocyanicacid obtainable from individual types, although subject to seasonalvariations, falls within limits sufficiently narrow to permit differ-entiation among different types?', 48 H. 0. Askew 49 also describesa mtisfactory method for the preparation and analysis of thematerial.Among new examinations of glucosides in plants may be mentionedthose of H. Colin and A. Chadun,SO who isolate a fructoside fromScilla, of G. Tanret 51 on the nature of coronillin from Coronilhseeds, the preparation of kzempferol rhamnoside from leaves ofPueraria h i r s ~ t a , ~ ~ and a further examination of salireposide 53 byM.W a t t i e ~ , ~ ~ who now describes this substance a8 a benzoate ofa heteroside yielding p-glucose on hydrolysis.Mineral Nutrition.-Potassium. That the efficiency of potassiumin plant nutrition is influenced by the nature of associated cationshas long been recognised. E. Blanck and W. Heukeshoven 55 makethe rather unexpected observation that the yield response of beansto potassium is higher for the oxalate, citrate, and formate than forthe sulphate. Moreover, the anionic influence shows itself notonly in affecting the total intake of calcium, phosphorus, and nitro-gen, but also in the distribution of these elements between seed and46 Planta, 1934, 21, 676; A., 1048.47 B. W. Doak, New Zealand J .Sci. Tech., 1933, 14, 369; B., 1933, 839.4 8 T. Rigg, H. 0. Askew, and E. B. Kidson, ibid., 1933,16, 222; A., 1934,4s I b d . , p. 227; A., 229.50 Bull. SOC. Chim. biol., 1933, 15, 1520; A., 1934, 464.5 1 Cornpt. rend., 1934, 198, 1637; A., 709.52 T. Ohira, J . Agric. Chem. SOC. Japan, 1933, 0, 337; A., 1933, 1216.53 Ann. Reports, 1931, 28, 244.54 Bull. Acad. roy. mkd. Belge, 1932, 12, 433; A., 1934, 1276.5 5 J . Landw., 1933, 31, 291.229STEWART AND POLLARD. 355straw. This is perhaps related to the fact that the utilisation ofcalcium phosphate by plants to which the various salts were appliedis paralleled by the solubility of calcium phosphate in the respectivesalt solutions. In an examination of the distribution of potassiumin potato plants W.0. James and N. L. Penston 56 indicate acontinuous circulation of potassium in the plant in the form ofsalts of amino-acids. Deficiency of potassium lowers the activityof enzymes in plants and also the relative order of activity invarious plant organs. Thus M. Cattle 57 establishes that lowereddiastatic activity is more pronounced in young and in old leavesthan in those of intermediate age. Similarly, invertase activityin upper leaves is diminished and that of lower leaves increased bypotassium starvation. The decline in invertase activity has alsobeen observed in sugar cane by C. E. Hartt.58 The well-knownrelationships between the formation of carbohydrates in plants andthe supply of potassium suggest, that light intensity may exerta controlling influence in potassium nutrition.K. Scharrer andW. S ~ h r o p p , ~ ~ working with peas, show an inverse relationshipbetween the optimum potassium requirement and the period ofexposure of the plants to light. Impoverished growth due todeficiency of light is to a considerable extent counteracted by in-creased feeding with potassium. Similar conclusions are reachedby R. Schwartz 60 in the course of field manurial trials of the effectof potash fertilisers on the seed yield of Lolium italicurn.Iron and mnganese and their relation to chlorosis. The appearanceof chlorosis in plants is frequently traced to deficiencies within theplant of iron or manganese and since the solubility of many soilminerals containing these elements is lowered by reaction changestowards alkalinity, the explanation of lime-induced chlorosisseemed a simple one.Recent investigations indicate that otherfactors are concerned. The intake of manganese 61, 62+and iron 83by plants is conditioned by the nature of the compounds concernedand by the presence of other substances. Thus the absorption ofmanganese from the dioxide by wheat seedlings is depressed byadditions to nutrient media of sodium nitrate and calcium carbonate.No depression occurs when manganese chloride is used. Thereduced intake caused by additions of phosphate is especially marked56 Ann. Bot., 1933, 47, 279; A., 1933, 649.67 New Phylol., 1933, 32, 364; A., 1934, 337.58 Hawaiian Planters Rec., 1933, 37, 13; B., 1934, 340.5D 2.Pfinz. Dung., 2934, A , 35, 186; B., 1075.6o Ernahr. P&nze, 1934, 30, 293; B., 1027.61 J. Davidson, Proc. 2nd Internat. Cong. Soil Sci., 1933,11,84; A,, 1934,337.62 C. Oleen, Compt. rend. Trav. Lab. Carlsberg, 1934,20, No. 2 ; A., 1048.63 W. Schropp, 2. Pfinz. Dung., 1934, A, 33, 38; B., 340366 BIOCHEMISTRY.in the case of manganese chloride and sulphate. Similar influenceof anions on the absorption of iron by plants is also described.The availability to plants of organic compounds of iron is found tobe much less affected by reaction changes than that of manyinorganic forms .Extensive investigations in Germany 641 65, 66 of chlorosis in theyellow lupin indicate that the effect of lime is not limited to restrictingthe intake of iron by plants, but extends within the plant systemitself, where the iron is rendered immobile in the older leaves.Young leaves become chlorotic even when the total amount of ironwithin the plants may be sufficient t o meet their requirements.In the case of the blue lupin iron deficiency is related to the presenceof manganese, which is an essential nutrient for this plant.W.Scholz 67 shows that excessive proportions of manganese in theblue lupin plant produce no injury provided a sufficiency of iron ispresent. If the iron supply is restricted, as in lime-chlorosis,manganese intensifies the injury. Deficiency of manganese in theplant, however, may cause a characteristic manganese-chlorosisdistinct from that resulting from shortage of iron.Lime-inducedchlorosis is also observed in flax,68 and in this case also, the customarytreatment of the soil with iron salts is rendered inoperative if muchmanganese is present in the plant.A magnesium-chlorosis may occur as the result of excessiveapplications of magnesium carbonate to soils.69 It resembleslime-chlorosis in being associated with iron deficiency in leaves, andin its response t o additions of iron salts to soil.Copper. F. G. Anderson 70 cites a case of chlorosis due to copperdeficiency in leaves, remedied by spraying with copper sulphatesolution, and B. D. Wilson and G. R. Townsend 71 record the re-markably improved productivity of peat soils following treatmentwith copper sulphate. Similar effects were cbtained by R.V.Allison 72 and co-workers, in the saw-grass pea.ts of Florida. It issuggested in this case that in addition to its nutritional function,copper protects the root surface from injury by toxic organic64 R. Reincke, 2. Pjihnz. Dung., 1931, A , 23, 77; B., 1932, 126.68 W. Scholz, ibid., 1932, A , 25, 287; 1933, A , 28, 257; A, 29, 59; 1934,A , 33, 340; B., 1932, 907; 1933, 567, 599; 1934, 517.66 S. Triwosch, ibid., 1933, A , 31, 14; B., 1933, 884.67 Ibid., 1934, A, a, aa; B., 937.W. Scholz, ibid., 1934, A , 34, 296; B., 901.6D S. Triwosch, ibid., 1934, B, 13, 155; B., 597.70 J . Pomology, 1932, 10, 130; B., 1932, 907.71 J . Amer. Soc. Agron., 1933, 25, 523; B., 1933, 1073.7 2 Proc. 4th Cong. Int. SOC. Sugar Cane Tech. Bull., 1932, No. 112; B.,1933, 759; Proc.2nd Interat. Cong. Soil Sci., 1930, VI, 257; €3, 1933, 243STEWART AND POLLABD. 357substances known.to exist in these soils. Deficiency of copper isshown to be the cause of exanthema in pears 73 and of pecanrosette. 74Boron. Considerable interest centres on the essential nature ofboron for the growth of plants. The actual requirement (and alsothe minimum lethal dosage) varies considerably with species.Boron-deficiency affects the development of plants in a variety ofways, prominent among which is the curtailment of root growth,e.g., in flax,75 maize, and p ~ t a t o e s . ' ~ This effect is probably relatedto the influence of boron on thc calcium-intake of plants.K. Warington 77 shows that the addition of boron to nutrient solu-tions for Vicia fuba increases the gross amount of calcium .absorbed,and also prolongs the period during which the rate of intake ofcalcium is increasing.The direct relationship between the amountof calcium absorbed and that supplied is maintained aft,er borontreatment, but the general level of intake is raised. The normaldecline in the ratios N/Ca and K/Ca in plants with advancingmaturity appears to be accelerated by the presence of boron.Symptoms of boron deficiency appear more slowly in spring andautumn as a result of the shorter period of daylight. Temperatureis a less important factor in this re~pect.~8 The stimulatory actionof boron on root growth is shown by its ability to minimise theinjury to sugar beet caused by root and crown rots.7B Accordingto 0.Kaufmann,*o borax is much more efficacious than boric acidfor this purpose, and its protective action persists in soil for severalyears. E. V. Bobko and M. A. Bt:lvoussov81 record optimumgrowth of beet in nutrients conta.ining 5 mg. of boric acid per litre.The ill effects of heavy liming on the growth of beet in field soilsare counteracted by relatively small dressings of boric acid. In anexamination of a boron-deficiency disease of lettuce, J. S. McHargueand R. K. Calfee 82 show that boron may be added to culturemedia in the form of simple borates (that of manganese avoids toxiceffects) or as borosilicate in the form of powdered glass. Boric73 J. Oserkowsky and H. E. Thomas, Science, 1933,78, 315; A., 1934, 122.7 4 A.0. Alben, J. R. Cole, and R. D. Lewis, Phytopath., 1932, 22, 595;75 M. Skolnik, Bull. Acad. Sci. U.S.S.R., 1933, 7 , 1163; B., 1934, 463;76 K. Scharrer and W. Schropp, Z. PJEanz. Dung., 1933, 28, A , 313; B.,7 7 Ann. Bot., 1934, 48, 743; A., 1274.7 8 K. Warington, Ann. Bot., 1933, 47, 429; A., 1933, 989.79 K. Scharrer and W. Schropp, Landw. Jahrb., 1934, 79, 977; B., 1026.80 Deut. Zuckerind., 1934, 69, 305; B., 852.81 Ann. agron., 1933, 3, 493; B., 1933, 1073.82 Plant Physiol., 1933, 8, 304; B., 1934, 645.B., 1932, 954.Cornpt. rend. Acad. Sci. U.S.S.R., 1934, 2, 104; B., 597.1933, 518358 BIOCHEMISTRY.acid is essential to the development of pollen grains in certain tropicalwater lilies,a3 and borax is associated with marked stimulatoryeffect on the yield of beans.s4Plant Crowth-promoting Substances (Plant Hormones).It has long been realised that, although current knowledgeaffords an explanation in some detail of the chemical mechanismof nutrition, assimilation, metabolism, and synthesis in plants, themanner in which this mechanism is controlled and structural develop-ment regulated in response t o varied external conditions dependsupon characteristically different factors.Indeed it is only duringthe last few years that the activity of growth-regulating substances,hormones, auximones, etc., has been regarded as possibly due tospecific chemical substances. The spectacular work of F. Kogl andhis colleagues 85 in elucidating the chemical nature of auxin marksa very important stage of development in the subject and may wellform the source of inspiration of an enormous field of investigationfor the biochemist.Auxin- A.*Many aspects of plant development involve the elongation ofindividual cells (as distinct from their multiplication), and numerousinvestigators 86 have indicated that this process is influenced by ahormone-like substance which increases the plasticity of the cellwall and, possibly as a direct result of cell turgidity, thus facilitateselongation.The active material, auxin (so-named by K0gl),87 is usuallydetected and determined by its ability to promote renewed growthof Avena coleoptiles following decapitation.I n Went’s nowgenerally adopted technique, the auxin, prepared in agar, is appliedasymmetrically to the cut surface of the coleoptile stump and bycausing proportionally greater extension on the treated side, pro-duces a curvature which increases with the potency of the prepara-tion.The cuticle of the coleoptile is not readily permeable t o auxinin agar. Auxin readily penetrates the external tissue of roots,however, and here produces a restriction of elongation.S3 T. Schmucker, Naturwiss., 1932, 20, 839; A , , 1933, 105.8* M. Gorski, Polish Agric. Porestal Ann., 1932, 28, 27; B., 1934, 597.85 Ann. Reports, 1933, 30, 105.*6 Among others, see F. W. Went, Rec. Trav. bot. ne‘erl., 1928, 2 5 , l ; A. N. J.87 F. Kogl and A. J. Haagen-Smit, Proc. K. Akad. Wetensch. Amsterdam,* In the following pages the word “ auxin ” refers always to Auxin-A.Heyn, ibid., 1930, 28, 113; N.Cholodny, Biol. Zentr., 1927, 47, 604.1931, 34, 1411; A., 1932, 661.Auxin-B is so written in all casesSTEWART AND POLLARD. 359Occurrence in Phnts.-The rapidly growing apical tissues of rootsand shoots of plants contain relatively large accumulations of auxin ,and Went originally extracted this by standing freshly cut tipson an agar block, into which a portion of the hormone diffused.Extraction by solvents has now been adopted, auxin being separatedin the ether-soluble fraction from various plant organs.The proportion of auxin in coleoptiles decreases from the tiptowards the base 89 and, according t o observations of K. Koch,gOthe hormone is concentrated very largely in the 0.2 mm.of the apex.I n sections distant more than 3 mm. from the tip, scarcely detectableamounts are found. The growing tips of young plants have moreauxin than those of older plants 91 and in Avena the amount formedtends to decrease with rising temperature of germination. I napical tissues of seedlings, light influences the production of auxin,and in the case of Lupinus albus cited by A. E. Navez 92 approxim-ately twice as much was found in lighted seedlings as in those grownin darkness. Auxin occurs in similar proportions in the tips ofboth young and old roots of Zeu muisY93 but in a number of otherspecies examined, Cholodny (loc. cit.) was unableeto detect the hor-mone by the Avena method. Cholodny favours the view that auxinis actually formed in the root tip, but doubt on this point isexpressed by E.BiinningYg4 C. J. GorterY95 K. V. ThimannYgs andothers, who assume the translocation of the substance from aerialparts of the plant.Cholodny also records a marked decline in the activity of excisedroot tips after 5-6 hours unless an appropriate nutrient (in thiscase, gelatin) is provided. This apparent exhaustion of the hormonemay also explain the cessation of elongation of sections of Avenacoleoptiles after immersion in water for a similar period!7 Underthese conditions growth continues on further treatment with auxin.Pollen Hormone.-F. Laibach and colleagues have made an exten-sive examination of the growth-promoting substances occurring inthe pollen of certain orchids and of Hibiscus.Pollen produces thecustomary effect of auxin on Avena coleoptiles,98 causes enlargementof the gynosternium, accelerates or renews the growth of floweringLOC. cit. K. V. Thimann, J . Gen.. Physiol., 1934, 18, 23; A., 1272.Planta, 1934, 22, 190-220; A., 1272.91 H. G. van der Wey, Proc. K . Akad. Wetensch. Amsterdam, 1931, 34,a* Proc. Nat. Acad. Sci., 1933, 19, 636; A., 1933, 987.N. Cholodny, Planta, 1934, 21, 517; A., 1044.*4 Plantu, 1927, 5, 635.06 Dissert., Utrecht, 1932.87 J. Bonner, J . Gen. Phyaiol., 1933, 17, 63; A,, 1933, 1214.875; A., 1932, 201.86 L O C . cit.Ber. deut. bot. Gee., 1932,50,383 ; 1933,51,336; A., 1933,103; 1934,120360 BIOCHEMISTRY.stems and tendrils, restricts the growth of side-shoots when amppliedto cut main stems, and stimulates root production on cut stems of anumber of plants.99 The hormonal potency of pollen is very highand is retained for long periods.This is ascribed 1 to the fact thatthe substance does not occur actually in the pollen grains but in thecaudicle, the plastic nature of which affords a protective action.By incorporation of lanolin with pollen extracts Laibach producesa concentrated auxin preparation of persistent activity, whichin addition to the ordinary effect on decapitated coleoptiles iscapable of producing curvature in intact coleoptiles when paintedon one side, and a corresponding (reverse) curvature in similarlytreated aerial roots of Cissus gongyloides.Occurrence in Fungi, etc.-Auxin was isolated from cultures ofRhizopus suinus by N.Nielsen and its properties were studied byH. E. Dolk and K. V. Thimann.3 According to these authorshormone production is favoured by aeration, is influenced by thestate of purity of the peptone used in media, and is paralleled bythe formation of carotenoid colouring matter. The occurrence of thehormone is associated with the production of sporangia and isprobably concerned in the germination of spores. Nielsen alsoobtained auxin from Boletus edulis, but was unable to detect it inPsallista mrnpestri~.~ The production of active material fromAspergillus niger has been examined by P. Boy~en-Jensen,~ whoregards auxin merely as a metabolic product of the fungus and asplaying no part in its growth, Formation in the organism dependson the nature of its nitrogen nutrient.The presence of certainamino-acids of high molecular weight appears necessary. Peptoneand hzemoglobin are effective in this respect. Inorganic forms ofnitrogen are unsuitable.6 T. Sakamura and T. Yanagihara 7 confirmthe formation of auxin in media containing peptone and also sodiumnucleate, but show that alanine, asparagine, aspartic and glutamicacids are ineffective sources of nitrogen. The growth substance isproduced in a,erobic and in anaerobic cultures. I t s formationis inhibited by the presence of glucose (contrary to Boysen-Jensen’sobservation), sucrose, fructose, maltose, galactose, and glycerol,but not of mannitol or lactose.OD F. Laibach, A. Muller, and W. Schiifer, Natumuiss., 1934, 22, 588; A , ,1272.This effect is also produced by Kogl’s auxin.F. Laibach, Ber. deut. bot. Ges., 1933, 51, 386.Jahrb. wiss. Bot., 1930, 73, 125; Biochem. Z., 1931, 23, 244.Proc. Nat. Acad. Sci., 1932,18, 30, 692; A., 1932, 549; 1933, 327.Ibid., 1932, 250, 270; A., 1932, 1065.Ibid., 1931, 239, 243; A., 1931, 1334.Proc. Imp. Acad. Tokyo, 1932, 8, 397; A,. 1933, 197.* Biochem. Z., 1932, 249, 196; A., 1932, 887STEWART AND POLLARD. 361The hormonal preparation from yeast recorded by N. Nielsenalso contains auxin, which is presumably the active agent of yeastextracts concerned in stimulating the blossoming of peas.9Various species of bacterialowhen grown on peptone media produceauxin, which has also been isolated from the marine alga Valoniamrophysa, in which it is mainly concentrated in the cell walls.llOccurrence in Animal Orguns.-The distribution of auxin is notlimited to the plant kingdom.It has been found in the blood,liver, and kidneys of guinea pigs, in rabbit lungs, in pig’s thyroid,in human and mouse carcinoma, and in a wide variety of otheranimal tissues.12 Much larger proportions were obtained by Kogland colleagues l3 from urine, the source of the material used in theirinvestigations of the chemical constitution of the hormone. Urinaryexcretion of auxin seems very largely controlled by the dietaryintake and is not influenced by age, sex, pregnancy, or menstruation,or by carcinomatous or tuberculous conditions.14Geotropism, Phototropism, and Electrotropism in Plants as relatedto Auxin-A.-The relationships between the action of auxin andgeotropic and phototropic curvature in plants have provided aninteresting field of investigation.F. W. Went (Zoc. cit.) and alsoE. Seubert l5 have shown that decapitated Avena coleoptiles whenplaced horizontally can be stimulated to further growth with normalgeotropic curvature if the hormone is placed symmetrically on thestump. H. E. Dolk l6 assumed that under these conditions auxinwas translocated preferentially to the under side of the coleoptileand induced the normal upward curvature. If the auxin is placedasymmetrically on the horizontal stump, geotropic response in thenew growth is retarded or enhanced according as the auxin is applied* Biochern. Z., 1931, 236, 205; A , , 1931,1091.* A.I. Virtanen and S. von Hausen, Nature, 1933, 132, 408; 1934, 133,383 ; A., 1933, 1093 ; 1934, 463 ; V. Subrahmanyan and G. S. Sidappa, ibid.,1933, 132, 713; A., 1933, 1342.lo P. Boysen-Jenaen, Biochem. Z . , 1931 , 236, 205; A,, 1931, 1091.11 H. G. van der Weij, Proc. K . Akad. Wetensch. Amsterdam, 1933, 36,769; A , , 1934, 120.l2 E. Maschmann, Natumoiss., 1932,20, 721 : A., 1932,1156; E. Maschmannand F. Laibach, ibid., 1033,21, 517; A., 1933, 1213; F. Kogl, A. J. Haagen-Smit, and B. Tonnis, 2. physiol. Chem., 1933, 220, 162; A., 1933, 1213.13 Summaries of this work are t o be found in Angew. Chem., 1933, 46, 469;A., 1933, 987; Rep. Brit. ASSOC., 1933, 600; Naturwiss., 1933, 21, 17; A.,1933, 435.1 4 F. Kogl, A.J. Haagen-Smit, and H. Ersleben, 2. phyaiol. Chem., 1933,220, 137; A., 1934, 1213.1 5 2. Bot., 1926, 17, 49.16 Proc. I<. Akad. Wetensch. Amsterdava, 1926, 29, 1113; Dissert., Utrocht,1930.M 362 BIOCHEMISTRY.to the upper or the lower side of the st~mp.1~1 1% l9 In corre-sponding experiments with roots it has been shown that in theseorgans also geotropic response is associated with the movement ofauxin to the lower side of the tip with consequent restriction ofelongation on this side.201 21Similarly the normal phototropic response of a coleoptile which isweakened or eliminated by decapitation is restored by symmetricalplacement of auxin in agar on the stump. Asymmetrical applic-ation causes a restricted or accentuated response according asactivation is on the near or the far side of the stump with respect tolateral illumination. Similar effects are produced by removal ofone half of the coleoptile tip (Koch, Zoc.cit.). It is concluded thatin the normal coleoptile auxin tends to move towards the shadedportions of the stem.The conception of the existence of a potential gradient in theplant system, and of its variation with the rate of growth, is of longstanding. The known growth response of plants to an artificiallyapplied electric field has been shown by Dolk, Went, Cholodny(Zocc. cit.) and other workers to be explicable by the translocation ofauxin within the plant towards positive polarity. Kogl (loc. cit.)observed that the potency of his auxin preparation a.s measured bythe Avem method varied hourly, and from day to day.He finallytraced this effect to variations in the electrical condition of the atmo-sphere. Further, by passing very small currents through theagar-auxin block and the coleoptile stump on which it was placed,he demonstrated that the curvature per auxin unit could be variedat will by changing the polarity of the system, to accelerate or retardthe normal basal movement of the hormone. The acid character ofauxin indicates a tendency to migrate towards the positive pole, andKoch 22 explains the electrotropic curvature of stems towards thepositive by the impermeability of the cuticle to aqueous solutions.The cuticle acts as an insulating medium, and an externally appliedpotential difference induces a reversed polarity within the tissueitself.As a result, auxin moves toward the internal positive(i.e., towards the external negative) pole, producing curvaturetowards the external positive. The view is confirmed by insertionof needle electrodes through the cuticle directly into the tissue ;curvature towards the negative pole then occurs. Epidermal tissuesW. G. du Buy, Rec. Trau. bot. nierl., 1933, 30, 1.E. Nuernberk, Flora, 1933.lS K. Koch, Plantu, 1934, 22, 190; A., 1272.2O N. Cholodny, Ber. deut. bot. Qes., 1932, 60, 317; 1933, 61, 85,21 P. Boysen-Jensen, Plantu, 1933, 20, 688; A., 1934, 334.22 LOC. C i t STEWART AND POLLARD. 363of roots are freely permeable and no question of induction arises.Curvature is always toward the positive pole.More recent observations of K.Ramshorn 23 confirm and adddetail to earlier records of the electrical conditions obtaining inplants, and demonstrate the electropositive character of zones ofrapid growth with respect to the more slowly growing parts.Tropic movements in general, therefore, are such as to indicatemovement of the growth-promoting substance toward the positivepole, towards gravity or away from light and, in general, the effectsof artificially applied auxin are superimposed on normal tropic re-sponses.Cell Extension and Plant Metabolism in Relation to Auxin-A.-Various workers have observed that auxin stimulates cell elongationby increasing the plasticity of the walls. The mechanism by whichthis is effected would appear to be somewhat complicated.Thework of K. V. Thimann and J. Bonner 24 indicates that the action ofauxin is not on the formation of cell wall material, nor does it modifythe permeability of the wall, but is primarily directed on the proto-plasm. They also observe that the respiration of coleoptile sectionsincreases when the proportion of auxin present is small.S. Strugger 25 shows that the growth of seedling shoots of HeEianthusannuus is stimulated by immersion in acid buffer solutions and thatif a longitudinal strip of the epidermis is removed a permanentcurvature away from the wound is produced in decapitated (andsupposedly auxin-free) hypocotyls. A similar effect is induced inanaerobiosis in which the internal acidity of the cells is automaticallyincreased.Strugger suggests that auxin promotes cell elongationby regulating the course of cell metabolism to produce acid condi-tions, and in this way influences the rate of growth, which is relatedto the difference between the pH and the isoelectric point of theprotoplasm,I n a later paper J. Bonner 26 confirms the increased plasticity ofcell walls following acid treatment in the case of Avena coleoptilesand further shows that the resulting growth increase is inhibited byconcentrations of hydrogen cyanide of the same order as those whichinhibit the action of auxin. Auxin itself does not increase cellacidity. The stimulative action of acids on decapitated coleoptilesis ascribed to the conversion of inactive salts of amin remaining inthe stump into the active non-dissociated form.23 Planta, 1934, 22, 737.z4 Proc.Nut. Acad. Sci., 1933, 19, 714; A., 1933, 1093; Proc. ROY- S0C.sz 6 Ber. deut. bot. Om., 1932, 50, 77; A., 1933, 102; {bid., 1933, 51, 193.z6 Protopkssma, 1934, 21, 406; A., 1272.1933, B, 113, 126; A., 1933, 757364 BIOCHEMISTRY.The association of geotropic influence with chemical differences inplant tissues is brought out by observations of T. Warner,27W. Gundell,28 and P. Metzner.29 The undersides of horizontallyplaced shoots have a markedly increased sugar content and hydrogen-ion concentration, a small increase in catalase activity, and, in theexpressed sap, only small differences in osmotic pressure, conduc-t i ~ t y , viscosity, and surface tension as compared with the uppersides.In so far as these observations can be interpreted in relationto Strugger's views they are of a confirmatory nature.Auxin-B.Associated with auxin-A from a number of sources is anothergrowth-substance, differing from it in physiological activity butrelated to it chemically. The chemical constitution of these sub-stances is more appropriately dealt with in the Organic Chemistrysection of these Reports. Apart from physiological distinctions,it is usually sufficient in biochemical work to differentiate betweenthe two substances by the solubility in ether ( A is soluble) and byresistance to heat and oxidation (B is resistant).Auxin-B has no influence on Avem coleoptiles, but is usuallyc haracterised by accelerating the growth of Aspergillus niger.Auxin-B occurs with -A in Rhizopus suinus, in amounts which appearto be related to the pH of the medium.30 E.Biinning31 recentlyconfirmed the presence of auxin-B in A . niger and examined itsaction on the growth of the fungus. Auxin-A does not affect theweight of mycelium produced or the numbers of conidia, but causesa slightly accelerated formation of conidia and subsequent degener-ation. Auxin-B, however, produces a very marked increase inmycelium production. Both hormones favour the resorption ofnitrate from media and retard that of ammonia. The resultingtendency towards increased % in the media may explain the slightlyearlier formation of conidia in the presence of auxin-A. N. Nielsenand V.Hartelius 32 indicate that auxin-B acts on A . niger as a resultof modification of %-growth relationships as in the case ofRhizopus (above). Thus optimum growth in the presence ofa.uxin-B occurs at pH 6-7, and in its absence, at pH 3.The rate of regeneration of yeast is increased by auxin-B to anextent proportional to the amount present. The size of the27 Jahrb. wiss. Bot., 1928, 88, 431.2D Ber. deut. boi?. Ge.., 1934, 52, 506.so N. Nielsen and V. Hartehe, Compt. rend. Trav. Lab. Carlaberg, 1932,19,31 Ber. deut. bot. Ges., 1934, 52, 423.32 Compt. rend. Trav. Lab. Carlsberq, 1933, 19, No. 15; A , , 1933, 1205.28 Ibid., 1933, 78, 623.No. 8; A., 1932, 661STEWART AND POLLARD. 366individual cells is unaff eoted. Turbidity measurements of yeastcultures are suggested by E.Almoslechner 33 for the quantitativedetermination of auxin-B.reports the presence of auxin-B in maize oil and in malt.It is also found in Boletus edulis and in urine, blood, milk, and anumber of vegetables. Examination of preparations from the latterproducts shows that auxin-B, in order to exert its full activity,requires the presence of a complementary substance (" Co-B "),which is deficient in a number of the above products. Filter-paperash and zinc salts appear to fulfil this requirement.35KoglOther Growth-regulating Substances.Since Wilder first recorded the existence of a growth-regulatingsubstance, " bios," a number of hormone- or vitamin-like substancesaffecting growth in the plant world have been described. Morerecently it has been shown that animal hormones and vitamins mayalso exert a growth-promoting action on plants.Certain simil-arities in the physiological activities of these substances, or in thoseof various constituent fractions into which a number of the crudematerials have now been resolved, tend towards the view that amongplant and animal hormones and vitamins there may well exist closerfundamental relationships than are as yet apparent. This con-ception is illustrated by such investigations as that of Williams etal.36 Yeast-stimulating preparations, obtained from a wide rangeof plant and animal products, all contained a polyhydroxylic acid,panthothenic acid, very closely related to vitamin-B,. Wilder's" bios," now shown t o contain probably three constituents, inducesincreased growth in certain fungi. Auxin-A preparations fromAvena and from fungi stimulate yeast growth but do not contain the" Z "-factor regulating fermentati~n.~' T. Philipson 38 also recordsa yeast-stimulating complex in green peas. Two constituents areindicated, neither of which alone shows any activity in this respect.The growth of Nematospora gossypii, especially as related to theassimilation of nitrogen compounds, depends on the presence of agrowth factor, shown by H. W. Buston and co-workers 39, 40 to be33 Planta, 1934, 22, 515.34 F. Kogl, H. Erxleben, and A. J. Haagon-Smit, 2. phyewl. Chem., 1934,35 V. Hartelius, Biochem. Z., 1933, 261, 76, 89; A., 1933, 751.36 R. J. Williams, C. M. Lyman, G. H. Goodyear, J. H. Truesdail, and D.3 7 H. von Euler and T. Philipson, Biochem. Z., 1932,245,418; A., 1932, 550.38 Ibid., 1933, 258, 244; A., 1933, 427.39 H. W. Buston and B. N. Pramanik, Biockern. J., 1931, 25, 1656, 1671;40 H. W. Buston and S. Kasinathen, ibid., 1933, 27, 1859; A., 1934, 230.225, 215; A., 1044.Holiday, J. Amer. Chern. SOC., 1933, 55, 2912 ; A., 1933, 982.A., 1931, 1458366 BIOCHEMISTRY.a complex related to (' bios," and to contain i-inositol as a necessaryconstituent factor. Among bacteria, Micrococcus eykrnunii isstimulated by a substance occurring both in plants and in animals,which in some respects resembles but is not identical with auxin.This growth substance depends for its activity on the presence ofpeptone .41For example,W. Schoeller and H. Goebe142 have demonstrated the action offolliculin in accelerating the development and flowering of hyacinths.Apparently the hormone requires conversion into more readilyabsorbed sodium salt before becoming effective. Similar results arerecorded by M. J a n ~ t , ~ ~ who also finds that equilin, equilenin, anddihydrofolliculin are even more active. Injection of thyroidmaterial into bulbs increases the rate of flGwering and the numberof flowers produced.44 Thyreoidin stimulates the germination offungal spores, improves vegetative growth, and accelerates alcoholicfermentation by yeast, but has little influence on bacterial develop-ment.45 The action of thyroxine on plants is mainly directedtowards leaf development, whereas adrenaline and hypophysin actprincipally on roots.46More extensive examinations of the action of vitamin-B on fungiare recorded and serve to illustrate the trend of opinion towards theview that this vitamin has much in common with the typical planthormones. W. H. Schopfer4' has prepared from wheat germ,yeast, and pollen a growth substance accelerating vegetative growthand zygote formation in Phycomyces blakesleeanus, closely resemblingauxin and differing from the vitamin-B complex only in heat re-sistance and in adsorption by animal charcoal. Vitamin-B, andto a lesser extent -B, produce similar effects on the fungus, althoughin this case the action is dependent on the nature of the carbohydratesupply. E. Bunning,48 working with A . niger, shows thatvitamin-Bl has little action on growth except in alkaline media(cf. folliculin, above), whereas -B, markedly increases dry matterproduction. Like auxin-A and -B, the vitamin-B complex favoursthe absorption of nitrates by the fungus and restricts that ofL. E. den D. de Jong, Arch. Mikrobiol., 1934, 5, 1 ; A., 699.42 Biochem. Z., 1931, 240, 1; A., 1931, 1337; ibid., 1932, 251, 223; A.,1932, 1068; ibid., 1934, 272, 215; A., 1145.43 Compt. rend., 1934, 198, 1175; A., 463.44 E. E. Davies, Plant Physiol., 1934, 9, 377; A., 1272.46 A.A. Imschenetzki, Bull. Acad. Sci., U.R.S.S., 1932, 1559; A., 1933, 868.40 D. V. Hykes, Compt. rend. SOC. Biol., 1933, 113, 629; A., 1934, 934.47 Arch. Sci. phya. nat., 1934, [v], 16, Suppl., 23, 26, 29; A., 1035; Ber.Many animal hormones also influence plant growth.deut. bot. Qea., 1934, 52, 308.Ber. deut. bot. Qes., 1934, 52, 423STEWART AND POLLARD. 367ammonium. Also its action is similarly related to respiratoryactivity and to reaction changes in the media. W. G. Solheim etaZ.4Q find that vitamins-B, and -B, stimulate fructification in anumber of fungi and increase yellow pigmentation in A . niger andPenicillia. These physiological similarities among different hor-mones and vitamins are tending to intensify investigations of thechemical nature of the substances concerned, and provide anenormous stimulus to research in this fascinating branch ofbiochemistry .C. P. STEWART.A. G. POLLARD.49 W. G. Solheim, S. S . Sears, and R. C. Robbins, Phytopath., 1933, 23,929; A., 1934, 220