BIOCHEMISTRY.CONTINUED interest in the chemistry of the vitamins, and theconsiderable advances achieved during the past year, justify theattention which is again given to this field. Of outstanding inter-est, too, is the development of the recent work on the secondarysex hormones, more especially in relation to the striking advancesin the chemistry of the cholane series. Of the other subjects dealtwith this year in the section of animal biochemistry, particularattention is directed to the new and promising work on urea form-ation in the liver, and to the progress made in the investigation ofcarcinogenic hydrocarbons.In the section of plant biochemistry the general arrangement ofthe last Report has been retained. Undiminished interest in themineral nutrition of plants is again apparent, and the steadyadvancement in the elucidation of the varied metabolic processesof moulds is continued.A section dealing with certain plantenzymes and their activation is necessitated by the activity ofworkers in this field. The admirable work of R. and G. M. Robin-son on the anthocyanin pigments marks a very great advance inthis subject. The scope of the published work is too wide foradequate treatment here and reference to detail should be madein the original papers.ANIMAL BIOCHEMISTRY.Secondary Sex Hormones.The last few years have witnessed a steadily increasing activityin t,he investigation of the internal secretory products responsiblefor the control of the'development of the secondary sex charactersof the male and the female animal.In particular, during the pastyear, considerable advances have been made in the elucidation ofthe structures of these interesting substances. These advancesare mainly due to the new structural conception of the cholaneseries (bile acids and sterols) introduced by 0. Rosenheim andH. King and developed by them 2 and by the German workers,notably H. Wieland and E. Dane.3 At the time of writing itseems clear that two substances, or types of substance, have beenreasonably well characterised. The first of these is the follicularhormone which in animals produces oestrous, and influences thegrowth and course of development of the secondary female sex1 J . SOC. Chern. Id., 1932,51, 464.3 2. physiol. Chem., 1932, 212, 41, 263.Ibid., p.954340 BIOCHEMISTRY.characters. It is also claimed that this substance accelerates theformation of flower and fruit buds in plant^,^ and it has been statedthat the hormone, or substances closely resembling it in structureand properties, may be obtained from plant sources. Thus, acrystalline substance of this type has been isolated from palm-kernel extracts.5 The second sex hormone is the testicular hormoneisolated in a crystalline condition from male urine. It wouldappear to be closely related structurally to the follicular hormoneand it is suggested that both have the same basal constitution asthat of t'he members of the cholane series, namely, a tetra-nuclearstructure comprised of a three-ring phenanthrene system with anadditional five-membered ring.The FoZZicuZar Hormone.-In the Reports for the last two yearsthe isolation and initial steps in the investigation of this hormonehave been recorded.It has now become possible, in view of thenewer work on the cholane series, to suggest a structure for thehormone and for the related inactive alcohol, pregnandiol, isolatedalong with the hormone from the urine of pregnancy. A. Buten-andt suggests that many substances with a similar hormoneactivity and closely related to one another may exist, but on thebasis of present work it is clear that the most important are thea-follicular hormone (ketohydroxyoestrin, C,,H,,O,) and its hydrate(trihydroxyoestrin, C,,H,,O,). The investigations of Marrian andof Butenandt and their respective colleagues have led to thefollowing conclusions.The hydroxy-ketone is formed from thehydrate by loss of water from two neighbouring hydroxyl groups.The third hydroxyl is acidic. Catalytic hydrogenation yields ahexahydro-derivative in the form of a saturated hydrocarbonC&,O. Zinc distillation yields the aromatic hydrocarbon C18H1,.The three double bonds inferred t o be present are in the same ringand this also carries the acidic hydroxyl, whereas the alcoholichydroxyls of the hydrate are in a saturated ring. It is thereforesuggested that the hormone is a condensed system consisting of abenzene ring and three saturated rings. X-Ray crystallographicmeasurements by J. D. Berna1,lo and an investigation of unimole-* W.Schoeller and H. Goebel, Biochem. Z., 1932, 251, 223; A., 1068 (seealso A., 1931, 1337).A. Butenandt, Angew. Chem., 1932, 45, 655.Ann. Reports, 1930, 27, 271 ; 1931, 28, 236.LOG. cit. See also A. Butenandt and I. Stomer, 2. physiol. Chem., 1932,208, 129; A., 781.G. F. Marrian and G . A. D. Haslewood, Biochem. J., 1932, 26, 25; A.,655.LOG. c i t . See also Nature, 1932, 130, 238; .4., 971.lo J. SOC. Chem. Ind., 1932, 51, 269POLLARD AND PRYDE. 241cular surface films by N. K. Adam and his colleagues,ll and byJ. F. Danielli l 2 show that the saturated ring carrying the ketonicgroup and the benzene ring carrying the acidic hydroxyl are atopposite ends of the molecule.The facts outlined in the foregoing are expressed in the formuhappended.(I) is the hydrocarbon C,,H,,, (11) is. ketohydroxy-oestrin, C18H2202, first obtained by Butenandt, and (111) is tri-hydroxyoestrin, C,,H2,0,, first obtained by Marrian.Me Me(1.1 (11.1 (111.)Testiculur IIormone.-This hormone in a crystalline state hasbeen obtained by A. Butenandt 13 (with K. Tscherning) from maleurine. Its isolation from testes is also claimed in an earlier com-munication by B. Frattini and M. Maino,14 whose preparation wasless well characterised and probably much less pure than that ofButenandt.15 The testicular substance is closely related to thefollicular hormone and is also a hydroxy-ketone. It is saturatedand possesses no acidic properties. Its melting point is given as178" and its composition is C18H2,02 or C,,H,,O,, although itshomogeneity is still in doubt.Butenandt 16 has tentatively sug-gested the formula (IV). Its activity would seem to be of a highorder, since a total quantity of 1 to 1.2 y administered in four dosesover a period of two days produced a 30-35% increase in combgrowth in cocks.Me Me Me0UV.1 (V.) Pregnandiol (VI.) CI,H,,O, Alcohol11 N. K. Adam, J. F. Danielli, G. A. D. Haslewood, and G. F. Marrian,l2 J . Soc. Chern. Ind., 1932, 51, 1075.13 LOG. cit.14 Arch. 1st. biochim. ital., 1930, 2, 639; A., 1931, 398.Biochem. J., 1932,26, 1233; A., 1173.See also 2. angew. Chern., 1931, 44, 906; A., 96.See also Bwchern.Z., 1932,258, 202; A., 1173.16 Angew. Chem., 1932,45, 324; A,, 781. 16 LOG. cit242 BIOCHEMISTRY.The Origin of the Xex Hormones.-The present state of the in-vestigations just outlined leads to the conclusion that the sexhormones are oxidation products of the bile acids and sterols, theprocess involving the breakdown of side chains and the conversionof a saturated into an aromatic ring with associated loss of a methylgroup (compare the conversion of ergosterol, C,,H,O, into neo-ergosterol, C27H,00).17 This view of the relationship of the sexhormones to the cholane series gains in probability from the isolationof pregnandiol, C21H3602, together with two other inactive pro-ducts, an alcohol, C1,H,,O2 (m.p. 232"), and a hydroxy-ketone(m. p. 176.5") isomeric with the testicular hormone. These sub-stances, for which the formuh (V) and (VI) are suggested, mayreadily be fitted into the general scheme as intermediate steps inthe biological formation ol the active products.G. F.Marrian and G. A. D. Haslewood18 have recorded theisolation from pregnant mare's urine of a dihydroxyphenol,C,,H,,O(OH),, m. p. 189-190*5", to which they have given thename equol. It is physiologically inactive. If this compound isrelated to those already discussed, it would seem to be a derivativein which the three-ring phenanthrene nucleus alone persists withone additional carbon, but at the time of writing evidence of therelationship of equol to previously described compounds is lacking.Isomeric Follicular Ho7mones.-Reference has already been madeto the possible existence of closely related substances with similarhormone activities.A. Butenandt and I. Stormer l9 state that,when water is eliminated from trihydroxyoestrin by distillationover potassium hydrogen sulphate, there are formed 01- and p-hydr-oxy-ketones which form mixed crystals and are both physiologicallyactive. The a-isomeride is the original hormone of Butenandt, andthe existence of the two forms is ascribed to a cis-trans isomerism.E. Schwenli. and F. Hildebrandt 2o have isolated from mare's urinea further isomeride for which they claim a physiological activityhigher than that of the a-hormone of Butenandt. A. Girard andhis collaborators 21 have also described isomeric crystalline prepar-ations isolated, in addition to the a-hormone, from mare's urine.These appear to have an oestrogenic activity about one-eighth thatof the a-follicular hormone. Whether these substances are pureisomeric hormones of lower physiological act'ivity, or mixed crystalsof active and inactive components, remains an open question, but1' H.H. Inhoffen, Annalen, 1932,497, 130.20 Natumuiss., 1932, 20, 658; A., 1173.21 A. Girard, G. Sandulesco, A. Fridenson, and I. J. J. Rutgers, Compt. rend.,18 Biochem. J., 1932, 26, 1227; A., 1156. 19 L O C . cit.1932,194,909, 1020; A., 433, 547POLLARD AND PRYDE. 243there seems t o be little doubt concerning the existence of isomeridesin this interesting group of substances.A Substance Present in the Testicle which Increases TissuePermeability,F. Duran-Reynals in 192822 and 192923 described a testicularextract which much increased the lesions produced by intradermalinoculation of vaccine virus.D. hlcClean z4 made further observ-ations on this interesting substance, and showed that it increasedthe permeability of the dermis so much that the bleb following theinjection immediately disappeared and the inoculum diffused throughan increased a'rea of the dermis, Extracts of spermatozoa were shownt o possess the same activity as extracts of the whole testicle. Thissuggested that the phenomenon is connected with the germinalactivity of the testicle and is not a fortuitous property of the inter-stitial tissue of the gland. A possible association of the activesubstance with the sudden change in the permeability of the ovaon fertilisation was suggested. W.T. J. Morgan and D. McClean 25have now described the purification of the active testicular extracts,and the preparation of a highly active dry material. The latterhas [.ID - lo", nitrogen lo%, and amino-nitrogen 1.5%. TheMolisch test is negative and all protein tests are positive. Theactive substance is not adsorbed from aqueous solution by benzoicacid. The minimal dose producing definite diffusion in the skin ofthe rabbit is 0.00001 milligram.Secretin.During the past year two papers dealing with the preparationand chemical properties of secretin have appeared. In 1928,J. Mellanby26 described a method for preparing the hormonesecretin which gave a product free from the depressor substancealways present in acid extracts of the duodenal mucosa. He nowdescribes an improved and simpler method : 27 the product containsC, 49.1; H, 6.9; N, 13.8; S, 1.5; ash, 1.17; 0 (by difference),28.6%, but no phosphorus.These figures, together with thephysical properties of secretin, suggest to Mellanby that secretinis a polypeptide. It is rapidly destroyed by trypsin-kinase, but isnot acted upon by enterokinase or by trypsin in freshly secretedpancreatic juice. The yield is about 10 milligrams from 500 gramsof intestinal mucosib. Secretin does not dialyse from aqueous22 Compt. rend. SOC. Biol., 1928, 99, 1908.2s J . Exp. Med., 1929, 50, 327.24 J. Path. Bact., 1930, 33, 1045; 1931, 34, 459.25 J. SOC. C h m . Ind., 1932, 51, 912.47 Proc. Roy. SOC., 1932, [B], 111, 429; A.: 1171.26 A., 1928, 1403244 BIOCHEMISTRY.solution through a collodion membrane.Its physiological actionsare : (1) the production of a large volume of pancreatic juice;(2) the contraction of intestinal muscle; (3) the secretion of asmall qxantity of bile.A paper by R. N. Cunningham 28 also describes the preparationof secretin free from depressor substances, insoluble proteins, andinorganic salts. Cunningham’s conclusion is that secretin is asecondary proteose. It passes through cellophane, but is retainedby collodion membranes permeable to peptones. The results ofMellanby and Cunningham are, therefore, subst antially in agree-ment, especially in regard to the peptide structure of their respectivematerials. There is, howcver, some divergence of opinion regardingthe molecular dimensions of secretin.Urea Formation in the Animal.An important paper by H.A. Krebs and K. Henseleit 29 dealingwith the formation of urea in the animal body opens up new aspectswhich may well prove to be fundamental. The authors havestudied the rate of synthesis of urea from carbon dioxide andammonia in surviving tissue sections of rat’s organs. The methodsemployed reveal the liver as the sole organ in which this synthesisoccurs, and in this organ the rate of synthesis is greatly increasedby the presence of ornithine. This amino-acid acts as a catalyst,since it is not used up and small amounts effect a large synthesis.Similar results are obtained with arginine in virtue of its conversioninto ornithine in the liver.On the othcr hand no increased urcaformation was observed in the presence of glycine, dl-alanine,d-valine, Z-leucine, d-cysteine, asparagine, aspartic acid, glutamicacid, phenylalanine, tyrosine, putrescine, cadaverine, lysine, creatine,guanidine, choline, tryptophan, or histidine. Apart from ornithineand arginine, the only other amino-acid effective in producingincreased urea synthesis is citrulline. This acid was isolat’ed byY. Koga and S. Odake 3O in 1914 from the water melon (CitruEEusvulgaris). Its constitution was established by M. Wada31 bysynthesis. It is a-amino- 8-carbamidovaleric acid,NH,*CO*NH*CH2*CH2*CH2*CH( NH,)*CO,H.Its formation has been observed by D. Ackermann 32 when arginineis acted on by putrefactive bacteria in a suitable medium, and in28 Biochem. J., 1932, 26, 1081; A., 1171.29 2.physiol. Chem., 1932, 210, 33; A., 1059.30 J. Tokyo Chem. Soc., 1914, 35, 519.31 Proc. Imp. Acad. Tokyo, 1930, 6, 15; A., 1930, 1224.33 Z. physiol. Chern., 1931, 203, 66 ; A., 196.See also Biochcrn.Z., 1930, 224, 420POLLARI) AND PRYDE. 245general the formattion of carbamido-acids from amino-acids is awell-established biological process.33In accelerating urea formation citrulline is consumed in theprocess and furnishes one atom of nitrogen per molecule of urea.The respective actions of ornithine and citrulline in promoting ureasynthesis in the liver are explained by Krebs and Henseleit asfollows : (1) The formation of citrulline by the condensation ofone molecule of ammonia and one of carbon dioxide with the6-amino-group of ornithine; (2) the condensation of one moleculeof citrulline with a second molecule of ammonia to form arginine;(3) the decomposition of arginine by arginase to form ornithineand urea.NH2*[CH2],*CH(NH2)*CO2H + NH3 + CO,IOptimal formation of urea in the liver sections is observed in thepresence of 10 mg.per cent. of d-ornithine and 200 mg. per cent.of dl-lactate. Urea is not formed in liver pulps in which the cellstructure is destroyed, and although its synthesis does not involvean increased oxygen consumption by the liver, the process is closelydependent upon respiration. It is of interest to note, in view ofthe currently accepted structural relationship of urea to ammoniumcyanate, that the latter does not increase the urea formation in theliver.The next step in this interesting series of investigationswould appear t o be the attempted isolation of citrulline, or a suit-able derivative, from a liver preparation actively forming urea.D. Ackermann% has failed t o observe the fission of arginine intocitrulline and ammonia on subcutaneous injection of arginine intoa dog. Nor was this process observed in ox liver, kidney, muscle,spleen, goose liver, or liver and muscle of the crayfish. It will,however, be borne in mind that this conversion is the reverse ofthat postulated by Krebs and Henseleit.These new observations are obviously of the greatest importance,but it must not be forgotten that the animal does not form urea fromammonia but from amino-acids, and it has not been possible, sofar, t o correlate the deaminisation process with ammonia production.33 H.D. Dakin, J . Biol. Chem., 1909, 6, 240.34 2. physiol. Chem., 1932, 200, 12; A., 9G2246 BIOCHEMISTRY.Dyes and Urease.I n the Report of last year reference was made to the suggestivework of J. H. Quaste135 on the toxic action of dyes and relatedcompounds on various enzymes. These investigations have nowbeen extended to u r e ~ s e . ~ ~ In general it is found that acidic dyesare entirely inert and that most basic dyes are toxic. The toxicityof such dyes as brilliant-green is enhanced by substances, appar-ently of the nature of the unsaturated glycerides, present in soya-bean oil, which act as highly specific mordants between urease andthe basic triphenylmethane dyes.Urease may be protected fromthe t'oxic action of the various dyes by urea, a-amino-acids, sar-cosine, et hylenediamine, met hylamine, dimet hylamine, hydrazine ,and hydroxylamine. No protection is afforded by trimethylamine,betaine, urethane, methylurea, diethylurea, and oxamic acid. Ofparticular interest is the observation that potassium cyanate pro-tects urease from the toxic action of brilliant-green, whereasammonium carbamate has little or no effect. The underlyingtheory being that protection indicates a combination between theenzyme and the protective substance, this observation is evidencefor the contention that cyanic acid, rather than carbamic acid, isproduced from urea by urease.It should, however, be noted thatsodium cyanate is entirely without protective action against toxicdyes.Carcinogenesis by Pure Hydrocarbons.The varied physiological activity of the cholane series and relatedpolycyclic compounds lends added interest to an already interestingproblem, the production of cancerous new growths by applicationto the skin of pure polycyclic hydrocarbons. That hydrocarbonscan produce cancer in mice followed from the work of E. L. Kenna-way,37 who investigated the carcinogenic mixtures, which couldconsist only of hydrocarbons, produced by heating acetylene orisoprene in an atmosphere of hydrogen. The strong fluorescenceof these and of other carcinogenic mixtures well recognised inmedical practice (e.g., gasworks tar, shale oil, heated petroleum,and products of the action of heat on various substances of bio-logical origin such as cholesterol, yeast, skin, muscle, and hair)suggested that such hydrocarbons were of the polycyclic aromatictype.5. W. Cook, I. Hieger, E. L. Kennaway, and W. V. May-neord 38 have prepared and examined the following compounds,35 Ann. Reports, 1931, 28, 226.36 J. 13. Quastel, Biochem. J., 1932, 26, 1G85.3 7 J . Path. Bact., 1924,27, 234; Brit. Med. J., 1925, 2, 1 ; Biochem. J., 1930,38 PTOC. Roy. Xoc., 1932, [ B ] , 111, 455; A., 1166.24, 497; A., 1930, 807POLLARD AND PRYDE. 247composed entirely of condensed benzene rings, with the view ofascertaining their carcinogenic action if any : (I) all the six possible4-ring compounds; (2) all the ten known compounds out of thefifteen possible 5-ring compounds ; (3) some compounds containingsix and eight rings, and others.The work has been extended byJ. W. Of these compounds it is found that 1 : 2 : 5 : 6-dibenzanthracene (I) 4O alone shows carcinogenic power. It retainsthis undiminished even when very highly purified; thus, it hasbeen shown t o be active in nine different media, and has producedcancer when applied to the skin of mice in a concentration of 0.00370in benzene. Less active are its 2’-methyl, 3’-methyl, 9-amino-,9-methoxy, and 9 : 10-dibenzyl derivatives, and phenanthra-ace-naphthene (11). The production of mesoblastic tumours in ratsand mice following the intraperitoneal injection of 1 : 2 : 5 : 6-dibenzanthracene in a fatty medium has been described by H.Burrows.41 The hydrocarbon was dissolved in lard in a concen-tration of O.lyo, and the tumours conformed to the usually acceptedcriteria of malignancy.More active even than 1 : 2 : 5 : 6-dibenzanthracene is 5 : 6-cyclo-penteno-1 : 2-benzanthracene (111), and the carcinogenic activityof 6-isopropyl-1 : 2-benzanthracene (IV) has also been established.Met,astases in the axillary glands and lungs were obtained in fivemice t o which the cyclopenteno-compound was applied.Theevidence so far obtained suggests that a molecular structure cpn-sisting of ring substituents attached to the 1 : 2- and 5 : 6-positionsof the anthracene ring system is particularly efficacious in pro-moting carcinogenic activity.1 : 2-Benzanthracene is itself in-active, and the inactivity of 2’ : 3’-phenanthra-1 : 2-anthracene39 Proc. Roy. SOC., 1932, [El, 111, 485; A., 1156.40 Ann. Reports, 1931, 28, 126. 41 Proc. Roy. SOC., 1932, [B], 111, 238248 BIOCHEMISTRY.(a six-ring compound) and of 4 : 5-benz-10 : 11-(1’ : 2’-naphtha)-chrysene (a seven-ring compound) indicates that the molecularcomplexity must be restricted within fairly narrow limits for themanifest ation of carcinogenic activity.Vitamin A .Up to the present no crystalline preparation of vitamin A, orof a derivative of the vitamin, has been prepared, so that it is stillnot possible to state with certainty that the vitamin has beenobtained in a state of purity.The characteristics of highly activepreparations have been examined by I. M. Heilbron, R. N. Heslop,R. A. Morton, E. T. Webster, J. L. Rea, and J. C. D r ~ m m o n d , ~ ~and, after a comprehensive review of the position, it is concludedthat the most potent preparatioiis obtained by them and by Karrerand his colleag~es,~~ both from mammalian and from fish-liver oils,are qualitatively and quantitatively indistinguishable in respect ofultra-violet absorption. If the products are not homogeneous,then either the non-vitamin material is relatively diactinic, or thepreparations contain substances so closely alike that both exhibitthe 328 mp band. It is pointed out that it would indeed be curiousif exactly the same proportion of material exhibiting negligibleabsorption were present in the products derived from variousspecies of animals.But, on the other hand, the discovery thatthe isomeric a- and @-carotenes may both be transformed in vivointo vitamin A-like substancesu points t o the need for dernon-strating, rather than assuming, strict homogeneity. Heilbron andhis colleagues find their own observations are a t least as consistentwith the formula of Karrer 45 as with any alternative, and regardthe weight of evidence as impressively, though not conclusively,in its favour. It is true that the experimental data on molecularweights46 lead to a va’lue of 320 & 15 as against 286 on the basisof the Karrer formula. This is a small discrepancy, but doubtsconcerning purity are strengthened when the results of a verylarge number of ultimate analyses uniformly indicate values notquite consistent with the C,oH,oO formula.The analysis of hydro-genation products from both crude and distilled vitamin A con-centrates points to the presence of some contaminant in even thepurest preparations. The question is not, however, one of grossimpurity, and it is suggested that there may be present a smallquantity of an alcohol more saturated than vitamin A.4y Biochem. J . , 1932, 26, 1178; A., 1174.43 P. Karrer, R. Morf, and K. Schopp, Ann. Reports, 1931, 28, 221 ; Helv.Chim. Acta, 1931, 14, 1431 ; A., 200.44 Ann. Reports, 1931, 28, 219, 222. 46 Ibid., p. 221. 46 Ibid., p. 221POLLARD AND PRYDE. 249A further communication from Heilbron, Morton, and Webster *'records the extraordinary ease with which vitamin A (concentrate)is transformed into a cyclic derivative in the presence of hydro-chloric acid and alcohol.Dehydrogenation of this product withselenium yielded 1 : 6-dimet'hylnaphthalene. On the basis of theKarrer formula (q.v.) these changes may be formulated as follows :CMe, CH MeI - - - - - - - -Me &H:CHCMe:CH*CH2*OHCyclic product formed from vitamin A. 1 : 6-Dimethylnaphthalene.Similar substituted napht'halenes are obtained from many bicyclicsesquiterpenes by dehydrogenation with sulphur or selenium. Thusthe terpenoid nature of vitamin A is established, and there seemst o be present in the richest concentrates a material which possessesas far as the 14th carbon atom the const,itution advanced by Karrer,Mod, and S ~ h O p p .~ ~Reference was made in the Report of last year to the claim ofH. S. Olcott and D. C. McCann49 regarding the conversion ofcarotene into vitamin A on incubating the hydrocarbon with freshliver tissue in vitro. Further negative results have been recordedby J. L. Rea and J. C. Drumrn~nd,~o and a critical survey, byB. Woolf and T. Moore,51 of the technique upon which the claimwas based leaves little doubt that further proof of the transformationis desirable.An interesting observation regarding the action of antimonytrichloride on carotene has been made by A. E. Gillam, I. M. Heil-bron, R. A. Morton, and J. C. Drumrn0nd.~2 These workers findthat on pouring into water the stable blue solution (A max.583-590 mp) obtained by the interaction of the trichloride and carotene,the organic matter may be recovered free from antimony. Theproperties of the substance recovered agree with those of isocarotene,which is devoid of growth-promoting activity and is derived exclus-ively from the optically inactive @-carotene.= A study of theabsorption spectra of the products of the action of antimony tri-chloride on carotene has led J. R. Edisbury, A. E. Gillam, I. M.4 7 Biochem. J., 1932, 26, 1194; A., 1174.4y Ann. Reports, 1931, 28, 222; A., 97.2. Vitaminforsch., 1932, 1, 177; A., 973.81 Lancet, 1932, 223, 13; A., 1174.52 Biochem. J., 1932, 26, 1174; A., 1174.53 R. Kuhn and E. Lederer, Ber., 1932, 65, [B], 637; A., 782.48 LOC.c i t 250 BIOCHEMISTRY.Heilbron, and R. A. Morton 54 to suggest the partial conversion ofthe vitamin into hydronaphthalene derivatives.Vitamin B,.The identity of the substance, or substances, comprising thecrystalline preparations of vitamin B, still remains in doubt.S. Otake 55 has claimed that his anti-neuritic preparation (oryzanin)from rice bran yields an active hydrochloride, C6H,02N2,HC1,m. p. 250°, resembling Jansen’s crystalline vitamin B,. A. Windausand his collaborators 56 have obtained from yeast a highly activecrystalline anti-neuritic preparation in which they revealed thepresence of sulphur. On the basis of analyses of the crystallinepicrolonate, they tentatively suggested the formula C,,H,,ON,S forthe vitamin. The earlier work of A.G. van Veen 57 led him to sug-gest a formula very similar to that of Otake, namely, C,H,,O,N,,HCI,but further investigation 58 has confirmed the presence of sulphur,and van Veen now suggests as the most probable formula for thehydrochloride, C1,H2,0,N,S,2HC1. The preparation agrees inmelting point, chemical properties, and anti-neuritic activity withthat of Windaus and his colleagues. The present position hasbeen reviewed by R. Tsche~clie,~~ who concludes that the crystallinehydrochloride obtained from the Windaus preparation is identicalwith that of Jansen and Donath, which likewise contains sulphur.The question of the purity of these preparatlions must still remainopen, however, to judge from an examination of the problem byH. W.Kinnersley, J. R. P. O’Brien, and R. A. Peters.6o Theseworkers find that the crystalline hydrochloride, obtained from yeastby the charcoal adsorption methods of Kinnersley and Peters,61contains sulphur which cannot have been introduced during theisolation procedure. The crystalline hydrochloride has been pre-pared with an activity of 2-4 y per diem (pigeon dose). A similarmethod of test showed the Jansen and Donath crystals from rice tohave an activity of 5-8 7 per diem, and it was suggested that thepreparation obtained by Peters and his colleagues was more potentthan that of Windaus.62 This has now been confirmed by directfi4 Riochem. J., 1932, 26, 1164; A,, 1174.5 5 J . Agric. Chem. Soc. Jupun, 1931, 7, 775; A., 657.56 A.Windaus, R. Tschesche, H. Ruhkopf, F. Laquer, and F. Schultz, 2.6 7 Rec. trav. chim., 1932, 51, 265; A., 433.5 8 A. G. van Veen, Z. physiol. Chern., 1932, 208, 125; A., 782.69 Chem.-Ztg., 1932, 56, 1 6 6 ; A., 547.6o J . Physiol., 1932, 76, 17P.O1 “Chemistry a t the Centenary Meeting of the British Association,”E2 A. Windaus, et ul., loc. cit.physiol. Chem., 1932, 204, 123; A., 310.Heffer and Son, Cambridge, 1932, p. 131POLLARD AND PRYDE. 251test,63 the vitamin B, units 64 per milligram being, for the Windauspreparation 260-280, and for the Peters preparation 470. Thetest used was the curative pigeon method. Moreover, Peters andhis colleagues have found it possible to fractionate their crystallinepreparation still further and they conclude that the Windauspreparation cannot be the pure vitamin B,.Vitamin C.The year under review has seen a considerable extension of thechemical problems arising from the investigation of the anti-scorbutic vitamin.At the present time there seem to exist reason-able grounds for the belief that the vitamin has been isolated in apure crystalline state.First, reference must be made to a series of publications byRygh and his collaborators. Results published early in 1932 by0. Rygh, A. Rygh, and P. Laland 65 made the interesting claimthat ethereal extracts of neutralised orange-juice, possessing a highanti-scorbutic activity, yielded a syrup and crystalline narcotine.The latter is inactive against scurvy, but it was claimed that afterirradiation with ultra-violet light it developed anti-scorbuticproperties.It was suggested that this activity developed as aresult of demethylation of the alkaloid, and it was further claimedthat the o-diphenol resulting from this demethylation (methyl-nornarcotine), obtained by heating narcotine with concentratedhydrochloric acid for eight days a t loo", was highly active in pro-tecting guinea-pigs from scurvy. 66 P. Laland G7 stated that narcotinewas present in unripe tomatoes, cabbages, and potatoes, and theunderlying theory seemed to involve the formation of activedemethylated products during ripening. Further investigation ofthis interesting theory by R. L. Grant, S. Smith, and S. S. Zilva,680. Dalmer and T. Moll,69 W. A. Waugh and C. G.King,70 3. Till-mans and P. HirschY7l E. Ott and K. Pa~kendorff,~2 and J. Briigge-mann 73 has failed to corroborate the original claim. A recentpaper by 0. Rygh and A. Rygh '* seems to imply a modification of63 H. W. Kinnersley, J. R. O'Brien, and R. A. Peters, Nature, 1932, 130,774.64 Medical Research Council, Pharm. J., 1932, 129, 5 ; A,, 886. (12 mg.Jaiisen acid clay = 1 pigeon dose.)Z.physio1. Chem., 1932, 204, 105, 114; A., 310.6 6 See also 0. Rygh, Z. Vituminforsch., 1932, 1, 134; A . , 783; A. W. Owe,6 7 Z.physio1. Chem., 1932, 204, 112; A , , 311.c8 Biochem. J., 1932, 26, 1628; J . SOC. Chern. Ind., 1932, 51, 166.69 2. physiol. Chem., 1932, 209, 211; A., 1069.70 J . Biol. Chem., 1932, 97, 325; A., 973.'i2 2. physiol. Chem., 1932, 210, 94.T'idsskr.Kjemi Berg., 1931, 11, 120; A., 201.71 Biochem. Z . , 1932, 250, 312.74 Ibid., p. 275. 73 Ibid., 211, 231252 BIOCHEMISTRY.the original claim in that it is now suggested that the active sub-stance may be a combination of methylnornarcotine with a uronicacid.A much more general acceptance has been accorded to strikingnew results for which A. Szent-Gyorgyi 75 is responsible. In 1928Szent-Gyorgyi, 76 in studying peroxidase systems, isolated fromthe cortex of the suprarenal glands a crystalline substance, CsHsOc,isomeric with the lactone of glucuronic acid, and apparently belong-ing to the uronic acid group, which he referred to simply as hexuronicacid. The substance possessed strong reducing properties and waspresumed to be, on very good evidence, the same substance as thatresponsible for the reducing power of many plant extracts.Thepure compound has been studied by E. L. Hirst and R. J. W.Reynolds 77 and, on the basis of their results, W. N. Haworth 78ascribed to it the constitution of a 6-carboxylic acid of a keto-hexose. A crystalline monoacetone derivative has been described, 79and further chemical investigations by E. G. Cox, E. L. Hirst, andR. J. W. Reynolds 80 have led them to suggest the following tauto-meric structures :CO,H*CO*C( OH):CH*CH( OH)*CH,*OH SCO,H*CO*CO*CH,*CH( OH)*CH,*OHThe double bond in the enolic modification readily accounts forthe strong reducing properties, similar behaviour having beenobserved in an unsaturated trimethyl derivative of glucurone (thelactone of glucuronic acid), prepared synthetically by J.Prydeand R. T. Williams.81 The hexuronic acid of Szent-Gyorgyi doesnot form a lactone, so that, although it is isomeric with the lactoneof a typical hexuronic acid, strictly speaking it does not belong tothis group of compounds. Szent-Gyorgyi and Haworth havesuggested the name “ascorbic acid” in place of the previouslyused “ hexuronic acid.” 81aHexuronic acid can be oxidised reversibly and irreversibly, andit is to the double function of oxidation and reduction in the re-versible change that the acid probably owes its biological activity.The parallelism between the reducing power and the anti-scorbuticpotency of plant extracts has been investigated in a series of well-documented researches by s.s. Zilva and his collaboratorsYs2 andmore recently by J. Tillmans, P. Hirsch, and J. J a c k i s ~ h . ~ ~ The7 B Nature, 1932, 129, 943; A., 886.77 Nature, 1932, 129, 576; A., 648. Ibid., p . 576; A . , 548.79 L. v. Vargha, ibid., 130, 847. Ibid., p. 888.81u Ibid., p . 24.82 Ann. Reports, 1927, 24, 247; 1928, 25, 270.83 2. Unters. Lebensm., 1932, 68, 241, 267, 216; A., 658.76 Biochem. J., 1928, 22, 1387.Ibid., 1933, 131, 67POLLARD AND PRYDE. 253latter workers claim t'o have shown that the vitamin C content andthe reducing capacity are, under many conditions, strictly parallel.Moreover the reducing substance studied by them may be oxidised,like hexuronic acid, in a reversible and an irreversible manner.Biological feeding experiments by J.L. Svirbely and A. Szent-Gyorgyi 84 show that guinea-pigs have been completely protectedfrom scurvy for a period of ninety days by the administration of1 mg. daily of pure hexuronic acid. This claim has been confirmedby W. A. Waugh and C. G. King S5 (who place the dose at 0.5 mg.per day), by 0. Dalmer and T. M011,8~ by S. S. Z i l ~ a , 8 ~ and byL. J. Harris and J. R. M. Innes.88 The protective amount of thepure acid, 0-5 to 1.0 mg. per day, is approximately that of thereducing substance present in the protective dose of plant extracts(e.g., 1 to 1.5 C.C. of lemon juice). Harris and Innes 8s find that1 mg. of hexuronic acid has an activity slightly greater than thatof 1 C.C. of orange juice, and state that the raw suprarenal cortexhas a high anti-scorbutic activity approximately equal to itshexuronic acid content.Hirst and his associates have examinedthe absorption spectrum of de-citrated lemon juice and have estim-ated that the hexuronic acid content corresponds with the recordedevaluation of the anti-scorbutic activity of the isolated acid.This body of evidence seems to afford reasonably conclusiveproof that Szent-Gyorgyi's hexuronic acid is vitamin C, but onemust reserve a final judgment. In particular one would call atten-tion to the fact that, the identity of hexuronic acid and the vitaminbeing assumed, the protective dose is of a considerably higher orderthan that of the other vitamins as far as these are known.Vitamin D.Full details of the properties of the crystalline preparation ofvitamin D obtained by the German workers have been publishedby A.Windaus, 0. Linsert, A. Luttringhaus, and G. W e i d l i ~ h . ~ ~There is general agreement that the product obtained by them isidentical with the calciferol of F. A. Askew, R. B. Bourdillon, et al.,which was described in the Reports of last year.s2 The Germanworkers have also obtained the pyrocalciferol, first described bythe British workers, together with an additive compound of pyro-calciferol and an isomeric alcohol. The structure of vitamin D84 Biochern. J., 1932, 26, 865; A., 886. See also Nature, 1932, 129, 576,690 ; A., 548, 657.86 LOC. cit. S6 2. physiol. Chmn., 1932, 211, 284; A., 1069.Nature, 1932, 129, 943; A., 887.Lancet, 1932,223, 235; A., 1175.89 LOC. cit.9O E. G. Cox, E. L. Hirst, and R. J. W. Iteynolds, Zoc. cit.9 1 Annakn, 1932, 482, 226; A., 311. 92 Ann. Reports, 1931, 28, 215254 BIOCHEMISTRY.now remains to be elucidated and there is little doubt that withthe help of the Rosenheim-King cholane formula, and the conse-quent advances in our knowledge of the chemistry of the sterols,this problem will soon be materially advanced.With regard to the toxicity of large doses of vitamin DYg3 theview that this is an inherent property of the vitamin and is notdue t,o a contaminant is supported by new results of Sir H. H.Dale, A. Marble, and H. P. Marks.94 It is shown that pure calci-ferol has in dogs the same toxic action, in excessive doses, as thecrude product obtained by irradiating ergosterol.The toxic actionis obtained by intravenous injection as well as by oral administration,and the congestion of the alimentary mucosa, produced by suchfatal doses, is equally pronounced with either method of adminis-tration. Of particular interest, in view of the association of theparathyroid gland with calcium metabolism, is the observationthat complete parathyroidectomy does not prevent, or significantlyhinder, tjhe fatal intoxication produced by large doses of calciferol.At most it lowers the level of concentration reached by the bloodcalcium before death. The results, therefore, do not lend supportto the view that vitamin D in excessive doses acts by promotingsecretion of the parathyroid hormone, or by rendering the organismmore responsive to its action.PLANT BIOCHEMISTRY.Mineral Nutrients and the Growth of the Higher Plants.Meclzanism of Nutrient Intake.-Considerable attention continuesto be given to this important but complicated problem, moreespecially from the viewpoint of the mutual influence of ions onthe rate of their absorption by the plant roots.Individual casesof inter-ionic effects are recorded in considerable number, but thesehave tended to emphasise a multiplicity of factors concerned ratherthan to yield data of a constructive nature. It would appear thatmore definite progress is likely to result from the careful applicationof the electro-physical concepts of the nature of electrolyte solutionsand of membrane diffusion to the problems of plant nutrition.Thus, W.Thomas,95 in a very suggestive discussion of the reciprocalaction of nitrogen, phosphorus, and potassium in plant assimilation,illustrates the manner in which conflicting views of several aspectsof ion absorption may be reconciled. The " selective " absorptionof ions and the influence thereon of the hydrogen-ion concentration93 Ann. Repovts, 1931, 28, 218.94 Proc. Roy. SOC., 1932, [R], 111, 522; A., 1176.95 Soil Sci., 1932, 33, 1 ; B., 276POLLARD AND PRYDE. 255of the nutrient solution find explanation on the basis of the Donnanequilibrium and the physical factors controlling the mobility ofions. Diverse views of the physiological balance of nutrients inrelation to Liebig's " Law of Nnimum " and its corollary may beexpressed as variants of the same basic phenomena.In a some-what analogous attempt to link the nutrient absorption of plantswith fundamental physico-chemical phenomena, H. P. Cooper 96observes the close correlation between the proportions of elementsin the ash of plants and their positions in the electromotive series.Since also there is a qualitative relationship between the order ofremoval of cations from soils by electrodialysis and their absorptionby plants, it would seem that considerations of electrode- andionisation-potentials of the elements concerned may afford a basisof explanation of the mechanism of the nutrient intake of plants.The effect of homologous series of ions on the growth of seedlingshas been examined by K.Pir~chle,~' who shows that growth responseis related to the position of the ion in the series and to the structureof the corresponding atoms. The current tendency to ascribe thecontrol of the absorption of minerals to more purely physicalforces and to minimise the effects of inherent physiological activityor adaptability of the plant is shown in a variety of papers. Thus5. Mucco 98 indicates the potential difference between plant leavesand the soil adjacent to the roots to be a controlling factor inthe ratio of absorption of nutrient ions. Pirschle (Zoc. cit.) andF. C. Steward gg show that temperature and light intensity, whileaffecting physiological activity in general, do not alter, to anymarked extent, the relative order of intake of ions by plant cells.Both writers, too, emphasise the part played by carbon dioxide inmaintaining a suitable hydrogen-ion concentration in the nutrientmedium, thus compensating for the unequal penetration of anionsand cations.Nitrogen Assiwdution and Growth.-Diff erences in the responseof plants to ammonium salts and to nitrates again form the basisof much experimental work.Although under conditions prevailingin normal soils the problem seldom arises, it becomes of considerableinterest in the physiological aspect of internal nitrogen economyand metabolism. In many plants examined, the growth responseto ammonium salts is optimal from nutrients having a neutral orslightly alkaline reaction, whereas nitrates are the more effective in96 Soil Sci., 1930, 30, 421; B., 1931, 175; also Proc.2nd. Internat. Cong.97 Jahrb. wiss. Bot., 1930, 72, 335; 1932, 76, 1 ; A., 1931, 174; B., 695.9 8 2. PJlanz. Dung., 1932, 24A, 334; A., 183.99 Protophzsmu, 1932, 15, 29; A,, 664.Soil Sci., 1932, 4, 164256 BIOCHEMISTRY.acid-to-neutral media.l, 293 It is pointed out by V. Tiedgens andW. A. Robbins2 that while ammoniacal nitrogen is absorbed bytomatoes over wide ranges of pH, effective utilisation and elabor-ation into more complex bodies does not occur unless the mediahave pa >7*0. It is possible that the accumulation of ammoniawithin the plant may become toxic in cases where the physiologicaldetoxicating capacity of the plant (W. Ruhland and K. Wetzel*)is exceeded.The injurious effect is attributed by A. B. Beaumontand his colleagues to a disturbance of normal metabolic processes.This view receives some support from observations of the alteredintake of other nutrients occurring when nitrogen is supplied as anammonium salt. ascribes the failure of kugar-cane plants grown with ammonium sulphate to a deficiency ofadsorbed calcium. I n experiments with cotton K. T. Holley,T. A. Pickett, and T. G. Dulin show that the use of ammoniacalnitrogen in culture solutions leads to a much reduced intake ofbases, especially calcium and magnesium.Differences in growth response and composition of plants sup-plied with ammonium salts or with nitrate are recorded by R. M.Addoms and F. C. Mounce * in the case of cranberries.Ammoniumsalts produced much greater runner growth than did nitrates, lowconcentrations of which stimulated, and higher concentrationsrestricted, vegetative growth. Where urea was supplied to sugar-cane plants there was a characteristic increase in the number ofsuckers produced (Purdo, Zoc. cit.).That nitrogen may be assimilated from more complex substancesis shown by Beaumont (Zoc. cit.) in the case of tobacco, which canutilise urea, asparagine, and cystine but not arginine, alanine,glycine, leucine, acetamide, or cyanamide. A. I. Virtanen recordsthat aspartic acid is utilisable by legumes but not by cereals.Potassium and the Growth and Composition of Plants.-The associ-ation of potassium in plants with respiratory activity and carbo-hydrate synthesis is of long standing, but in many instances attemptsto elucidate definite numerical relationships have led to contra-dictory results.The gradual acceptance of the idea of “luxuryconsumption,” i.e., the intake of nutrients in amounts in excess ofThus 5. H. PurdoAnn. Reports, 1931, 28, 241.R. M. Addoms and F. C . Mounce, Plant PhysioZ., 1932, 7, 643.Ann. Reports, 1930, 27, 245.J . Agric. Res., 1931, 43, 559; A., 1932, 101.4th Gong. Internat. SOC. Sugar Cane Tech., 1932, Bull. 13; B., 1932, 1047.Georgia Agric. Exp. Sta. Bull., 1931, No. 169.Plant PhysioZ., 1931, 6, 653; B., 1932, 201.S u o m n Kern., 1932, 5, 67; A., 975.a New Jersey Agric. Exp. Sta. Bull., 1931, No. 536; B., 1932, 567POLLARD AND PRYDE.257physiological requirement, has brought to light one source ofmisunderstanding in the potassium-carbohydrate relationships.This phenomenon, effectively demonstrated by F. Sekera lo in 1928and by other investigators since that time, would appear t o beparticularly marked in the case of potassium. Thus A. E. V.Richardson, H. C. Trumble, and R. E. Shapter l1 observe thatduring the migration of mineral substances from the leaves ofgrasses during maturation, nitrogen and phosphorus accumulate instem bases and roots, but about one quarter of the total potashintake actually returns t o the soil. This is possibly related to thefact that a very large proportion of the total potash content ofplants remains in a soluble condition (G. Janssen and R.P. Bar-tholomew 12) and as a result of rapid translocation, full physiologicalactivity may be maintained by a minimum proportion of potassium.F. J. Richards l3 adds further evidence of the solubility of plantpotash by indicating the probable leaching by rain of potash fromleaves rich in that element but not from those of potash-deficientplants.The function of potassium within the plant is not limited to itseffect on carbohydrate metabolism. Relationships between potass-ium and nitrogen metabolism are indicated not only by the growthresponse of plants to suitably balanced nutrients, but also byexamination of the nitrogen distribution of plants with regulatedpotash supply. This aspect of potash nutrition is discussed byJanssen and Bartholomew (loc.cit.), Richards (Zoc. c i t . ) , and byG. Gassner and G. Goeze.l*The action of potassiuni in increasing the stiffness of cerealstraws is examined by W. Acker,15 who shows by physicalmeasurements that the stability of barley straws increases with thepotash supply t o a maximum value corresponding to a definiteN : P : K ratio. This maximum differs from that associated withthe maximum crop yield. That definite modifications in the struc-ture of the mechanical tissues (especially of the nodes) can beascribed t o variations in potash nutrition is shown by C. Blattnyand V. Vukolov.16Boron and Plant Growth.-Evidence of the necessity of thiselement for the growth of plants continues to be forthcoming.lo 2. Pflanz. Dung., 1928, 7B, 633; B., 1929, 67.l1 J.Counc. Sci. Ind. Res. Australiw, Bull. 66 (1932) ; B., 1096.l2 J. Agric. Res., 1930, 40, 243; B., 1930, 387; also J . Amer. SOC. Agron.,l3 Ann. Bot., 1932, 46, 367; A., 660.lo Ber. &ut. bot. Ges. (Pestschr.), 1932, 50A, 412; A., 890.l5 PfEanzenbau, 1932, 9, 104; B., 1096.16 Erkhr. PJlanze, 1931, 27, 355; A., 1932, 204.1932, 24, 667.REP.-VOL. XXIX. 258 BIOCHEMISTRY.J. S. MacHargue and R. K. Calfeel’ show that in lettuce borondeficiency results in a type of leaf-burn and later in the death oftissues a t the growing point. I n plant metabolism boron cannotbe replaced by any other element. Borax, but not boric acid,stimulates the growth of wheat l8 and of clover,lg red cloverappearing to be more sensitive than the grasses in this respect.Boron injury to wheat occurs in nutrient solutions containingmore than 5 p.p.m.of this element,20 but F. M. Eaton21 recordsthat cotton plants do not produce maximum growth in nutrientscontaining less than the surprisingly high concentration of 10 p.p.m.Boron accumulation is most marked in leaves. Citrus leavesnormally contain 100 p.p.m. of boron, but in trees injured byirrigation with water containing boron this value was increased tomore than 1000 p.p.m.22Calcium and Plant Growth.-The relatively few papers to beconsidered under this heading are concerned with the confirmationor elaboration of existing data rather than with the introduction ofnew subject matter. Calcium deficiency in tomatoes results in achlorosis beginning in the upper stems and leaves, and, in additiont o the characteristic decay of root tips, there is a sloughing of cellsin the upper portions of the root.This is ascribed to the incom-plete development of calcium pectate in the middle lamella.Deficient plants fail to assimilate nitrate and accumulate carbo-hydrates, although sugar translocation and starch digestion appearto be undisturbed. Granular proteinaceous inclusions also appear.The calcium in deficient plants is almost entirely insoluble and itsrate of utilisation so small that normal cellular structure in thetissues cannot be n~aintained.~~ In an examination of calcium-deficient apple trees M. B. Davis a4 records increased shoot growthand production of enlarged leaves in the early stages of deficiency,followed by tissue breakdown later.The total ash content of suchtrees is abnormally low and contains a relatively high percentage ofpotassium and magnesium but low proportions of calcium andphosphorus. W. A. Albrecht and H. Jenny25 show that calciumdeficiency is an important factor in the appearance of “damping1’ Plant Physiol., 1932, 7, 161 ; B., 441.18 H. S. Morris, Bull. Torrey Rot. Club, 1931, 58, 1; A., 1938, 664.l9 R. E. Guilbert and F. R. Pember, Plant Physiol., 1931, 6, 727; B., 1932,Zo H. S. Morris, Zoc. c i t .22 C. S. Scofield aid L. V. Wilcos, U S . Dept. Agric. Tech. Bull., 1931, No.23 G. T. Nightingale, cl al., f’1a)j.l f’hysiol., 1931, 6, 605; A . , 1932, 205.24 J . Pomology, 1930, 8, 316; d., 1931, 273.2G Bot.Gaz., 1931, 92, 263; U., 1932, 200.200.31 Soil Sci., 1932, 34, 301; B., 1129.264; B., 1932, 317POLLARD AND PRYDE. 259off ” o€ soya bean seedlings, and at all ranges between pH 3.8 and6-9 an increased calcium supply in the nutrient reduces the numberof diseased plants. In this respect calcium is a much more activeagent than magnesium or potassium.Intake and Distribution of Mineral Substances during &ow.&-Among a number of investigations on this subject reference shouldbe made to the work of H. Wagner on oats 26 and sugar beet.Z7 Inoats the general decline in the percentage of nitrogen, phosphate,and potassium with growth is temporarily interrupted at the periodof shoot production and again at the blossoming stage.The totalcontent of the nutrients in the stems and leaves reaches a maximumprior to the period of active seed formation and is followed by amovement of nitrogen and phosphorus from stems and leaves andof potassium from leaves only, into the maturing seed. In generalthe .maximum intake of nutrients by stems and leaves precedesthat of organic matter production, but in flowers and seeds the twomaxima are practically simultaneous. In the case of sugar beetthe general decline in percentage nutrient contents of leaves withgrowth was limited largely to nitrogen until the period of rapidsugar accumulation ; the percentage of potassium then decreasedsomewhat and that of calcium considerably. The percentage ofphosphorus did not change appreciably throughout the first yearof growth.The rates of nutrient intake and organic matter pro-duction in the first-year leaves were closely parallel, but in thesecond-year leaves mineral intake preceded the formation of organicmatter. In this respect and also in the translocation of mineralsfrom leaves to seeds the second-year growth of beet resembled thatof oats. Along analogous lines the assimilation and translocationof minerals in the wheat plant are examined by F. Knowles and5. E. Watkin.28 The total intake of nutrients by wheat plantsincreased steadily until after the emergence of the ears. Thesubsequent intake declined in rapidity and the individual elementsattained their maximum values at varying periods prior to harvest-ing, uix., potassium, 7 weeks ; calcium, chlorine, 5 weeks ; nitrogen,3 weeks ; carbon, phosphorus, silica, 2 weeks.The translocationof nutrients from stems to grain ceased one week before harvesting.Indications of a downward movement of potassium, calcium, andchlorine with approaching maturity are recorded. The nitrogencontent of the organic matter of the ear decreased as maturityapproached. An investigation of the soya bean by H. L. Borstand L. E. Thatcher 29 reveals a genera1 similarity in the nutrient2 6 Z. P’anx. Dung., 1932, %A, 48; B., 696.28 J . Agric. Sci., 1931, 21, 612; B., 1932, 38.39 Ohio Agric. Exp. Sta. Bull., 1931, No. 494; A,, 1932, 436.2 7 Ibid., p. 129; B., 811260 BIOCHEMISTRY.intake but with certain characteristic divergences.Thus thegeneral decrease in the percentage of nutrients in the plants withadvancing age is interrupted, in the case of nitrogen by a period ofincrease toward maturity. The mature seed contained more thanone half of the plant’s total nitrogen, a great proportion of potassiumand a smaller proportion of calcium than any other organ of theplant.Plant Saps.-Continued interest in this subject is manifestduring the period under review. Variations in composition resultingfrom diverse methods of extraction have been clarified to someextent by further work of J. D. Sayre and V. H. Morris.30 I nsuccessive fractions of sap obtained from maize by increasingpressure the definitely soluble materials, total- and reducing-sugars, nitrate, and inorganic phosphate remain constant.Otherconstituents which may be presumed to occur, a t least partially, ina colloidal condition, e.g., total solids, total nitrogcn, and phos-phorus, tend to decrease as the pressure rises. It is concluded thatin so far as the constituents in true solution are concerned the sapcomposition is representative of that of the whole plant. The re-action of saps has been examined by K. Biining and E. Boning-SeubertY3l who show that in tobacco the nature of the nutrient doesnot markedly affect the pH of leaf juices but does produce a consider-able effect on the buffer capacity. Correlation between buffer indexcurves of the sap and the composition of the sap are indicated.Calcium-deficient plants show a rather high pB and low buffercapacity in the sap, and vice versa.Deficiency of potassium andphosphate produces a highly buffered sap, and an excess of acidicions in the nutrient a poorly buffered sap. Iron distribution inplants and the p , of the sap appear to be closely related,32 high pHbeing associated with low soluble- and high total-iron contents, aidlow p, with high soluble- and low total-iron contents. The sensi-tiveness of the condition of iron and presumably of its physiologicalfunctions in plants to changes in light intensity is revealed by theobservation that diurnal changes in the pII of the sap due to lightchanges are reflected in variations of the proportion of soluble iron.Translocation of iron occurs principally in the xylem and precipit-ation, which takes place over a wider p,-range than in purelyinorganic systems, is most marked in tissues of high pa lying adjacentto others of low pH.I n plants whose tissues have a low and fairlyuniform pR, precipitation of iron is small and the element remainsevenly distributed throughout all tissues. The parallelism between30 Plant Physiol., 1932, 7, 261; A., 1180.33. Biochem. Z., 1932, 247, 35; A., 786.32 C, H. Rogers and J. W. Shive, Plant Physiol., 1932,7, 227; A., 1181POLLARD AND PRYDE. 261high iron contents and high acidity in pear sap is also recorded byJ. Oserk~wsky,~~ who observes that these conditions are mastmarked in the early part of the growing season, but the period ofhigh acidity is prolonged after the seasonal decrease in iron contentcommences.It is also significant that among trees of the sameorchard there is no appreciable difference in the pH or iron contentof the sap of normal and chlorotic branches. In an examination ofsap from mulberry leaves, Y. Imamura and M. Furuya34 indicatethe nature of the gradient in sap concentration in various celllayers, vix., upper epidermis > lower epidermis > spongy paren-chyma > palisade tissue. The sap concentration generally, increaseswith the age of the leaves up to maturity and subsequently declines.Changes in the sugar content of saps corresponding with alterationsin physiological activity are recorded by a number of workers.R. C. Cole 35 records greater proportions of glucose than of sucrosein stems and leaves of potato plants.Regular diurnal changes tookplace in the glucose concentration, but the sucrose content remainedat approximately the same level throughout the day. The totalsugar content increased with growth, but was not directly affectedby the manurial treatment of the soil. Fertiliser applications,however, definitely influence the mineral constituents of sap, therebeing a general tendency to associate higher sap concentrations withhigher proportions of the respective ions in the soil. A somewhatunexpected instance is, however, recorded by N. A. Pettinger,36working with maize. Pertilisers containing chloride increased thechloride concentration of the sap to a considerable extent, the effectbeing persistent for a long period of time.Sulphate-containingferfilisers, however, caused little, if any, change in the proportion ofsulphate in the sap. An association between the crop-producingpower of vines and the sap concentration of carbohydrate andasparagine a t the period when buds are breaking is shown to existby L. Moreau and E. Vinet,37 and the relationship is confirmed bythe fruit yield increase, following artificial injection of glucose intothe stems immediately prior to the budding period.Changes in, and Distribution of Certain Plant Constituents.Climate and the Nature of Plant Substances.-Reference shouldbe made here to a series of papers by J. B. McNair 3* in which are33 C. H. Rogers and J. W. Shive, Plant Physiol., 1932,7, 253; A., 1180.34 Bull. Sericult. Silk I n d .Japan, 1932, 4, 7; A., 977.35 Soil Sci., 1932, 33, 347 ; B., 697.36 J . Agric. Res., 1932, 44, 919; A., 1181.37 C m p t . rend. Acad. Agric. France, 1932, 18, 193 ; B., 362.38 Amr. J. Bot., 1931,18,416; 1932,19, 168, 255, 518; A., 99, 663, 665;also Science, 1932, 76, 8 3 ; A., 1177262 BIOCHEMISTRY.traced relationships between the chemical composition and, in somecases, physical properties of the principal plant constituents, and theenvironmental conditions of growth. Thus the acid-, alcoholic-,ester-, and hydrocarbon-constituents of volatile oils, saponins, andcarbohydrates from plants occurring in temperate regions have, ingeneral, higher molecular weights than those from tropical plants.Dextrorotatory compounds are more general in the volatile oils oftemperate-region plants, and laevorotatory in tropical plants.Amongsaponins, those of tropical areas are the less toxic. Acetone is morecommon among the products of hydrolysis of cyanogenetic glucosidesof temperate zones, and benzaldehyde among those of tropical plants.Nearly all plants containing cyanogenetic glucosides also containcalcium oxalate depositions, but in temperate plants the trihydratedand in tropical plants the monohydrated crystals predominate.Temperate-plant starches are the more reactive and have widerranges of polarisation values and lower gelatinisation temperaturesand are less " saturated " towards iodine than tropical starches.Essential oils, resins, and calcium oxalate crystals are far morecommonly found in tropical than in temperate plants.The essentialoils and resins occur in most cases in similar anatomic structures andthere is a general tendency for resins to be formed by the condensationor polymerisation of the constituents of the essential oils.Similar relationships are traced among plant waxes and alkaloids,although these are perhaps a little less clearly defined.It is further recorded by S. Ivanov 39 that plant fats from northernareas always contain a larger proportion of unsaturated glyceridesthan those from southern areas, except among those containing oleicacid, in which a climatic influence is not appreciable. Other generalrelationships of a similar nature are shown by the work of N. N.Ivano~.*~Nitrogenous Constituents. -Among investigations of factors in -fluencing the transformation of nitrogen compounds in plantsinterest attaches to that of S.H. E ~ k e r s o n , ~ ~ who traces the effectsof nutrient deficiencies on the reduction of nitrates. Reducaseactivity, while largely influenced by light conditions, practicallyceased in plants deficient in potassium or phosphate. Calcium orsulphate deficiency produced a similar effect but much more slowly.In the case of sulphate deficiency there is a tendency for the main-tenance of a minimum rate of reduction over a period of some weeks.39 Chem. Rund. Mitteleuropa Balkan, 1930, 7, 115; A., 1931, 535; alsoN. N. Ivanov, M. N. Lavrova, and M. P. Gepochko, Bull. appl. Bot. Russia,1931, 25, 1 ; A., 1932, 663.40 Biochem. Z., 1932, 250, 430; A., 1181.dl Contr.Boyce Thompson Inst., 1932, 4, 119; A., 890POLLARD AND PRYDE. 263Regulation of protein metabolism is examined by K. mot he^,*^ whohas isolated from the onion an active substance which under reducingconditions stimulates protease activity and under oxidising condi-tions inhibits it. The material is rich in amino-acid constituents,when reduced responds to the nitroprusside test for the thiolgroup, and is probably glutathione or a closely related substance.T. Schulze43 also records the isolation by a similar process of acystine-like substance which influences proteolytic activity in leavesin a similar manner to the above. The view is also expressed thatthe protein balance in leaves is such as to maintain a characteristic'' stability value " of protein per unit dry weight or per unit surfacearea of leaf.Hydrocyanic acid from cyanogenetic glucosidesactivates protein exchange. Following up earlier investigation^,^^G. Klein and his colleagues 45 have examined the distribution of ureain plants. I n the higher plants a large proportion of urea exists inthe form of ureides (probably of aldehydes), but in many fungi freeurea predominates. In the former urea appears to result from thedestruction of arginine by enzymes. The same author, applyinghis recently developed methods of analysis, examines the distributionof choline in plants, especially in seedlings.46 The choline contentsof fresh seeds is small, leguminous seeds containing more than others.During the germination of maize lecithin- and free-choline (especiallythe latter) increase considerably, the preponderance of lecithin-choline appearing in the cotyledons. Etiolated seedlings have morefree choline and less lecithin-choline than normal.Nitrogen ex-change in seedlings is also examined by P. M c K ~ ~ , ~ ' who observes aclose relationship between the disappearance of insoluble- andprotein-nitrogen during germination of lupin seeds and the increasedproportion of asparagine. Later, as the seedling becomes estab-lished, protein synthesis begins and there occurs a decline in theamount of asparagine and a corresponding increase in proteose.The simpler forms of nitrogen, nitrate, ammonia, amides, and amino-acids appear immediately growth begins and after maintaining amaximum value for a few days decline to a low maintenance level.In germinating soya beans 48 the initial protein decomposition resultsin the production of ammonia and urea.Subsequent changes in the42 Naturwist?., 1932, 20, 103; A., 436.43 Planta [Z. wiss. Biol.], 1932, 16, 116; A., 549.43 Jahrb. wiss. Bot., 1930, 73, 174; A., 1931, 990.4b Ibid., 1931, 74, 429; A., 1932, 313; Biochem. Z., 1931, 241, 413; 1932251, 10; A., 1932, 101, 313, 1179.4 6 G. Klein and H. Linser, Biochem. Z., 1932, 250, 220; A., 1179.4 7 Biochem. J., 1931, 25, 2181; A., 1932, 202.46 W. S . Tao and S . Komatsu, Mem. Coll. Sci. Kyoto, 1931, 14, 287, 293 ;A., 1932, 202264 BIOCHEMISTRY.seedling are due to the action of proteases on glycinin.The ureaseactivity of seedlings is greater than that of the seeds.Seasonal variations in the nitrogen distribution in fruit trees haveagain received attention. A. S. Mulay 49 continues his examinationof Bartlett pear shoots. 50 Among the non-protein nitrogenous con-stituents of current-year shoots, amides and amino-acids in bothwood and bark reach a maximum prior to the commencement ofnew growth. During growth the amide-nitrogen of the wood exceedsthe amino-nitrogen. Basic-nitrogen is low a t mid-summer, butsubsequently rises to attain a maximum some time before newgrowth begins. The insoluble-nitrogen of bark and wood is alsoexamined. In the bark small increases in amide- and humin-nitrogen a t the expense of basic- and residual-nitrogen occur in theearly growing season and later there is a decrease in amino-nitrogenand a corresponding rise in residual-nitrogen as growth proceeds.The principal changes in the wood are shown in a considerable declinein amino-nitrogen and an increase in melanin.Residual nitrogenincreases somewhat after the actual cessation of growth. Similarthough perhaps rather less detailed examinations of apple trees arerecorded by J. T. Sullivan and €1. It. Kraybill 51 and by A. E.Murneek and J. C. Logan.j2 The nitrogen and carbohydrate meta-bolism of celery plants is examined by H. P l a t e n i ~ s . ~ ~ Duringgrowth there is an inverse relationship between the nitrate- andthe amino-nitrogen contents and between the amino- and protein-nitrogen.The proportion of ammonia in the plants was consistentlysmall. Temperature changes had little effect on the rate of amino-acid synthesis. I%elationships between metabolic changes and thcpremature seeding of celery are discussed.Carbohydrates in Plants.-Apart from papers dealing with purelysystematic chemistry, a relatively small number of investigationsfall to be reported here. H. Belval 53a discusses the process of sugarformation in leaves and from a consideration of the wheat plantindicates that in leaves sucrose formation precedes that of the simplehexoses. Sucrose is alsothe first recognisable sugar formed in the banana leaf. Wheat stemsdiffer from those of maize and rice by the presence, along withglucose and fructose, of an alcohol-soluble non-reducing lzevorotatorysugar other than sucrose.Prom the roots of Lycoris squamigeraA fructoside is also formed from sucrose.49 Plant Physiol., 1932, 7, 107, 323; A., 436, 1180.5O Ann. Reports, 1931, 28, 251.61 PTOC. Amer. SOC. Hort. Sci., 1931, 2’7, 220; A., 1932, 99.62 Missouri Agric. Exp. Sta. Res. Bull., 1932, No. 171.53 Cornell Un$v. Agric. Exp. Sta. Mem., 1932, No. 140; A., 1295.5% Bull, SOC. d’Encour., 1931, 130, 605; A . , 1932, 100; see also Chinese J .physiol., 1930, 4, 365; A . , 1031, 130POLLARD AND PRYDE. 265Belval has isolated two fructosides, one of which, Zycoroside,(CaH8,,O4J, [a] - 34", is not hydrolysed by invertase and thesecond 54 has [a] - 19" and is slowly hydrolysed by the enzyme.Carbohydrate changes during seed formation in peas show varietaldifferences in the period of formation of s t a ~ h y o s e .~ ~ In one caseappreciable amounts of sucrose with reducing sugars but no stachyosewere present initially. With the development of starch accumulationreducing sugars disappeared, sucrose decreased in amount, andstachyose appeared only in the later stages. In a second variety theformation of stachyose and of starch occurred simultaneously.Significant differences in the carbohydrate exchange of sterile and ofinoculated soya beans are recorded by E. Ruffer.56 I n the earlystages of growth the greater assimilation by inoculated plantsresulted in relatively greater accumulations of sugars and starch,which were subsequently reduced below the level of sterile plants,presumably as a result of utilisation by the nodule organisms.I nthe Carbohydrate metabolism of Crassulucece, J. Wolf 57 shows thatthe carbohydrate consumption is not exactly balanced by the form-ation of malic acid and carbon dioxide. It is considered that highconcentrations of carbon dioxide in the tissues reduce decompositionby inhibiting the first stage of oxidation (oxalacetic acid formation)and also by causing an increased acetaldehyde accumulation,which in turn restricts the decomposition of a-ketonic acids bycarboxylase. 58Carbohydrate and other changes during the ripening and storage offruits. In the development of apples, starch formation commences(in this country) about, mid- June and after reaching a maximum stage(July-August) the starch content declines steadily until the endof October.In apple tissue there exist a hydrolysable poly-saccharide other than starch or pectin and a polyuronide. Bothsubstances yield arabinose on hydrolysis and are classed as hemi-celluloses.59 Hemicelluloses do not act as reserve carbohydrates,but resemble pectin in structure and function. In peaches, G. T.Nightingale, R. M. Addoms, and M. A. Blake 6o show that pectatesare absent from the flesh cells during ripening and are only to befound in the thick-walled cells adjacent to the epidermis. Proto-pectin, although irregularly distributed, occurs in all cell walls and54 Compt. rend., 1931, 193, 891; A., 1938, 802.55 M. Bride1 and C. Bourdouil, ibid., p. 949; A., 1932, 100.5 6 2.Pflanx. Dung., 1932, HA, 129; A., 1180.57 Planta [Z. wiss. Biol.], 1931, 15, 572; A., 312.58 See also K. Wetzel and W. Ruhland, ibid., p. 567; A., 31%.59 E. M. Widdowson, Ann. Bot., 1932, 46, 597; A., 1070.60 New Jersey A.gric. Exp. Sta. Bulls., 494 and 507; A., 1931, 273.1266 BIOCHEMISTRY,is closely associated with cellulose. In the soft ripe stage there is agradual decline in the protopectin content. The ripening of theJapanese medlar is associated with an accumulation of fructose,sucrose, and malic acid in, and the disappearance of maltose,tartaric acid, and amygdalin from, the pericarp. With advancingripening, dextrin, starch, cellulose, hemicellulose, protein- and non-protein-nitrogen decline steadily in amount. Changes of the reversetype occur simultaneously in the seed, where amygdalin, starch,cellulose, and hemicellulose increase in proportion and maltose dis-appeam61 R.Nuccorini 62 in confirmation of earlier work 63 recordsdifferences in the distribution of organic acids in ripe cherries,peaches, and plums according t o the period of ripening. In general,the proportion of malic acid formed during ripening is smaller, andthat of tartaric acid greater, in warmer than in colder seasons.Characteristics of ripening oranges recorded by P. R. v. d. R.Copeman 64 include a steady increase in total solids and sugars anda decrease in the acids of the juice and also a decrease in the propor-tion of cell-wall material in the pulp. The interesting observation isalso made that spraying with lead arsenate induced a definite de-crease in the acid and a slight decrease in the sucrose (but not reduc-ing sugar) content of the juice and also a significant increase in theproportion of cell-wall material in the pulp.The sugar thataccumulates during the colouring period of fruit is considered byE. W. Allen 65 to consist mainly of sucrose in stone fruits, sucrosepZus reducing sugars in apples, and reducing sugars in pears. Duringthe storage of apples there occurs an inversion of sucrose and hydro-lysis of starch, if any is present. Sucrose inversion and sugaroxidation do not always take place a t parallel rates. Differencesin hexose content thus brought about were shown in fructose but notin glucose, which remained in almost constant proportions.Storagealso involves a steady loss of acid and of insoluble matter. Theextent of the above changes varies somewhat with the period ofpicking. Generally speaking, late gathering results in a low rate oftotal-sugar loss, a high rate of sucrose inversion, a higher level ofconcentration a t which sucrose inversion ceases, and wider variationsin the content of reducing sugars.66 Artificial ripentng induced bytreatment with ethylene appears to follow the normal course inrespect of acid and carbohydrate changes in so far as these have61 ,4. L. Kurssanov, Planta LZ. wiss. Biol.], 1932, 15, 752; A., 435.62 Ann. Chim. Appl., 1932, 22, 10; A., 435.63 Ibid., 1930, 20, 302; A., 1930, 1482.84 Trans.Roy. SOC. S. Africa, 1931, 19, 107; A . , 1931, 882.Hilgardia, 1932, 6, 381 ; A . , 973.ea H. K. Archbold, Ann. Bot., 1932, 46, 407; A., 1070POLLARD AND PRYDE. 267been 68y 699 70 although the changes involved varyconsiderably with conditions of treatment, period of picking, andindividual varieties. Treatment with acetylene 71 has similareffects.Ant7tocya;nirns.-Reference must be made here to the valuable workof Mrs. G. M. Robinson and R. Robinson. Detailed considerationof the synthesis of anthocyanin pigments 72 and the examinationof their distribution in a vast number of plants 73 are beyond thescope of this section of the Report. The simplest type of antho-cyanin is represented by chrysanthemin (cyanidin 3-monoglucoside),which forms the basis of consideration for the structure of morecomplex types.Pelargonin, peonin, cyanin, and malvin conformt o a 3 : 5-di-monoside type and are not classed as biosides. Among3-biosides are included mecocyanin, prunicyanin, etc. No evidenceof the existence of 5-monosides. has been obtained.Colour variations, especially among varieties of the same speciesof plants, are not ascribed to variations in the pB of saps but dependupon differences in the nature of " co-pigments," i.e., substancesforming weak addition compounds with the anthocyanin andeffecting a modification of colour. Such additive compounds usuallydissociate at high temperatures or may be separated by partitionbetween solvents. Co-pigmentation appears to be most marked innatural pigments containing anthoxanthins. Commonly occurringco-pigments include tannin, flavone, and flavonol glycosides. Theexistence of tannin complexes is also recorded by A.G~illiermond,~~who indicates the oxyflavonols as precursors of anthocyanins inflowers of Iris germanim. The rate of formation of anthocyaninpigment in plants appears to vary with the growth rate, the photo-synthetic activity, and the accumulation of nutrient substance^.^^In green fruit during chlorophyll assimilation the formation ofanthocyanin is retarded owing to the production of anthocyanidinsfrom the corresponding oxyflavones. In picked unripe fruit,reduction processes prevail and flavone compounds are converted67 W. B. Davis andC. G. Church, J . Agric. Res., 1931, 42, 165; A., 1931,774.E.H. Kohman, l n d . Eng. Chem., 1931, 23, 1112; B., 1932, 79.69 F. W. Allen, Zoc. cit.70 H. S. Wolfe, Bot. Qax., 1931, 92, 337.R. Hartshorn, Plant Physiol., 1931, 6, 467; B., 1931, 992.7' J., 1931, 2665-2742; 1932, 2221, 2293, 2299, 2488; A., 1931, 1423;1932, 1038, 1140; A. Lebn, R. Robinson, et al., Anal. 2%. Quim., 1932, 30,267 ; A., 859.73 Biochem. J . , 1931, 25, 1687; 1932, 26, 167; A., 1932, 101, 1296.'4 Compt. rend., 1931, 192, 1581 ; 193, 112; A., 1931, 1099.7 5 H. Kosaka, J . Dept. Agric. Kyushu, 1931, 3, 29, 99; A., 1931, 660;1032, 101268 BIOCHEMISTBl*.into antho~yanins.~6 I n wheat and rye, but riot in oats or barley,the anthocyanin colour is of sufficient intensity t o be utilised as anindication of quality."Certain Plant Enzymes and their Artijkial Activation.-Theassociation of certain growth characteristics in plants with enzymeactivity has become very general in recent years.Investigations ofcatalase activity are prominent, and in general indicate that thisenzyme is most markedly active a t the end of a period of dormancyor at the initiation of certain definite stages in growth, e.g., duringseed germination, sprouting of tubers, breaking of buds in fruit ant1other t r e e ~ . ~ ~ a Thus M. N. Pope 78 records maximum periods ofactivity in barley during early germination, during development ofcrown roots, and during early jointing.A. Niethammer 79 correlates germinative capacity in seeds withtheir catalase content. Frost resistance in wheat plants appears t obe related to the catalase activity of the leaf and in partiallychlorotic leaves variations in catalase and in chlorophyll contentsare closely parallel.81 This association of functional with enzymicactivity in plants is commonly reported in the case of amylase andother enzymes and much interest centres round this aspect of thcartificial stimulation of growth processes and especially the intcr-ruption of the normal dormant period.Thc obvious horticulturalvalue of such practice has doubtless been an incentive to numerousinvestigators. Probably the most widely examined activator isethylene chlorohydrin. Potato tubers dipped in a 5% solution ofthis substance show a much accelerated sprouting and an increasednumber of sprouting eyes.This effect is greatest in least mature" seed." Subsequent growth, however, is greatest when well-matured tubers are treated.82 The action of the activator decrease::as the period of treatment approaches the end of normal dormancy.Similarly, ethylene chlorohydrin vapour breaks the dormancy ofmaple and chestnut seedlings *3 and of seeds of maples andTreatment with certain sulphur-containing substances, e.g., thio-J. C. Politis, Praktika, 1928, 3, 440; A., 1933, 312.7 7 G. Gassner and TV, Straib, Pjlanzenbau, 1930, 4, 169; R., 1931, 507.S. Manskaja and M. Schilina, Biochem. Z., 1931, 240, 276; A., 1932, 99.J . Agric. Res., 1932, 44, 343; A., 784.R. Newton and W. R. Brown, Canadian J . Res., 1931, 5, 333; A,, 1931,81 H.von Euler, et al., Arkiv Kemi, Min. Geol., 1931, lOB, No. 13; A . ,82 H. 0. Werner, Nebraska Agric. Exp. Sta. Bull., 1931, KO. 67; B., 1932,83 W. C. Bramble, Science, 1932, 75, 193; B., 396.79 2. Pj-lanx. Dung., 1931, 21A, 69; B., 1931, 776.1465.1931, 1102.318.C. G . Deubner, ibid., 1931, 73, 320; E., 1931, 857POLLARD AND PRYDE. 269semicarbazicle, t8hioglycollic mid, methyl disulphide, t hioglpcol,and derivatives of thiocarbamic acid, is shown by L. P. Miller *5 t ohave a like effect on potato tubers. This stimulation of dormantmaterial to active growth is associated in nearly all cases with asimultaneous increase in enzyme activity notably of catalase oramylase or both. Subsequently, numerous investigators havesought to decide whether enzyme activity is caused directly by theactivator, or whether it is the indirect result of the action of theactivator on other material, or whether the enzyme is actually anecessary intermediary between activator and growth response.Workers from the Boyce Thompson Institute have contributed anumber of papers on this point.J. D. Guthrie8& shows that,among a number of activators examined, those inducing successfulsprouting effects on potatoes either contained sulphur, or producedan increased proportion of thiol derivatives in the tuber as a, resultof an increased pR or reducing power in the potato juice. Stimul-ative effects, however, are not closely correlated with changes incntalase or peroxidase activity nor with the pH or reducing power ofthe juices.In a similar examination of gladiolus corms it is shownE6that treatment with ethylene chlorohydrin increases the catalaseand peroxidase activity and the thiol content of the expressed juiceand of aqueous extracts of dried tissue. F. E. Denny 87 observesthat sprouting response, while closely related to the increased amylaseactivity of the potato, is not directly dependent on the activationof the enzyme by ethylene chlorohydrin or thiocyanates. Later,5. D. Guthrie 88 reports the isolation of glutathione from theexpressed juice of tubers treated with ethylene chlorohydrin but notfrom untreated tubers. The presence of glutathione in plant organsduring active vegetative growth is also recorded in an examinationof Obelia by E.J. L ~ n d , ~ ~ who reports maximum accumulations inthe active tips of stems and roots. The distribution of glutathione inthe plant is paralleled by variations in oxygen consumption, carbondioxide production, methylene-blue reduction, and electric potential.L. Binet and J. MagrouW also regard the glutathione content ofplants as a function of rapidity of growth. The direct activation ofisolated amylase by various sulphur compounds is examined byK,. H. Clark, F. L. Fowler, and P. T. Black.s1 Potassium thio-8 5 Contr. Boyce Thompson Inst., 1931, 3, 309; B., 1931, 85s.85a Ibid., p. 499; A., 1932, 201.8 6 J. D. Guthrie, F. E. Denny, and L. P. Miller, ibid., 1932, 4, 131 ; A., 889,8 7 Ibid., p. 53; A., 661.88 J. Amr. Chern. SOC., 1932, 54, 2566; A., 889.89 Protoplasma, 1931, 13, 236; A , , 1932, 98.90 Compt.rend., 1931, 192, 1415; A., 1931, 989.91 Trans. Roy. SOC. Canada, 1931, [iii], 25,111, 09; A . , 1932, 497270 BIOCHEMISTRY,cyanate, thiourea, and also ethylene chlorohydrin stimulate theaction of malt diastase on starch, each activator exhibiting a specificoptimum concentration giving maximum activity. H. Pringsheim,H. Borchardt, and H. Hupferg2 show that glutathione activatesboth yeast and pancreatic amylases. Dithioglycollic acid, however,is without effect. Reduced glutathione resembles cystine in itsactivating action on proteolytic enz~mes,~3 and it would appear thatthe activity of plant [and animal] cells is largely dependent on theS*S =+= S*H equilibrium. Thus, while fairly definite knowledgehas been obtained of the several factors involved in the activationof dormant plant tissue, the exact position of the enzymes as acausal or resultant factor in vegetative stimulation requires stillfurther elucidation.Growth and Metabolism of Moulds.Nutritional Factors.-The alleged parallelism between the pro -duction of mycelial tissue by Aspergillus niger and the supply ofcertain mineral nutrients, together with the utilisation of thisorganism as an indicator of the nutrient value of soils,94 has lead toa number of investigations of the effect of inorganic materials onthe growth of the mould.I n soil analysis the many externalinfluences on growth are stabilised as far as possible by the use ofmedia containing 1% citric acid and any calcium carbonate in thesoil is previously neutralised with citric acid.Extensive changesin the px of the nutrient are thus prevented. The proportion ofsoluble calcium in the nutrient nevertheless influences the growthof the organism 95 irrespective of the supply of potassium, andhighly calcareous soils yield excessively high values for availablepotassium. Mycelium of A . niger, grown under conditions whichmake the supply of potassium a limiting factor, contains 0.120/,(dry weight) of K,O and production is closely proportional to theamount of this element a~ailable.9~ Correlation between mycelialgrowth and the phosphate supply is less close. This observationmay be related, however, to the fact that A. niger is able to utilise,in addition to alkali and soluble calcium phosphates, the insolublephosphates of iron andof calcium and also the phosphorus of ~ h y t i n .~ '92 Biochem. Z., 1932, 250, 109; A., 1063; also Naturwiss., 1932, 20, 64;A., 304.93 W. Grassmann, et al., Z. physiol. Chem., 1931, 194, 124; A . , 1931, 393.94 H. Niklas, H. Poschenrieder, and J. Trischler, Z. Pflanz. Diing., 1930,g5 H. Niklas, G. Vilsmeyer, and H. Poschenrieder, ibid., 1932, 24A, 167;9 G J . Trischler, Wiss. Arch. Landw., 1931, 7, 39; B., 1932, 617.9 7 T. Simakova aid C . Bovschik, Z . Pflanz. Diing., 1932, 24A, 341; B.,18A, 129; B., 1931, 37.B., 566.74327 I POLLARD AND PRYDE.That mycelium production is also directly related to the mag-nesium supply is indicated recently.g8 The effect of heavy-metalsalts on the growth of fungi, with its obvious bearing on industrialfermentation industries, has again been the subject of considerableinvestigation.J, Talts 99 has examined the acidity produced inmedia by the growth of P. gluucum and distinguishes two phases,v k . , an initial stage characterised by rapid tissue production and amarked increase in hydrogen-ion concentration, followed by asecond stage in which the growth rate is smaller and aciditydiminishes. Salts of zinc, cobalt, nickel, and cadmium in 0.005M-solutions markedly affect the pDH changes in the medium and aretoxic in the order Ni<Zn<Co<Cd. Toxicity is ascribed to aretardation of absorption of nufrients rather t,han to coagulation ofthe colloids of the plasma, and is associated with reduced sporegermination.The effect c,f zinc sulphate on the growth of A . nigeris also examined by K. H . Stehle.9ga Stimulation by small con-centrations of zinc occurs only when the sugar in the nutrient isabove a definite proportion and does not appear if sta,rch or inulinis supplied instead of sugar. The addition of a colloid (agar-agar)prevents the stimulative action of small concentrations of zinc andprotects the fungus from the toxic effect,s of larger ones. Accordingto R. A. Steinberg zinc is to be regarded as an essential nutrientfor AspergiZEus rather than as a stimulant. The apparently irregularaction of iron in the culture of fungi is emphasised and discussedby A. Quilico and A.Di Capua,2 who record the isolation of twostrains of A . niger, one of which gave hardly any citric acid in thepresence of traces of iron, and a second which produced citric acidin proportion to the amount of ferric chloride supplied. Thepresence of cellulosic material also influences the growth of thisorganism, additions to the medium of filter paper, washed peat,powdered barley roots, etc., markedly increasing the amount ofmycelium pr~duced.~Metabolism and Acid Production.-Gaseous exchange in A . oryxceis examined by H. T a m i ~ a , ~ who records the view that the respir-atory quotient is greater or less than the combustion quotient ofthe substrate according to whether the latter is greater or less than98 H. Niklas, H. Poschenrieder, and A.Frey, ErmGr. Pflanie, 1931, 27,99 Protoplasma, 1932, 15, 188; A., 968.99a Bull. Torrey Eot. Club, 1932, 59, 191 ; A., 11GS.1 Zentr. Bakt. Par., 1932, 11, 86, 139; A . , 1168.2 Giorn. Chim. i n d . appl., 1932, 14, 289; A., 968.3 L. E. Kiessling, %. Pjlanz. Diing., 1931, 21A, 86; B., 1931, 774;4 Acta Phytochint., 1932, 6, 227, 265; A., 1167.465; B., 1932, 276.Pjanzenbau, 1932,9,293; A., 1259272 RIOCHENlTSTRY .0.1375 (the combustion quotient of t’he mould constituents taken asa whole). Energy resulting from respiration during vital synthesisappears to be utilised for the maintenance of enzymic and struc-tural energy, balancing heat exchanges in various stages of synthesis,and for activation of the substrate for the acceleration of syntheticreactions.Following up earlier work, the same author records themanner of utilisation of a large number of organic substances forgrowth and/or respiratory purposes, and associates certain specificatomic groupings with their utilisation by the mould.5 It wouldappear that the presence of more than one specific grouping isnecessary for growth. Typical “ chief radicals ” include the groups*CHMe(OH), :CH*C(OM):, and -CH(OH)*CH,*. These must bejoined a t least once with a “ residual radical ” such as will preventp-degradation and fission of the “chief radical.” Fission of di-and poly-saccharides and of glucosidcs precedes their utilisation.The utilisation, as sole source of carbon, of the higher paraffins bya strain of A . versicolor is recorded by S.J. Hopkins and A. C.Chibnall.G Both odd- and cvcn-paraffins up to C,,H,, are attacked,and also higher ketones hut not secondary alcohols. Contrary to theview of W. 0. T a u s s ~ n , ~ the first products of the action on paraffinsappear to be ketones, which gives rise subsequently to shortcr-chain fatty acids. I<. Rernhauer and his colleagues,s in a con-tinuation of their investigations of the various acids produced byA . niger, show that glycollic acid produced from acetates is rapidlyreplaced in cultures by glyoxylic acid as the action proceeds. Bothacids may be converted into oxalic acid. Quantitative examinationof the process in comparison with the conversion of succinates intooxalic acid leads to the view that acetic acid is transformed intooxalic acid by way of succinic rather than through glycollic acid.I n the conversion of ethyl alcohol into citric acid by A .niger thesimultaneous production of oxalic, malic, and tartaric acids is alsore~orded.~ The yield of citric acid is greater and that of oxalicacid smaller than the corresponding amounts formed from acetates.According to T. Chrzaszcz, D. Tiukov, and M. Zakomorny lo the con-version of alcohol into citric acid by PeniciZZium takes place throughthe stages, alcohol + acetic -+ glycollic + Z-malic -+citric acids. The extent of the side reaction, glycollic --+ oxalicacid, varies with the strain of organism used and with the natureof the substrate. In another paper dealing with this point T.Acta Phytochim. 1032, 6, 129; A., 651.Biochem. J., 1932, 26, 133; A., 653.Biochem. Z., 1928, 193, 85; A., 1928, 447.K. Bernhauer and Z. Scheuer, ibid., 1932,253,ll; A., 1168.‘3 K. Bemhauer and N. Bockl, ibid., p. 25; A., 1168.lo lbid., 250, 254; A., 1065POLLARD AND PRYDE. 273Chrzaszcz and D. Tiukov l1 show that for maximum accumulationof citric acid there is an optimum concentration of nitrogen in themedium which depends on the form of the nitrogen. For inorganicforms of nitrogen the optimum is lower than for organic forms.Further, some moulds produce citric acid by deamination withoutthe production of oxalic acid. Considerable yields of acetaldehydeproduced from sucrose by A . niger in media containing sodiumsulphite are recorded by K. Bernhauer and H. Thelen.12 It issignificant that neither citric nor oxalic acid is formed under theseconditions, but both are produced in the absence of sodium sulphite.The formation of itaconic acid by a new organism, A . itaconicus,grown on a sucrose-potassium nitrate medium is of interest. Thechange probably occurs in the order, sugar --+ gluconic acid +citric acid --+ aconitic acid + itaconic acid. The same organism,grown on sucrose-ammonium nitrate, produces 1-mannitol. Fruct-ose (but not glucose) undergoes similar changes.l3Among benzene derivatives obtained by the action of moulds onglucose must be mentioned puberulic acid, C8H606, m. p. 316"(decomp.), probably a dihydroxybenzenedicarboxylic acid, and anacid, C8H406, m. p. 296" (decomp.), not characterised. The acidsare produced by Penicillium puberulum, Bainier, and P. aurantio-virens, Biourge, when supplied with glucose as the sole source oicarbon.14 The course of the degradation, quinic acid --+ proto-catechuic acid -+ pyrocatechol -+ oxalic acid by A . niger isexamined by K. Bernhauer and H. H. Waelsch,15 who demonstratethe disappearance of pyrocatechol on the 6th day of the culture,but the appearance of oxalic acid is not shown until the 9--10thday. Oxalic acid is similarly produced from inositol, gallic, andsalicylic acids. Oxalic acid appears to be the only acid producedby A . niger from potassium salts of d-glycuronic, a-d-galacturonic,and tetragalacturonic acids.16 An extensive survey of the pro-duction of a number of organic acids by modern fermentationprocesses is given by 0. E. May and H. T. Herrick.17 The utilis-ation of olive oii by A . fravus and P. sylvaticzcm is reported byR. S. Katznelson l8 and of linseed and walnut oils by H. Oeffner.19In the latter instances evidence of the formation of y- andl1 K. Bernhauer and S. Biickl, Bioclmn. Z., 1931, 242, 137; +4., 1932, 93.l2 Ibid., 1932, 253, 30; A., 1168.l3 I<. Kinoshita, Acta Phytockim., 1931, 5, 271 ; A., 1932, 93.lC J. H. Birkinshaw and H. Raistrick, Biochem. J., 1932, 26, 441 ; rl., 651.Biochem. Z., 1932, 249, 223; A., 882.E. Hoffman, ibid., 1931, 243, 423; A., 196.17 Chem. News, 1932, 145, 81 ; A., 968.18 Arch. Sci. biol., Russia, 1931, 31, 385; A . , 1932, 1168.l9 Bot. Archiv, 1931, 33, 172; A., 1932, 93274 BIOCHEMISTRY,6-hydroxy-acids and their lactones is given. Intermediate productsinclude certain unsaturated compounds.Examination of fungal mycelium reveals differences in carbo-hydrate composition which are related to the nature of the nutrient.Thus on a glucose substrate A . niger contains a high proportion oftrehalose and a small amount of mannitol. On media containinginvert sugar or fructose the proportions are reversed.30 hlnnnitolis also present in the mycelium of A . $fischeri and A . oryxce. Theproduction of ergosterol by these organisms 21 and by Y. puberulum 22is also recorded.A. G. POLLARDJ. PRYDE?* Obaton, Conapt. Tend. SOC. Biol., 1930, 105, 6’13; A., 1932, 651.21 L. M. Pruess, W. H. Peterson, and E. B. Fred, J . Biol. Chefn., 1932, 97,22 J. H. Birkiiishaw, R. K. Callow. and C. F. Fischmann, B;orhcin. J . , 1931,483; A,, 1065.25, 1977; A ., 1932, 185