BIOCHEMISTRY.A SECTION has been devoted this year to certain aspects of thebiochemistry of bacteria. The extreme diversity of the chemicalactivity of these organisms renders impossible any approach to acomprehensive review of recent investigations. The more practicalaspects of bacteriology in relation to industry, pathology, agri-culture, sanitation, etc., receive attention elsewhere. It hasseemed more appropriate to consider certain selected topics, whichby reason of their fundamental or theoretical character are suit-able for inclusion here. Moreover, since no reference to bacteriahas appeared for some time in these Reports, the period underconsideration has been extended to cover the last three or fouryears. A short section on algze has similarly been introduced.In the section dealing with animal biochemistry the subjectsreviewed this year are the highly important new fermentationschemes of Embden and Meyerhof, deaminisation by survivingtissues, metabolic aspects of methionine, vitamin A , vitamin C(ascorbic acid), the physiology of vitamin B,, carcinogenesis bypure hydrocarbons, synthetic estrogenic substances, the cestringroup, and the new group of the Jlavin pigments.The morechemical aspects of the subjects italicised are dealt with in thealiphatic division of the organic chemistry report.PLANT BIOCHEMISTRY.Biochemistry of Certain Bacteria.The Nitrogen Assimilation of Azotobacter.-The relationshipbetween the nature of the nitrogen assimilation process and thegrowth of these important organisms has presented a fascinatingproblem to both the bacteriologist and the agriculturalist.It haslong been recognised that in the presence of nitrates the fixationof free nitrogen by Axotobacter is retarded or entirely prevented.The more precise information of the detail of this effect which hasbecome available during the last two or three years is to a largeextent due to the work of D. Burk and H. Lineweaver. Theirearlier investigations showed that the addition of small but increas-J . Bact., 1930,19, 389; A., 1930, 1219306 BIOCHEMISTRY.ing amounts of combined nitrogen to otherwise nitrogen-free mediarapidly reduced the amount of elementary nitrogen fixed. Fixationceased altogether when the medium contained 0.6 mg. of availableN per C.C.The growth and respiration rates of the organism alsoincreased rapidly with t,he supply of combined-nitrogen, reaching amaximum with 0.5-1.0 mg. of N per C.C. and declining slowly asthis value was exceeded. As far as could be ascertained, the principalphysiological functions of the bacteria were unaltered whether theassimilated nitrogen was free or combined. The nitrogen contentof the dry matter of the cells was likewise unaffected.It would appear that for normal metabolic processes Axotobacterutilise combined nitrogen if available and will resort to fixationonly when the alternative source fails. This view is confirmed,and more light thrown on the relative assimilability of variousnitrogenous compounds by J. E. Fuller and L.F. Rettger.2 Theeffect of a range of nitrogenous compounds varying in complexityfrom simple nitrates and ammonium salts to simple amides andamino-acids (urea, glycine, aspartic and glutamic acids) and morecomplex compounds such as tyrosine, nucleic acid, etc., on nitrogenfixation by, and the reproduction of, Axotobacter was comparedwith those in cultures from which all combined nitrogen was ex-cluded. I n general, the simpler compounds were the more easilyassimilated, produced the greater growth increases, and, at thesame time, caused the heavier reduction in the amount of freenitrogen fixed. Similar effects were observed by L. G. Th~mpson.~The clear differentiation between the course of N fixation and thegrowth process in Axotobacter is further emphasised in the nutri-tional requirements.Thus in media containing the customaryminerals (K, Mg, PO4”’, SOa”, etc.) and combined nitrogen littlecalcium seems necessary for optimum growth and normal meta-bolism.* During the fixation process calcium is necessary inconsiderable amount and becomes an important factor controllingthe amount of nitrogen fixed.5 The interesting point is broughtout by this investigation that strontium, but no other element,can replace calcium in this f~nction.~,The first product of nitrogen fixation so far identified is ammonia,and this appears only when an adequate energy (carbohydrate)source is available. If the organism is supplied with combinedforms of nitrogen, these are decomposed with the production ofSoil Sci., 1931, 31, 219; B., 1931, 557.J.Agric. Ites., 1932, 45, 149; A., 1932, 1169.I). Burk and H. Lineweaver, Arch. Mikrobiol., 1931, 2, 155; A , , 1931,D. Burk, Proc. 2nd Internat. C’ong. Soil Sci., 1932, 3, 67; A., 96.4 M. Schrtider, Zentr. Bakt. Par., 1932, 11, 85, 177; A., 1932, 306.1334POLLARD AND PRYDE. 307ammonia, which is then utilised. The deamination processapparently occurs only in the absence of carbohydrates (S. P.Kostytschev and A. Schelo~mov).~The examination of the effects of varying partial and totalpressures of oxygen and nitrogen on the fixation process, by Burk,8discussed in an earlier Report,g initiated a series of investigationsof the mechanism of the stimulatory action of humus, and later,of other substances on the activities of Axotobacter. In experimentson the respiratory exchange, K.IwasakalO showed that the re-productive process is associated with a steady increase in therespiratory rate, whereas the fixation process takes place withconstant respiratory conditions. A low oxygen pressure favoursfixation and inhibits growth, and respiratory activity. Humicmatter increases the respiration rate. The fixation of nitrogen isalso accelerated by molybdenum salts (Na,MoO,),ll, l2 but themechanism of the stimulation differs from that produced by soilextracts. Both materials increase the proportion of nitrogenfixed per unit of sugar consumed, but neither affects the ratio,nitrate assimilated : sugar consumed. Soil extract acceleratesthe growth rate of the organism, and in this respect, is effectivewhether free or combined nitrogen is being assimilated.Theaction of the molybdate, however, is confined to increasing theefficiency of the fixation process. That the growth-acceleratingeffect is a function of some organic constituent of soils is confirmedby the observation that the ash of soil extracts acts in a preciselysimilar manner to, and with approximately the same intensity as,the sodium molybdate. The activity of the latter apparentlydiffers from that of uranium (and in some cases of thorium), which,as previously reported by K. Hirai,13 stimulates both nitrogenfixation and growth. The action of humus is stated by J. Voicuand E. Lungulescu l4 to depend on its influence over the utilis-ation of energy materials by Axotobacter as well as on the actualfixation process.Thus, small amounts of humus accelerate theoxidation of sucrose but retard that of glucose. Larger proportionsin general retard oxidation to extents which vary with the natureof the carbohydrate present.7 2. physiol. Chem., 1931,198,105; A., 1931,986; Bull. Rcad.Sci. U.R.S.S.,8 J . Physical Chem., 1930, 34, 1174; A., 1930, 1068.S Ann. Reports, 1930, 242.10 Biochem. Z., 1930, 226, 32; A., 1930, 1622.11 H . Bortels, Arch. Mikrobiol., 1930, 1, 333; A., 1931, 1095.12 L. Birch-Hirschfeld, ibid., 1932, 3, 341 ; A., 1932, 1169.13 Proc. 1st Internat. Cong. Soil Sci., 1928, 3, 154..1 4 Bul. ChinL. SOC. Rodney 1930, 12, 71, 82; A . , 1930, 1478.1931, 661 ; A., 1931, 1459308 BIOCHEMISTRY.In a further investigation, Burk, Lineweaver, and G.K. Horner15associate the stimulatory action of humus with its iron constituents,since artificial iron-free humus is without action. Moreover theiron salts of certain organic acids (citrate, tartrate, malate) and, to aless extent, ferric sulphate may produce similar effects, the order ofefficiency being humus > organic salts of iron > ferric sulphate.Under cultural conditions such that natural humus fails to stimulate,the iron salts are also ineffective. It is indicated that the mainten-ance of an appropriate proportion of soluble iron in the medium is animportant factor in the production of stimulative effects.No confirmation was obtained by these workers of theories, fre-quently expressed, that the effect of humus results from its supposedaction in increasing the availability of nutrient materials, in detoxi-cating metabolic producls, or in improving the physical conditionof the substrate.Burk’s “ iron ” theory falls into line with observ-ations of many early workers and with that of J. Olsen,16 whoascribed the activity of Bottomley’s “ Bacterised peat ” to thepresence of iron compounds rather than to that of auximones.Bacterial Decomposition of Xugars and their Derivatives.-Thefermentation of various sugars has long formed the basis of testsused in characterising bacterial species. The consideration offermentability by bacteria as a property of sugars which might becorrelated with molecular structure is less commonly discussed.Bacteria as a class exhibit a specificity in this respect which is welldefined with regard t o individual sugars, but less easy to elucidatein reference to groups of sugars having common structural charac-teristics.An interesting variation of this theme is provided by thework of A. I. Kendall and C . E. Gross l7 in which the relative ease offermentability of sugars is compared with that of their correspondingsimple derivatives. Thus, while glucose is in general more readilyfermented than mannose, the reduction products sorbitol andmannitol show the reverse order of fermentability. Oxidation ofglucose to gluconic acid does not greatly restrict its utilisation bybacteria, whereas mannonic acid is attacked by relatively few organ-isms.The simple glucosides were rarely utilised, @-methylglucosidebeing more generally attacked than a-methylglucoside. a-Methyl-mannoside was very resistant. Alkylated sugars such as themethyl and isopropylidene derivatives of glucose, fructose andmannose, tetramethyl mannose, and y-trimethyl xylose were rarely,if at all, acted on by bacteria. Investigations of a somewhatsimilar character are more recently recorded by S. A. Koser andl6 Cornpt. rend. Trav. Lab. Carlsberg, 1930, 18, 1 ; B., 1930, 434.l7 J. Infect. Dis., 1930, 47, 249; A., 1931, 264.1 5 soil s~i., 1932,33,413, 455; B., 1932, 782POLLARD AND PRYDE. 309F. Saunders.l* A series of simple derivatives, selected as retainingthe essential spatial arrangement of atoms, were subjected to theaction of a series of bacterial species, and, incidentally, of two yeasts.Here again the refractory character of the glucosides is shown,d- a-met hylmannoside, Z- p- met hylarabinoside, and d - p - met hyl-xyloside being unattacked by any of the organisms which fermentedthe corresponding simple sugars, and a-methylglucoside beingfermented only in a few instances.On the other hand the intro-duction of the methyl group into the direct carbon chain as inrhamnose and fucose did not markedly reduce fermentability. Ofthe heptosa, a-glucoheptose and a-glucoheptulose were not utilisedby any of the bacteria examined. The hexose, sorbose, having asimilar spatial arrangement to a-glucoheptulose, was fermented onlyby a few species.Glucosamine and gluconic acid were attacked bythe majority of glucose-fermenting organisms, though not alwayswith equal readiness. It is significant that neither compound wasacted on by the yeasts. The sulphur derivative, glucose ethylmercaptal, and the acetyl derivatives of glucose and xylose provedunfermentable. The relative ease of fermentation of glucosamine isalso recorded by F. Lieben and L. LOwe,lg and the resistant characterof several glucosides towards certain organisms which attack thesimple sugars by S. Forssman.20 I n another paper, S. A. Koser andF. Saunders 21 show that a number of organisms ferment Z-arabinosemore readily than the d-form, although in a few cases the d- but notthe Z-form was utilisable.It is generally indicated that the spatialarrangement of atoms or the length of the carbon chain is by nomeans always a controlling factor in the fermentability of sugars bybacteria, even when the activities of a single species only are con-sidered.Production of Acetic Acid by Bacteria.-The very varied and oftencomplex transformations brought about by bacteria between carbo-hydrates, alcohols, acids and the intermediate products, togetherwith their control by the adaptation of cultural conditions to favouror inhibit the activity of individual bacterial enzymes, form thesubject of an ever increasing number of investigations. I n this, asin all other forms of the biochemical changes in bacteria, the im-portance of small variations in the primary or secondary nutrients,of the chemical form in which these are supplied, of the temper-ature, hydrogen-ion concentration and oxidation-reduction potentialof the media, of respiratory relationships and of the stage of develop-18 J .Bact., 1933, 25, 32; A,, 639; also ibid., p. 475.19 Biochem. Z . , 1932, 252, 70; A., 1932, 1066.20 Ibid., 1933, 264, 231; A., 1083.21 Proc. SOC. Exp. Biol. Msd., 1932, SO, 218; A., 1932, 1206310 BIOCHEMISTRY.ment of the cultures is repeatedly emphasised. Considerableattention attaches to the mechanism of the production of aceticacid, more especially to the final stage, aldehyde + acid.The dismutation theory initiated by Neuberg and Windisch in1926 postulated the change of two molecules of aldehyde t o onemolecule of acid plus one molecule of alcohol.The latter is furtherattacked to produce more aldehyde, thus obscuring the true courseof the change, when examined analytically.Later H. Wieland and A. Bertho 22 presumed the direct dehydro-genation of aldehyde to be the dominant reaction, CH,*CHO [orCH,CH( OH),] -+ CH,*CO*OH + H,O. The dehydrogenation ofisopropyl alcohol completely to acetone is cited as a, parallel action.23Further confirmation of the dismutation theory was furnished byE. 25 who established the occurrence of a keto-aldehydemutase in a number of species of acetic acid bacteria. K. Tanaka 26considered the dismutation and dehydrogenation of acetaldehyde astwo phases of activity of the same enzyme. Dehydrogenation withoxygen as the hydrogen-acceptor is more rapid in acid than inneutral media, the reverse being the case with dismutation.Thatdisrnutation can take place under aerobic conditions has further beenestablished by Simon.,'Kojic Acid.-Certain species of acetic acid bacteria which producegluconic acid from glucose, also yield kojic acid from fructose ormannitol. Among these are varieties of B. hoshigaki and, lessactive in this respect, B. xylinoides. According to T. Takahashiand T. Asai 28 the acid is formed only from fructose and mannitol,not from compounds containing less than six carbon atoms. Themechanism is represented as a progressive oxidation.-I€YH,*OH - H ~ O FH,*OH -~H,o (iH,*OH +- !.To [yH*OH], + o-+ F0 t oCH,*OH [(p*OHI,7Jo IG O O H 1CH,*OHCHKojic acid22 Annalen, 1928, 467, 95; A., 1929, 219; also A.Bertho and K. P. Basu,ibid., 1931, 485, 26; A., 1931, 394.z3 A. Bertho, ibid., 1929, 474, 1; A., 1929, 1492.24 Biochern. Z . , 1930, 224, 253; A . , 1930, 1477.25 C. Neuberg and E. Simon, ibid., 1932, 253, 2 2 5 ; A , , 1932, 1169.26 Acta Phytochim., 1931, 5, 239; A., 1932, 94.27 Biochem. Z . , 1931, 243, 401; A . , 1932, 196.28 Proc. Imp. Acad. Tokyo, 1032, 8, 364; A . , 189; also Zenir. Bakt. Par.,1933,11, 88, 286; A., 1206POLLAR,D AND PRYDE. 31 1It is of interest that kojic acid production by Aspergillus takesplace from simple compounds by an entirely different process(see p. 318).Xulphur Bacteria.-Some brief reference seems appropriate tothis widely distributed class of organisms, which are characterisedby the utilisation of relatively large proportions of elementarysulphur or its simple compounds.Only a very small percentage of the assimilated sulphur isrequired for purely nutritional purposes ; the major part serves inmost cases as an agent rendering available to the organism energyfrom other sources. The importance of these organisms in naturalwaters and water supplies, in sanitation, and in their relation tosoil fertility is well known.More recently their significance ingeological process is being explored. The possibility is also indicatedthat certain classes of sulphur bacteria are associated with, if notdefinitely concerned in, some forms of stone decay (Paine, et aZ).29Apart from these more specific activities, perhaps the chiefpoint of recent fundamental interest is the elucidation of the complexrelationship between the assimilation of hydrogen sulphide andthe respiratory process in these organisms.This relationship issomewhat obscured at times by the apparent ability of the bacteriato adopt an alternative metabolic process-so-called “ pleoener-gism.” 30 Many instances are recorded in which the normal develop-ment of the bacteria occurs in the complete absence of sulphur;e.g., the purple sulphur bacteria grow under anaerobic conditionsand in the presence of organic matter without any source of oxidis-able sulphur and in this condition utilise radiant energy.31- 32The final product of oxidation of sulphur under normal conditionsis sulphate, but the process occurs in stages which include theformation of thiosulphate, thionic and sulphite.Inter-mediate-stage oxidations may be intensified by use of particularspecies or by variation of growth conditions. As is to be anticipated,the reaction of the medium is an important factor influencingthe nature of the changes, and is utilised by some workers as abasis of classification. The particular stage or type of oxidationwhich is dominant in media of different reaction serves to dis-tinguish a number of main groups of organisms.34 For more29 S. G. Paine, l?. V. Linggood, F. Schimmer, andT. C. Thrupp, Phil. Trans.,1933, By 222, 97 ; B., 1933, 628.30 “ Sulphur Bacteria,” D. Ellis (Longmans), p. 47.3 1 C. B. van Niel, Arch.Mikrobiol., 1931, 3, 1 ; A., 1932, 884.33 F. M. Muller, ibid., 1933, 4, 131; A., 1207.33 Penta- and tetra-thionic acids were isolat’ed from soil cultures by G.34 I. P. Langc-Pozdeeva, ATkh. B i d . Naulc, 1930,30, 189; A., 1930, 1622.Guittonneau and J. Keilling (Compt. land., 1932,195, 679; B., 1932, 1128)312 BIOCHEMISTRY.detailed classification of the increasing number of species isolated,morphological characters, pigmentation, and the presence or other-wise of sulphur accumulations in the cells are considered.According to van Nie135 the development of the purple andgreen sulphur bacteria is largely controlled by the concentrationof hydrogen sulphide in the medium and in turn by its reaction.The metabolism is represented as a true photosynthetic processconforming to the equation CO, + 2H,S = CH,O + H,O + 2s.The sulphur is deposited within or without the cells and in turnreduces more carbon dioxide ; the complete equation becomingX O , + H,S + 2H,O + 2CH,O + H,SO,.I n the absence of sulphur the purple organisms utilise salts oforganic acids (lactate, pyruvate, acetate, malate, succinate, etc.).The work of F.M. Muller (Zoc. cit.) indicates that the preliminarytransformation of other acids to pyruvic acid is an essential stepin the building up of cell constituents. I n this case also the processmay be regarded as il particular instance of the general photo-synthetic rule. In another paper 36 Muller shows the similaritybetween the carbon assimilation of the red sulphur bacteria exposedto light and that of the higher plants.I n the presence of hydrogen-donors the organisms reduce carbon dioxide to formic acid andthence to formaldehyde. The red pigment is closely allied tocarotene and xanthophyll and appears to play a part in the assimil-atory function of these bacteria, similar to that of chlorophyll inplants.Carotenoid pigments are absent from the green sulphur bacteria,and in this species reduction of carbon dioxide occurs only in thepresence of hydrogen sulphide as a hydrogen-donor. Carotenoidsare associated with the utilisation of less active hydrogen-donors(e.g., water) than hydrogen sulphide.I n a recent monograph D. Ellis (op. cit.) gives a fairly comprehen-sive review of the morphology and chemical activities of the sulphurbacteria.Pigmentation and Luminescence in Bacteria.-Alt hough theformation of colour in bacterial cultures frequently serves a usefulpurpose in the identification or selection of organisms, little in-formation is available as to the nature of the pigments, their form-ation or purpose. The literature of the current year indicates arising interest in this aspect of bacterial biochemistry, and a briefreference to recent investigations seems desirable here, The verydefinite pigments of the sulphur bacteria (see above) have beenexamined spectrophotometrically but of their chemical compositionnot very much is known.The red pigment of B. prodigiosus has35 LOG. cit. ae Chern. WeekbZad, 1933, 30, 202; A., 429POLLARD AND PRYDE.313been more extensively studied. The production of the pigmentis largely influenced by growth conditions, particularly the natureof the carbon and nitrogen sources, and the reaction of the medium.37W. Moycho 38 indicates that the pigment is produced during auto-lysis of the cells in the presence of oxygen, and undergoes a colourchange (red --+ yellow) at p , 8.0 approx. In a series of papers3'. Wrede 39 and colleagues describe the isolation and examinationof the pigment prodigiosin, to which is ascribed the structure :R'--R MeQ-V-c==\/R 4 (l) 11 I (2) IThe position of the methoxy- group in ring(2) is uncertain. R + R' + R" = C,H1,. NH i /--IAn apparent inverse relationship between the active growthof bacteria and the formation of pigment is shown in an examin-ation of Grassberger's bacillus by G .Sandor and G. Rougebiefa40I n suitable media having a neutral or alkaline reaction the organismgrows rapidly, but for some time is colourless. When acid metabolicproducts have accumulated to lower t'he p E of the medium suffi-ciently, pigmentation is apparent. On the other hand, when theorganism is grown in slightly acid media, the red colour is apparentthroughout, but the culture develops only very slowly.The isolation of a highly toxic pigment from bacteria developingon coconut products is described by A. G . van Veen and W. K.Merten~.~l The yellow substance (m. p. 200") is dialysable, ampho-teric, and has a high nitrogen content. The minimal lethal dosewhen administered intraperitoneally to rats is less than 0.005 mg.The dependence of pigmentation on cultural conditions is demon-strated in a number of cases.For instance, s. Arakawa42 in anexamination of Axotobacter shows that, whereas A . chroococcum andA . vinelandii produce pigment in media containing the simplesugars, only the former organism is coloured when the carbonsource supplied is inulin, dextrin, or starch. I n both cases theaddition of potassium nitrate as a nitrogen source intensifies pig-mentation. Similarly, in the case of organisms producing fluor-37 G. Gorbach, Zentr. Bakt. Par., 1929, 11, 79, 26; A , , 1930, 1219.38 Compt. rend., 1930, 191, 497; A., 1930, 1478.39 F. Wrede and 0. Hettche, Ber., 1929, 62, [B], 2678; A., 1929, 1469;F.Wrede, Z. physiol. Chem., 1932, 210, 125; A., 1932, 1043; F. Wrede andA. Rotlihaas, ibid., 1933,215,67; 1933,219,267; A., 516, 1172.40 Bull. SOC. Chim. biol., 1933, 15, 415; A., 640.4 1 Proc. K . Alcad. Wetensch. Amsterdam, 1933, 36, 666; A , , 1206.42 Tottori Agric. Coll. Sci. Papers (10th Anniv.), 2 4 ; A., 1932, 652314 BIOCHEMISTRY.escent pigments, F. K. Georgia and C. 3'. Poe43 have shown thatpigment production necessitates the presence in the media ofmagnesium, phosphates, and sulphates. Quite small proportionsof these suffice, but the organisms are sufficiently sensitive to beutilised to detect, by their fluorescence, the presence of very smallamounts of these essential ions. The same organisms are also verysensitive to the nature of the nitrogen supply, different samples ofpeptone producing unexpectedly large variations in pigmentationA similarly intimate relationship apparently exists betweennutrient conditions and light emission by several luminescentbacteria.The work of F. F ~ h r m a n n ~ ~ shows that among mineralnutrients necessary to P. radians, both anions and cations of alkalihalides contribute separate effects to the intensity of the lightproduced. Various sugars increase luminosity not only in actualintensity but in the rate of increase to maximum intensity and thesubsequent decline. These effects differ with the nature of the sugarused and are modified t o different extents by variations in thesodium chloride concentration of the medium.The organism candevelop anaerobically, but free oxygen is necessary for luminescence.Light production and cell multiplication are apparently unrelatedprocesses. A. Mudrak,46 in an examination of several species ofluminous bacteria indicates that, while light emission occurs only inthe presence of free oxygen, the organisms can, in some instances,utilise sodium nitrate and chlorate as sources of respiratory oxygeii.Further, these bacteria can tolerate sodium and potassium chlorates(the former up to 9%) or sodium thiosulphate in considerable pro-portions, but growth is checked by sodium bromate, iodate or per-chlorate and by potassium iodate. As sources of nitrogen thenitrates of potassium and ammonium, urea, and tyrosine wereunsatisfactory. Glycine, asparagine, and aspartic acid were utilis-able, although in the case of the last-named, only the amino-nitrogenwas attacked.An instance of apparently normal development inthe absence of dissolved nitrogen suggests the possibility of the fix-ation of atmospheric nitrogen. Sodium is an essential element forthe growth of all species.S. E. Hill4' describes the effects of ammonia and of fatty acids indestroying the luminescence of B. Jischerei. This action is largelydue to actual penetration of the cell wall, and subsequent cytolysis.At a given pH in the medium the loss of luminescence, brought about43 J . Bact., 1931,22, 349; A., 1932, 198.44 Ibid., 1932, 23, 135; A., 1932, 430.45 Monatsh., 1932, 60, 69, 414; A., 1932, 652, 1066.46 Zentr.Bakt. Par., 1933, 11, 88, 353 ; A., 1334.47 J . Cell. Comp. Physiol., 1932,1, 145; A., 1932, 1290POLLARI) AND PRYDE. 315by fatty acids, increased in the order of the series, valeric -+ formicacids. I n certain ranges of pH, the cytolysis occurring on exposure toO6M-solutions of ammonium salts of the fatty acids increasedwith the molecular weight of the acid.AZgcr?.The chemistry of algae receives relatively small but persistentattention from research workers, and since little reference has beenmade to the subject in recent Reports a brief statement cf certainpoints arising during the past two or three years is included here.Our knowledge of the biochemistry of algze is of a somewhat frag-mentary character. Probably most consideration has been givento the nature of algae pigments.The elucidation of metabolicprocess is to a considerable extent restricted to the examinationof the commoner carbonaceous and nitrogenous constituents of thetissues. It is characteristic of the majority of alga? that they con-tain negligible amounts of sugars, the more persistent stage of car-bon metabolism being represented by the sugar alcohols, mannitol,sorbitol, and d u l ~ i t o l . ~ ~ , 49 According to Haas and Hill (Zoc. cit.) theformation of sugars in certain marine algae is a much slower processthan their subsequent conversion into alcohols, although the presenceof certain sugars is definitely recorded, especially from samplesobtained during the summer month~.~O, 51Two sugar derivatives, algin and laminarin (or laminaroloside)are very generally distributed in algae, the latter substance appear-ing in maximum quantities in the summer period. The laminarincontent of Laminaria appears to be closely related to sea temper-ature and the amount of sunshine.52 Laminarin yields only glu-cose on hydrolysis and is probably a condensation product of theformula (C,H,oO,), or ,.Algin is variously described as a glucosidecontaining a glycuronic acid residue 53 and as a mixed calcium-mag-nesium-aluminium salt of an acid resembling pectic acid.54 Theoxidation product, alginic acid, yields mannuronic acid on hydro-lysis and monosaccharic acid on oxidation. It occurs only to alimited extent in algae.I n an examination of the fatty constituents of seaweeds Haas48 H.Colin and P. Ricard, Compt. rend., 1930,190, 1514; A., 1930, 1072.49 P. Haas and T. G. Hill, Biochem. J., 1931, 25, 1470; A., 1932, 101;50 H. Colin and E. Gueguen, Compt. rend., 1930, 190, 884; A., 1930, 825.5 1 Z-Fucose and d-mannose isolated from sea weeds; R. H. F. Manske,52 P. Ricard, Bull. SOC. Chim. biol., 1931, 13, 417; A., 1931, 883.63 H. Colin and P. Ricard, ibid., 1930,12, 1392; A., 1931, 535.Ann. Bot., 1933, 47, 55; A., 436.J . Biol. Chem., 1930, 86, 571 ; A . , 1930, 825.J. Giral, Anal. Pis. Quim., 1929, 2, 144; A., 1930, 259316 BIOCHEMISTItY.and Hill (Zoc. cit., 1933) show that, although all species examinedcontain very similar fats, the more exposed species have, in general,a higher proportion of fat and also their fats are of lower iodinevalue.The unsaponifiable fraction of the fats tends also to becomegreater with increasing depth of average immersion of the species.In the same communication is described the distribution of cer-tain nibrogenous compounds. Again the influence of immersion isapparent. In most cases a high total-nitrogen content is associatedwith a relatively low proportion of fat. Ammonia occurs in allsubmerged species and rarely in those exposed. In the latter,amide-nitrogen predominates over amino-nitrogen, whereas insubmerged plants the order is reversed. An octapeptide of glutamicacid is recorded in Phaophycece and also in Pelvetia canali~ulata,~~and methylamine and trimethylamine in a number of species byR.Kapeller-Adler and F. Vering.56 In no case was dimethylaminedetected. The presence of nitrates in a number of green alga isshown by S. S~neson.~' Many brown and some red species do notaccumulate nitrate.The pigments of certain red alga have been the subject of muchinvestigation in recent years, and much of our present knowledge isdue to the work of R. Lemberg, who, from the chromoproteinsphycoerythrin and phycocyan, obtained the corresponding truepigments, which he named phycoerythrobilin and phycocyanobilin.He subsequently established their close relationship with the bilepigments.ss The chromoproteins contain approximately 2% of thetrue pigments, which resemble in many properties magnesiurn-free chlorophyll, and occur in the alga in proportions which varysomewhat with environmental conditions.The proteins of thetwo compounds apparently are not identical.Growth and Metabolism of Moulds.Further investigations of the r61e of zinc in the growth of moulds 59have been reported this year. A direct relationship between thezinc content of a number of fungi and their nucleolytic power isindicated by M. Mousseron and P. Fauroux.60 Where this valueexceeds 100 mg. of zinc per kilo. of dry matter, the organisms are5 5 P. Haas and T. G. Hill, Biochem. J., 1931, 25, 1472; A,, 1932, 101.56 Biochem. Z., 1931,243,292; A., 1932,204.5 7 2. physiol. Chem., 1932, 204, 81; 1933, 214, 105; A., 1932, 314; 1933,437.68 Annalen, 1928, 461, 46; 1930, 477, 195; A., 1928, 533; 1930, 488.With G.Bader, Naturwiss., 1933, 21, 206; A., 651.59 See also Ann. Repmi%, 1932, 271.Go Bull. SOC. Chim. biol., 1932, 14, 1235; A., 106POLLARD AND PRYDE. 317hsmolytic. The repeatedly observed inhibitory action of zincsalts on sporulation in Aspergillus niger is accompanied by modifi-cations in the metabolic and structural chemistry of this mould.61The addition to a sugar medium of 0.01% zinc sulphate resultedin increased utilisation of sugar and greater production of citricacid. The hemicellulose contents and the proportions of ether-and cold water-soluble matter of the mycelium were increasedand $hat of lignin was lowered. I n its stimulative effect on vegeta-tive growth in fungi zinc is associated with the " growth substance-B," probably acting as a co-catalyst of growth.62The importance of calcium as a nutrient for fungi, as distinctfrom the use of calcium carbonate as a neutralising agent in media,is further confirmed by A.Rippel and U. S t ~ e s s . ~ ~ I n A. niger andcertain other cases, the calcium concentration of the mediumexerts a marked effect on the growth of the mould only whenabnormally large proportions of magnesium are present. It thenappears to act as a regulator of intake (ion antagonism) and ofthe physiological functions. Similar effects are produced bypartial substitution of strontium (as in the case of Axotobactercited above) or tannin for calcium.The action of fungi on arsenic compounds is apparently morecommon than is generally supposed. C. Thom and K.B. Raper 64have isolated a number of organisms from soil. These producearsenical gases when grown in arsenic-containing media, andseveral strains of Penicillia grow well on such media withoutproducing gas. I n the case of P. brevicaule, reported by F.Challenger,65 trimethylarsine is formed from arsenites and fromorganic arsenical compounds, and quinquevalent arsenic com-pounds are reduced to tervalent .Organic Acids and Other Products of Fungal Metabolism.-Theproduction of formic acid by a number of species of Aspergillus andPenicillium is examined by T. Chrzaszcz and M. Zakomorny.66The complete chain of transformations from sugarwould appear to be :sugar + acetic --+ fumaric + glyoxylic --+ formic acid. Withfavourable growth conditions, formic acid accumulates beforeundergoing further decomposition to carbon dioxide and hydrogen.Under less favourable conditions formic acid may be convertedinto oxalic acid.The conversion of sodium and calcium formates6 1 N . Porges, Bot. Baz., 1932, 94, 197; A., 188.62 N. Nielson and V. Hartelius, Cornpt. rend. Trav. Lab. Carlsberg, 1932,19, No. 8; A., 1932, 661; Biochem. Z., 1933, 259, 340; 281, 70; A., 638, 751.63 Arch. Mikrobiol., 1932, 3, 492; A., 97.64 Xcience, 1932, 76, 549 ; A., 189.6 5 I n d . Chem., 1933, 9, 134; A., 638.66 Biochem. Z., 1933, 259, 156; 263, 105; A,, 536, 982318 BIOCHEMISTRY.into the corresponding oxalates and carbon dioxide is also recordedby K. Bernhauer and F. Slani~~a.~' It is not quite clear from aconsideration of the above papers whether oxalic acid is a necessaryproduct of oxidation of formic acid or whether it appears as theresult of a side reaction.Various species of Aspergillus which produce kojic acid fromsugars, inulin, etc., are examined by K.SakaguchL68 Acid condi-tions and a suitable source of nitrogen favour the productionof this acid,68, which is also stimulated by the presence of smallamounts of iron, on a glucose substrate,70 and by ethylene chloro-h ~ d r i n . ~ l A. J. Kluyver and L. H. C. Perquin 72 indicate thatkojic acid is a direct product of transformation from glucose,but when it is obtained from fructose, galactose, xylose, arabinose,mannitol, erythritol, or glycerol, a C,-carbohydrat,e is probably anecessary intermediate product.On the other hand Sakaguchi(loc. cit.), who obtained kojic acid from 3C-compounds and inone instance from ethyl alcohol, favours the conception that glucoseis first broken down to 3-carbon compounds, which by oxidativecondensation form the pyrone ring. This is in conformity withthe original view of Corbellini and Greg~rini,~~ who assumed theintermediate production of glyceraldehyde, thus,GH*OH H*QH*OH HG-CO-G-OH- 211 0 HO*H,C-C*OH + G*OH a+ HO*H,C--C-O--CHHO-CM + OKojic acid.and receives further support from the formation of kojic acid fromdihydroxyacetone by A . Oryxce reported by H. Katagiri and K.Kitahara 7* (compare bacterial production of kojic acid, p. 310).In a further series of papers H. Raistrick and his co-workersreport considerable extensions of their already very comprehensiveexamination of the metabolic products of moulds.From mediain which have grown Penicilliurn brevi-compactum and relatedspecies there have been isolated two mycophenolic acids6 7 Biochem. Z., 1933, 264, 109; A., 1082.6 8 J . Agric. Chem. SOC. Japan, 1932, 8, 264; A., 637.69 K. Katagiri and K. Kitahara, Mem. Coll. Agric. Kgoto, 1933, No. 26,70 A. di Capua, Gazzetta, 1933, 63, 296; A., 983.71 0. E. May, G. E. Ward, and H. T. Herric, Zentr. Bakt. Par., 1932, 86,72 Biochem. Z., 1933, 266, 82; A., 1332.73 Gazxetta, 1930, 60, 244; A., 1930, 959.74 Mem. Coll. Agric. Kyoto, 1933, No. 26, 1; A., 638.1 ; A., 638.IT, 129; A., 1932, 1168POLLAXD AND PRYDE. 319(C17H2006),753 77 3 : 5-dihydroxyphthalic 76 3 : 5-dihydroxy-2-carboxybenzyl methyl ketone, 3 : 5-dihydroxy-2-carboxyphenyl-acetylcarbinol, and a hydrated form of 3 : 5-dihydroxy-2-carboxy-benzoyl methyl ketone.77 Luteic acid, previously reported asproduced by P. Zuteum grown on glucose media, has now beenobtained from a number of other sugars, glycerol, and succinicand citric acids when used as sole sources of carbon for the organ-ism.78 On a glucose-sodium nitrate medium, P. griseo-fulvunaproduces 2-hydroxy-6-methylbenzoic acid, mannitol, fumaric acid,and the newly recognised product, gentisic acid (2 : 5-dihydroxy-benzoic acid). 79I n an examination of Helminthosporium gramineum, the organismresponsible for stripe disease in barley, there have been isolatedfrom the mycelium helminthosporin (4 : 5 : 8-trihydroxy-2-methyl-ant hraquinone) and hydroxyisohelminthosporin (probably 1 : S-dihydroxy-2-hydroxymethylanthraquinone).80 A related substance,cynodontin (probably 1 : 4 : 5 : 8-tetrahydroxy-2-methylanthra-quiiione), occurs in the mycelium of H .cynodontis and H . euchEmnmalBiochemistry of the Higher Plants.3IineraZ Nutrition.-The multiplicity of factors influencing therate at which nutrients pass from the external medium into plantroots continues to be emphasised Many workers have attemptedto discover direct relationships between the concentration of indi-vidual ions in the nutrient and the rate of entry into the plant.For very dilute solutions a straight-line relationship may possiblyexist, but under conditions obtaining in soils and still more in waterculture experiments the mutual effects of solute ions both within andwithout the plant root become operative. Further, the assimilativeand metabolic processes within the plant exert a powerful, if indirect,influence over the mechanism of intake by the roots.Thus S. A.Yaxinos 82 shows that with dilute solutions of individual nutrients theintake curves were of very similar form t'o the dry matter productioncurves in point of view of both gradient and time distribution. Whenall nutrients were supplied simultaneously in a necessarily more75 P. W. Clutterbuck, A. E. Oxford, H. Raistrick, and G. Smith, Biochnz.76 A. E. Oxford and H. Raistrick, ibid., p. 1907 ; A., 189.7 7 A.E. Oxford, H. Raistrick, and P. W. Clutterbuck, ibid., 1933, 2'7,7 8 J. H. Birkinshaw and H. Raistrick, ibid., p. 370; A., 752.79 H. Raistrick and P. Simonart, ibid., p. 628; A., 949.80 J. H. V. Charles, H. Raistrick, R. Robinson, and A. R. Todd, $bid.,81 H. Raistrick, R. Robinson, and A. R. Todd, ibid., p. 1170; A., 1082.82 2. Pjfanx. Diing., 1933, 28, [A], 1; B., 402.J . , 1932, 26, 1441; A., 1932, 1289.634, 654; A., 949.p. 499; A , , 752320 BIOCHEMISTRY.concentrated solution, nitrogen, phosphorus, and potassium enteredthe roots at rates which were relatively in advance of the dry matterproduction curves. The intake of magnesium and calcium laggedbehind the increasing dry matter yields, An interesting exceptionto the general nature of base intake appears in the case of sodium,which penetrated at a very much enhanced rate from the more con-centrated mixed nutrient.In experiments with a somewhatsimilar object G. Pfutzer 83 shows further confirmation of the lackof direct relationship between nutrient concentration and intakeexcept over small ranges of concentration. Working with tomatoplants in soils of different moisture content, E. M. Emmert andF. K. Ball 84 also illustrate the different extents to which the intakeof individual nutrients is affected by changing levels of water supply.Where moisture contents were low, the nitrate intake was notdepressed, but that of phosphate declined considerably. Thiseffect, combined with the depressive action of a lowered water supplyon the general metabolism of plants, resulted in a high nitrateaccumulation within the plant tissues.In an examination of thepotash nutrition of plants I). R. Hoagland and J. C. Martins5 indicatea general proportionality between the intake of potassium bycrops and the potassium concentration of the soil solution in any onesoil, i.e., under conditions in which the proportional effect of othersolutes would be generally similar. The relationship does not,however, apply to soils of different type in which the proportions ofother ions would differ very considerably and also would be affectedto different relative extents by natural changes in the water contentof the soils. That the intakes of water and of dissolved ions areindependent processes is shown by experiments of M.GrctEanin.8sMoreover the rate of water intake would appear to be approximatelyinversely proportional to the concentration of dissolved salts.Assirnilution and Utilisation of Nitrogen.-There has been noabatement of interest during the year under review in the problem ofthe relative effects of the intake of nitrogen as nitrate and as am-monia.88 A series of papers by J. W. Shive and his co-workersaffords good illustration of the fact that the growth stage of theplant is an important factor influencing the utilisation of these twoforms of nitrogen. the young plants Thus in the case of83 Landw. Jahrb., 1932, 76, 745; B., 403.84 Soil Sci., 1933, 35, 295; B., 403.86 Compt. rend., 1932, 105, 899; A,, 101.87 P. Mad, P.J. Maze, jun., and R. Axionnaz, Compt. rend. Soc. Biol.,8 8 Compare Ann. Reports, 1932, 255.89 A. C. Sessions and J. W. Shive, Soil Sci., 1933, 85, 355; B., 664.Ibid., 1933, 38, 1; B., 804.1933, 112, 852; B., 1027POLLARD AND PRYDE. 321grew equally well in nutrients containing high concentrations ofnitrate or of ammonia, but at a later stage the nitrate produced themore rapid growth. The ammonia and nitrate contents of the plantsin the respective cases varied with the concentrations in the media.Plants supplied with nutrients in which the ratio NH;: NO,’was high had relatively higher proportions of soluble organic-and total organic-nitrogen and lower amounts of inorganic-nitrogen,than wits the case when nitrates predominated in the nutrient.I nanother communication the effects of supplying approximatelyequal proportions of ammonia and nitrate are recorded. Underthese conditions also the intake of ammonia reaches a maximum inthe early stages and that of nitrate at the flowering stage of theplants. The maximum rate of absorption of ammonia is muchhigher than that of nitrate. The total nitrogen intake curve there-fore shows two maxima, the earlier being mainly dependent onammonia intake and the later on that of nitrate. Throughout thegrowth period there is no actual cessation of absorption of eitherform of nitrogen. Very similar conditions prevail in the case ofbu~kwheat,~l except that the ammonia maximum is not attaineduntil the beginning of flowering and that of nitrate after the floweringstage.Ammonia absorption is much more marked in buckwheatthan in oats, the maximum rate of absorption of ammonia exceedingthat of nitrate by about 600%.Differences both in dry matter production and in the distributionof nitrogen in cotton plants result from the use of nitrate and am-monia as nitrogen sources.Q2 The former induced the heavier cropproduction, but when concentrated media were used the totalnitrogen content of the “ ammonia-plants ” was the higher. Withdilute solutions there was little difference between the total nitrogencontents in the two series of planb, but significant differences in theaccumulation of nitrogen in roots, stems, and leaves indicated con-siderable differences in metabolic rates. H. C. M.Jacobson andT. R. Swanback03 record an alternating dominance of iiikake ofammonia and of nitrate by tobacco similar to that which is recordedabove for oats and buckwheat, from nutrients containing bothforms of nitrogen. During hot seasons the plants wilted morereadily as the proportion of ammonia in the nutrient increased.In cases in which ammonia formed the sole source of nitrogen,plants were stunted, were very susceptible to root rot infection, and90 A. L. Stahl and J. W. Shive, Soil Sci., 1933, 35, 375; B., 664.91 Idem, ibid., p. 469; B., 727.92 K. T. Holley, T. A. Pickett, and T. G. Dulin, Georgia Agric. Exp. Sta.93 Plant Physiol., 1933, 8, 340; B., 646.Bull., 1931, No. 169; B., 36.REP.-VOL. xxx. 322 BIOCHEMISTRY.contained abnormally low proportions of calcium.Ammonia,toxicity towards tobacco is shown by experiments of A. B. Beau-mont 9* to become apparent in media containing more than 6 p.p.m.of nitrogen in this form, and to be reduced by additions of sodiumnitrate; the effect iiicreases with the amount of nitrate supplied.In the early stages of growth, injury by ammonia may also bereduced by additions of calcium carbonate, but with increasing agethe effect of the ammonium ion again predominates and the drymatter yield is adversely affected. It is concluded that ammonia,toxicity is due to disturbed metabolism rather than to its physio-logically acidic action. In sand-cultured apple trees rapid ammoniaintake is associated with a lower reductase activity and also with ahigher hemicellulose content than when normal proportions ofnitrate are being as~irnilated.~~ The effect of light conditions onnitrogen assimilation is examined by L.S. L~barskaja,~~ whoseexperiments show that the reduced utilisation of nitrogen by sugarbeet seedlings in darkness is much more marked in the case of am-monia than of nitrates. Also the intake and utilisation of ammon-ium nitrate resembles more closely that of other ammonium saltsthan that of potassium nitrate.The biological process of detoxication of ammonia within the plantsystem is examined and brought into close relationship with thepH of the sap by M. Kult~scher.9~ The “ammonium plants ” ofRuhland and Wetzel are those with highly acid saps in which am-monia is neutralised directly by organic acids and stored largely inthe form of ammonium salts.In such plants deamination anddeamidation is marked. Saps having pH > 6.0 are associated with“ amide plants ” in which ammonia is largely converted into thestorage form of amide. The “mixed type” consists of plants ofintermediate ranges of pH associated with varying proportions ofammonia and amide in the stored nitrogen. No direct correlationbetween pH and the NH, : amide ratio is apparent, and it must be con-cluded that factors other than sap reaction are operative in decidingthe form of storage of nitrogen prior to elaboration into proteins.A number of recent investigations are concerned with the effectof external conditions on the nitrogen metabolism of plants and theclosely related process of carbon assimilation.In one of these,G. T. Nightingale 98 records the effects of temporary (10 day) changes94 Proc. 2nd Internat. Cong. Soil Sci., 1932, 4, 65; B., 85.s5 V. A. Tiedjens and M. A. Blake, New Jersey Agric. Exp. Sta. Bull., 1932,s6 2. Pflanz. Dung., 1933, [A], 28,340; A., 647.3 7 Plunta [Z. wisa. SWl.3, 1932, 17, 699; A., 197.OS Bot. Gaz., 1933, 95, 35; A,, 1215.No. 647; B., 981POLLARD AND PRYDE. 323of temperature on the metabolism of tomato plants. At the lowertemperature examined (13"), although nitrate absorption and trans-location took place freely, the reductase activity of the plants waslow and protein synthesis retarded. Simultaneously there was con-siderable accumulation of carbohydrates, notably starch, leaveswere deficient in chlorophyll, and stems showed anthocyanincoloration.In the absence of external supplies of nitrogen theseconditions were intensified. Increase of temperature to 21" per-mitted greater utilisation of carbohydrates and synthesis of proteineven in plants receiving no additional nitrogen. With externalsupplies of nitrate both processes were still more accelerated.Assimilation, translocation, and reduction of nitrate were muchmore rapid than at 13". At a still higher temperature (35") carbo-hydrate decomposition became extremely rapid and was associatedwith protein degradation even where external supplies of nitratewere available. Exhaustion of the plants soon occurred and wasaccelerated in those having access to nitrate supplies.The effect of varied rates of carbohydrate and nitrogen meta-bolism on fruit production in tomatoes is considerable.By con-trolling carbon assimilation through alt,ered exposure to light andsimultaneously varying the nitrogen supply, V. M. Watts 99 demon-strates that conditions producing an appropriate balance of carbo-hydrate and amino-acids within the plant system are necessary toensure optimum fruiting. Thus excessive exposure to light,with its accompanying rise in carbohydrate formation, induces anincreased dry matter production but lowers the nitrogen content ofthe plant and especially the smino-acid fraction : fruitfulnessdeclines and a weak woody growth of stems takes place. At theother extreme, in which the nitrogen supply is maintained at a highlevel, but exposure t o light is restricted, the ratio of amino-acid tocarbohydrate tends to become unduly high, and unfruitful plantsshowing a very succulent form of growth are produced.Differences in the level of nitrogen metabolism in highly manuredapple trees are manifest in differences in the lipin- and residual-nitrogen fractions in the leaves.Corresponding changes in carbo-hydrate metabolism are characterised by the nature of the reserve(insoluble) matter.lPotassium and Plant Growth.-The influence of the level of potashsupply to plants on their water economy is emphasised in severalrecent publications. Deficiency of potassium tends to lower the totaldry matter production of many plants but a t the same time to1 N.W. Stuart, New Hampehire Agric. Exp, Sta. Tech. Bull., 1932, No. 50;$0 Arkansas Agric. Exp. Sta. Bull., 1931, No. 267; B., 1931,243.A., 102324 BIOCHEMISTRY.reduce, to a greater extent, the water content of the tissues. Thisis ascribed by K. Schmalfuss2 to the effect of potassium in modi-fying the colloidal nature of protoplasmic constituents, and thusexerting a partial control over the proportions of “free” and“ bound ” water in the tissues. The practical result of this actionis shown by the increased drought resistance of plants receivingadequate supplies of potas~ium.~~Although it is generally recognised that a large proportion of thepotassium content of plants remains in a soluble condition, it wouldappear that considerable amounts have undergone elaborationinto complex compounds.Electrodialysis reveals that 35% ofthe total potash of young pea plants, and only 14% of that in olderplants, migrates to the cathode, and 16% and 18% respectivelyappear a t the anode, these proportions being independent of thenature of the membrane used. I n discussing these results S. L.Inosemzev indicates that the dialysable fraction of the potassiumcontains considerable amounts in organic combination. In re-cording the influence of the supply of potassium on the carbonassimilation of the leaves of cereals G. Gassner and G. Goetze drawattention to the fact that in curves relating potash supply withassimilation (rising with increasing potassium concentration to amaximum point and subsequently declining), the maxima aredifferent and characteristic for each cereal examined.Intake and Utilisation of Phosphorus by Plants.-In addition tothe known assimilation of inorganic phosphates by plants, manyworkers have obtained indications that a variety of organicphosphorus compounds of varying complexity may also be utilised.Recent work of J.Weissflog and H. Mengdehl adds definitionto this conception. Various types of phosphorus compounds areexamined, and, according to their effects on maize plants, classifiedinto three principle groups : (1) phytin group, which includesphytic and iiucleic acids ; (2) ortho-group, comprising phosphoricacid, hexose &phosphate, etc. ; and (3) ester group, which includeshexose monophosphates, glyceryl and sucrose phosphates.Group( 2 ) was the most effective as a source of phosphorus, as judgedby the gross weight of the crop and its phosphorus content : (1)and (3) produced similar dry matter yields, but the percentage ofphosphorus in the plants was greater with (3) than with (1). ThePhytopath Z., 1932, 5, 207; A., 545; 2. PJEanz. Dung., 1933, 28, [A],3 M. A. H. Tincker and F. V. Darbishire, Ann. Bot., 1933, 47, 27; A., 436.330 ; A., 649.0. Tornau and K. Meyer, J. Landw., 1933, 81, 175; B., 680.Ergebn. Veg. Lab.-arb. Prianischnikov, 1930, 15, 85; A., 650.Planta [Z. wiss. Biol.], 1933, 20, 391; B., 1026.7 Ibid., 1933,19, 182, 242; A., 648POLLARD AND PRYDE. 325order of efficiency of the organic substance was approximatelythat of the susceptibility to decomposition by the phosphatase ofthe maize.Plants utilising the ortho-group contained the greatestproportions of orthophosphates. Inferior utilisation of phytic andnucleic acids was indicated by accumulations of soluble organicand insoluble phosphorus in the plant roots. Among the inorganiccompounds examined, pyro- and meta-phosphates were absorbedbut were converted into ortho-phosphates before leaving the rootsystems. Hypophosphites and phosphites, although passing freelyinto the plant system, were not utilised.The nature of the phosphorus compounds in plants has beenexamined by a variety of methods. Earlier work frequently led toconfusing results, since the methods of isolation often involveddecomposition of the substances in question.To overcome thisdifficulty H. Magistris has examined the separation of organicphosphorus compounds by dialysis, and the effects of varioussalts on the process.8 The general association of seed formationwith phytin synthesis in plants is reflected in the number of recordsof investigation of the phosphorus distribution of seeds. In horsebeans 9 and in hemp seed lo the proportions of organic and inorganicphosphorus extractable with dilute acids aiid alkalis indicate thepresence of phytic, nucleic, and inorganic phosphorus in the seed.In sunflower seed more than three quarters of the total phosphorusexists as phytin.ll The phytin-lipin-nucleic- and inorganic phos-phorus of wheat grain is examined by T.S. Andrews l2 and of thewhole plant in various stages of growth by F. Knowles and J. E.VVatkin.13 The latter show that during the four months prior toharvesting the gross weight of lipin-phosphorus in the stems rosesteadily until ear emergence and subsequenkly declined to a fairlyconstant value. In the ear the lipin weight was small and re-mained a t a steady figure until shortly before harvesting. Therewas a steady transference of phytin and inorganic phosphorusfrom straw t o ear as the grain matured. A close parallelism existedbetween variations of the phytin-phosphorus and protein-nitrogenthroughout the period examined. In maize14 phytin is absentfrom all parts of the plant until after pollination, and then appears8 Biochem.Z., 1932, 253, 64, 81; A., 1932, 1181, 1203.9 E. Mnich, Bull. Acad. Polonube, 1931, [B], 123; A., 1932, 1181.10 E. Pischinger, ibid., 1932, [B], 37 ; A., 874.11 A. Goldovski and A. Bozhenko, iklasloboino Zhir. Delo, 1932, No. 7, 30;12 Ind. Eng. Chem., 1932, 24, 80; B., 1932, 574.13 J . Agric. Sci., 1932, 22, 755; A., 101.14 E. E. de Turk, J. 1%. Holbert, and B. W. Howk, J . Agric. Res., 1933,A., 1092.46, 121 ; A., 545326 BIOCHEMISTRY.only in the seed. During germination phytin disappears fromthe seed a t an early date. Little variation occurs in the percentageof lipin- and acid (0.2 yo HC1)-insoluble organic phosphorus duringgrowth of the plant. Translocation of phosphorus within theplant took place only with inorganic and acid-insoluble organicfractions.Mineral Nutrition and the Incidence of Disease in Plants.-Avery definitely increasing interest is being shown in this aspect ofplant pathology.Economic pressure has stimulated enquiry intothe quantitative as well as qualitative aspects of manurial problemsand brought in its train numerous incidental observations of thehealth condition of plants in relation to nutrition. Also the con-tinuously increasing outlay in curative measures has turned atten-tion much more emphatically to the possibility of minimising cropinjury by the often less costly method of adjusting nutrientconditions.For many years the use of potash fertilisers has been recognisedas a satisfactory method of reducing the susceptibility of plantsto certain diseases.It is becoming increasingly apparent that,whilst the influence of potassium in this respect is a very importantone, much more may depend on the relative proportions in whichvarious mineral nutrients are supplied to plants than on the specificpreventive capacity of any one of them.The effect of potassium in reducing injury by yellow rust inbarley, drought spot in oats, leaf roll in potato,15 rust in wheat,16stripe disease in potatoes,17 and wilt and rust in cotton 18 is recordedthis year. The effect of anions associated with potassium on itsaction in increasing disease resistance is shown by E. Lowig.lgThe infection of cereals by Erisiphe graminis is reduced to a greaterextent by applications of potassium silicate than by the carbonate,chloride, or sulphate, although no difference in manurial action isapparent.I n a number of instances, the proportion of calcium relative toother nutrients, and also in its relation to the reaction of the medium,appears to be concerned in the degree of infection by fungal diseasestogether with other diseases falling under the general heading of“ nutritional disorders.” The “ damping off ” of soya bean seed-lings appears to be influenced much more by calcium deficiency15 L.Kratschmer, Ernahr. PJlanxe, 1933, 29, 264; B., 981.16 W. Acker and F. Konig, ibid., p. 101; B., 518.17 Kostlin, ibid., p. 48; B., 439.18 V. H. Young, G. Janssen, and J. 0. Ware, Arkansas Agric. E’xp. Sta.19 Emiihr. PJanze, 1933,29, 162; B., 646.Bull., 1932, No.212; B., 201POLLARD AND PRYDE. 327than by the reaction of the nutrient.20 Calcium deficiency oran excessive MgO : CaO ratio is associated with severe attacksof root rot in tobacco,21 with " sand-drown " in tobacco and maize,22and with leaf wrinkle in soya beans.23 " Speck " disease in oatsis primarily the outcome of manganese deficiency, but is accentuatedby unbalanced ionic ratios in the nutrient, notably that of K : Ca.24Sulphur deficiency is the cause of, or at least a heavily contributingfactor in, the tea '' yellows " disease.25 An interesting investiga-tion of the effects of unbalanced proportions of the three principalfertilisers on the infection of tomato plants by Fusarium Zycopersiciis recorded by H. Ahmet.26 The susceptibility of the plants variedwith nutrient supplies in the decreasing order : potassium deficiency,low phosphate, excessive nitrogen, excessive phosphate, excessivepotassium, low nitrogen.An interesting resume of the effects ofunbalanced nutrition, of the absence of individual nutrients, andof other growth conditions on the distribution of plant disease isgiven by F. Labro~sse.2~ANIMAL BIOCHEMISTRY.Carbohydrate Utilisation in Muscle and Yeast.THE period under review has seen important developments inthe elucidation of the chemical processes of carbohydrate utilisationin both muscle and yeast. A new scheme of fermentation formu-lated by the lat,e G. Embden and his collaborators2* has beenaccepted and developed by 0. Meyerhof and his school, and shownto be equally applicable to the muscle and yeast processes.It will be remembered that 0.Meyerhof, K. Lohmann, and R.Meier,Z9 in an important paper dealing with carbohydrate form-ation during the recovery process in the intact muscle of the frog,showed that, apart from lactic acid, only pyruvic acid out of aconsiderable number of substances investigated, produced a recoverysynthesis of sugar with oxygen uptake. Later, E. Toeniessen20 W. A. Albrecht and H. Jenny, Bot. Gaz., 1932, 92, 263; B., 1932, 200.2 1 T. R. Swanback and H. G. M. Jacobson, Scknce, 1933,77, 169; B., 440.22 A. Gehring, Ernahr. Pjlanze, 1932, 28, 101 ; B., 1932, 569.23 E. W. Hopkins, Plant. Physiol., 1933, 8, 333; B., 644.24 H. Lumdegardh, Medd. Centralanst. Pcirscisksr Jordbruks., 1931, No.403 ;25 H. H. Storey and R. Leach, Nyassaland Dept. Agric. Bull., 1932, No. 3;26 Phytopath. Z., 1933, 6, 49; R., 840.27 Ann. Agron., 1932, 2, 774; A., 437-28 G. Embden, Deuticke, and Kraft, Klin. W O C ~ . , 1933, 12, 213.a@ Biochem. Z., 1925, 157, 469; A*, 1925, i, 7-27.B., 1027.B., 245328 BIOCHEMISTRY.and E. Brinkmann30 demonstrated that pyruvic acid was rapidlyremoved when perfused through the musculature of a rabbit.E. M. Case and R. P. Cook31 isolated pyruvic acid and methyl-glyoxal from frog and rabbit muscle and formed the opinion thatboth participated in some muscle process. The occurrence ofpyruvic acid was further investigated by Case,32 who found thatit was not an intermediate in lactic acid formation, nor was it aproduct of the oxidation of the latter acid.The possible r81e ofpyruvic acid was further emphasised by A. Hahn and W.Haarmann 33 when they found that thoroughly washed muscle(free from lactic dehydrogenase) readily produced pyruvic acidfrom fructosediphosphoric acid. Numerous well-known investiga-tions by C. Neuberg and his collaborators have established theimportance of pyruvic acid in many yeast and bacterial fermenta-tion processes.It was shown by K. Lohmann34 and by F. Lipmann and K.Lohmann35 that, when muscle tissue is minced in the presence offluoride and added glycogen or starch, only a part of the hexose-phosphoric acid ester which accumulates is the true Harden-Youngfructose-I : 6-diphosphoric acid. A considerable part, and some-times even the whole, is found to be present in the form of an esterof the same elementary composition but possessing a much greaterresistance to acid hydrolysis.In fact Lohmann called it the‘‘ unhydrolysable ” ester. The monophosphoric esters isolated byRobison, Neuberg, and Ernbden were converted into this resistantester by taking up one equivalent of phosphate from muscle extractscontaining fluoride.Early in the year under review G. Embden and his colIeagues36found that a constituent of the Lohmann “ unhydrolysable ” esterwas glyceric acid-monophosphoric acid (phosphoglyceric acid).This acid was first encountered by R. Nilsson 37 in 1930 and isolatedas the barium and strychnine salts during his studies of the enzymicbreakdown of carbohydrate by yeast.At about the same timeas Embden’s discovery another constituent of the Lohmann esterwas isolated in Meyerhof’s l a b ~ r a t o r y . ~ ~ This was Z-a-glycero-30 2. physiol. Chern., 1930, 187, 137; A., 1930, 637.81 Biochern. J., 1931, 25, 1319; A., 1931, 1184.32 Ibid., 1932, 26, 759; A,, 1932, 875.33 2. Biol., 1930, 90, 231; A., 1930, 1064; see also A. Hahn, ibid., 1933,34 Biochem. Z., 1930,222,324; A., 1930, 1210.37 Arkiv Kemi, Min., Geol., 1930, 10, [A], No. 7; A., 1930, 641.38 Nature, 1933,132, 337, 373; A., 1074; 0. Meyerhof and D. McEachorn,94, 97; A., 1194.35 Ibid., p. 389; A., 1930, 1210. 86 L O C . cit.Biochem. Z., 1933, 260,417; A., 742POLLARD AND PRYDE. 329phosphoric acid. Thus it appeared that the Lohmann ester con-sisted of equimolar proportions of phosphoglyceric acid and glyccro-phosphoric acid :( BO),PO*O*CH,*CH (OH) *CO,H + (HO),PO*O*CH,*CH( OH) *CM,* OH.Embden and his colleagues showed that phosphoglyceric acid istransformed into pyruvic acid by minced muscle.Moreover,although lactic acid was not produced by the addition of eitherpyruvic acid alone or a-glycerophosphoric acid alone to muscleextracts free from carbohydrate, it was found that the simultaneousaddition of phosphoglyceric acid and a-glycerophosphoric acid tomuscle brought about an increased formation of lactic acid. Purther-more the addition of pyruvic acid and of cc-glycerophosphoric acidtogether did lead to the production of lactic acid, the amount ofthe latter formed being equivalent to twice the pyruvic acid whichhad disappeared, thus :CH,*@O*CO,H + C€€,(OH)*CH(OH)*CH,*O~PO(OH), +2CH,*CH(OH)*CO,H + H3PQ,.Further insight into the probable course of the breakdown ofsugar in muscle is obtained from a study of the behaviour of triose-phosphoric acid.M. 0. L. Fischer and E. Baer39 have recentlysynthesised glyceraldehyde-y-phosphoric acid and the dextro-component of their racemic compound has been shown by C. V.Smythe and W. Gerischer*O to undergo fermentation by yeastwith an initial velocity a t least as great as that of glucose of thesame molar concentration. Moreover Embden has corroboratedthe formation of lactic acid on adding glyceraldehyde phosphoricacid to muscle tissue.0. Meyerhof and W. Kiessliiig 41 have shownthat in muscle extracts one half (Le., one optical component ofthe racemic compound) is transformed into phosphoglycesic andglycorophosphoric acids. When sulphite, but no fluoride, is addedto the muscle extract, pyruvic and glycerophosphoric acids areformed. Meyerhof 42 points out that glyceraldehyde-y-phosphoricacid is the first synthetic ester to be converted into lactic acid aseasily as and by the same path as the naturally occurring phos-phoric acid esters.Embden summarised his views regarding these anagrobic processesin a scheme which may be represented as shown. According tothese views the formation of lactic acid in muscle is a true oxidation-reduction process in which the mutation of hexosediphosphoric39 Ber., 1932, 65, [El, 337; A., 1932, 364.40 Biochern.Z., 1933,260, 414; A., 750.4 1 Ibid., 964, 40; A., 1080; Naturwias., 1933, 21, 223; A., 528.42 LOG. cit.L 330 BIOCHEMISTRY.acid is involved, the oxidised product being pyruvic acid and thereduced Z-a-glycerophosphoric acid.These new ideas are equally applicable to the scheme of yeastfermentation. Thus C. Neuberg and M. Kobe143 have shown thatyeast and the lactic acid bacterium (B. DeEbrucki) transform phos-phoglyceric acid into pyruvic acid. When yeast is used, someacetaldehyde is also formed. As has already been mentioned,Nilsson 44 in 1930 isolated phosphoglyceric acid from dried yeastacting on hexosediphosphoric acid in the presence of glucose,acetaldehyde, and fluoride.Meyerhof and Kiessling 45 have shownthat in yeast maceration juice containing fluoride, hexosediphos-phoric acid is converted, as in muscle extract, into Lohmann’s“ unhydrolysable ” ester, i.e., to the equimolecular mixture ofa-glycerophosphoric acid and phosphoglyceric acid. Glyceralde-hyde-y-phosphoric acid behaves in similar fashion. Phospho-glyceric acid in fresh yeast, extract is converted into acetaldehyde,carbon dioxide, and phosphoric acid. The acetaldehyde is reducedto alcohol, not by reacting with glycerophosphoric acid, but pre-sumably 46 with the triosephosphoric acid arising from the inter-action of glucose and hexosediphosphoric acid. Phosphoglycericacid is formed simultaneously, thus :(HO),PO*O*CH,*CH(OH).CHO + CH,*CHO + H,O --+(HO),PO*O*CH,*CH(OH)*CO,K + CH,*CH,*OH.Meyerhof states that, apart from the presence of carboxylase, thisreaction provides the main difference between alcoholic fermentationand lactic acid formation in muscle, the difference lying essentiallyin the fate of the pyruvic acid. The latter in muscle extract isreduced to lactic acid by interaction with glycerophosphoric acid,whilst in yeast i t is split (by carboxylase) into carbon dioxide andacetaldehyde, the latter then being reduced as already explainedto alcohol.It should be noted that acetaldehyde can be replacedby other reducible systems, for example, methylene-blue. Theseobservations can be summarised in a scheme similar to that alreadygiven for the muscle process. One may finally remark that sodiumfluoride presumably inhibits in both the muscle and the yeast processthe splitting of phosphoric acid from phosphoglyceric acid and theconsequent formation of pyruvic acid.In muscle, iodoacetic acidinhibits the interaction of glycerophosphoric acid with pyruvic48 Biochem. Z., 1933, 260, 241 ; A . , 637.44 L O C . cit.45 LOG. cit.4 6 See also F. Zuckerkandl and L. ,lilessiner-Klebermass, Biochem. Z., 1932,266, 330; A., 188P O U D AND PRYDE.1 Phosphoglyceric acid331IJ.1 a-Glycerophosphoric acid + Pyruvic acid + H,PO,4. Lactic: acid +Triosephosphoric acidRepresentution of Embden's Fermentution Scheme.1 Hexosediphosphoric acid + 1 Glucose + 2 Phosphoric acid4 Triosephosphoric acidv / \ 2 a-Glyceroyhosphoric acid + 2 Phosphoglyceric acid2 Pyruvic acid + 2 Phosphoric acid(2 Acetaldehyde + 2C0,2 Phosphoric acid1 Glucose + +I2 Triosephosphoric acid + 2 Acetaldehyde2 Ethyl alcohol + 2 Phosphoglyceric acidErnbden-Meyerhof Scheme of Yeast Fermentation332 BIOCHEMISTRY.acid and the consequent formation of lactic acid and triosephos-phoric acid.There can be no doubt that the developments just describedrepresent a very considerable advance in our knowledge of carbo-hydrate utilisation, and they provide a welcome basis for theco-ordination of many previously isolated observations.Theinitiation of the new fermentation scheme i s a fitting finale to thegreat services which Gustav Embden has rendered to biochemistry.Through his untimely death the science loses one of its most activeand inspiring leaders.Deuminisation.H. A.Krebs has continued his work described last year 47 on themechanisms of deaminisation. Me finds 48 that the kidney is a siteof this process no less important than the liver ; indeed in the kidneyof the rat deaminisation is more rapid than in the liver of the sameanimal. The ammonia formed by the kidney in this process is thesource of the urinary ammonia, and the mechanism plays an impor-tant part in regulating the acid-base equilibrium. A number ofoptical antipodes of naturally occurring amino-acids (e.g., Z-alanine,Z-valine, d-leucine, d-phenylalanine, and d-histidine) are deaminisedby rat kidney some 10-20 times as rapidly as the naturally occurringforms.Perhaps the most interesting aspect of this part of Krebs’work is his direct experimental corroboration, using liver and kidneyslices, of the Neubauer-Knoop theory of oxidative deaminisationwith formation of the corresponding a-ketonic acid. In 1922Y. Kotake 49 recorded the isolation of phenyl- and hydroxyphenyl-pyruvic acids from the urine of rabbits receiving large amounts ofphenylalanine and tyrosine respectively, and N. F. Shambaugh,H. B. Lewis, and D. Tourtellotte 50 have more recently made closelysimilar observations. But apart from the phenylamino-acids,proof of the direct conversion into the keto-acids was lacking for thebulk of the amino-acids. In the present investigations Krebsrecords the practically quantitative conversions : alanine ->pyruvic acid, a-aminobutyric acid --+ a-ketobutyric acid, phenyl-alanine + phenylpyruvic acid, valine + dimethylpyruvic acid,leucine --+ E-ketoisohexoic acid, norleucine --+ a-keto-n-hexoicacid.The keto-acids have been isolated in each of the cases quoted.For the deaminisation oxygen is necessary and the process is shownto apply also to glycine, to dicarboxylic acids, and to diamino-acids.47 Ann. Reports, 1932, 29, 244.48 Klin. Woch., 1932, 11, 1744; A., 1074; 2. physiol. Chem., 1033, 217,49 Ibid., 1922, 122, 241; A., 1922, i, 1218.60 J . Biol. Chem., 1931, 92, 499; A., 1931, 1185.191 ; A., 856POLLBRD AND PRYDE. 333In a further series of experiments, using dog, guinea-pig, andrabbit kidney slices, Krebs 51 describes the isolation of ketoglutaricacid from d-glutamic acid, and of oxalacetic acid (yielding pyruvicacid and carbon dioxide) from aspartic acid.I n these cases it wasnecessary, in order to prevent further degradation of the a-ketonicacids, to treat the kidney slices with M/lOOO-arsenious acid.H. Manderscheid 52 has investigated the process of urea formationin the livers of two of the lower vertehratea-23. esculentu andT. g r a m . There is revealed a mechanism similar to that of themammalian liver, synthesis of urea being markedly accelerated bythe addition of ornithine. It is concluded that when urea is formedin the vertebrates it is always by way of the ornithine -+ citrulline + arginine mechanism. Manderscheid has found that with theexperimental methods used in these investigations, in birds and fish(the selachimns are a possible e x c e ~ t i o n ) , ~ ~ there is no measurablesynthesis of urea from ammonia and carbon dioxide. In birds thesynthesis of uric acid is not influenced by ornithine.Citrulline has now been isolated by M.Wada 54 from the productsof tryptic digestion of caseinogen under conditions which do not leadto the conversion of arginine into citrulline. On the other handF. Horn 55 records the formation of citrulline from arginine byB. pyocyuneus but not by B. coli. He postulates an arginine-desimidase distinct from arginase and trypin.Methionine.During the past .few years there have appeared several studies ofthe most recently discovered amino-acid methi~nine.~~ That thisamino-acid probably plays an important rBle in metabolism isindicated by the fact that it is the principal sulphur-containingamino-acid of caseinogen and probably also of egg-alb~min.~'H. D.Baern~tein,~~ who assumes that proteins contain no methoxylcompounds and no methylthiol compound other than methionbe ,gives methionine-sulphur contents varying from 26% of the totalsulphur (in secalin) to 90% (in casein). It is probably justified,therefore, to regard methionine as quantitatively the most important51 2. physiol. Chem., 1933, 218, 157 ; A., 976.58 Biochem. Z., 1933, 263, 245; A., 976.53 See A. Hunter and J . A. Dauphin&, Proc. Roy. SOC., 1924, [B], 97,54 Proc. Imp. Acad. Tokyo, 1932, 8, 367 ; A., 172.s 5 2. physiol. Chem., 1933, 216, 244; A., 753.56 Ama.Reports, 1928, 25, 233 ; 1931, 28, 234.5 7 N. W. Pirie, Biochem. J., 1932, 26¶ 2041 ; A . , 305.68 J . Biol. Chem., 1932, 97, 669; A., 1932, 1149.227; A., 1925, i, 104334 BIOCHEMISTRY.sulphur-containing amino-acid in ordinary diet and in proteinsother than sclero -pro teins .In 1924 J. H. M ~ e l l e r , ~ ~ the discoverer of methionine, showedthat in man it was readily oxidised to sulphuric acid. N. W.Pirie 6O has recently extended these observations and finds that in thedog methionine is oxidised to the same extent as cystine. 8-Ethyl-cysteine and S-benzylcysteine are not appreciably oxidised, whilstX-methylcysteine is oxidised but is too toxic to permit of accuratemeasurements. R. W. Jackson and R.J. Block 61 have shown thatthe addition of methionine to a diet deficient in cystine produces amarked increase in the weight of rats, and the same observers 62state that equally effective growth responses are obtained in ratsreceiving d- or Z-methionine or Z-formylmethionine, whereas d-formyl-methionine is devoid of action. Similar conclusions regarding thereplaceability of cystine by methionine may be drawn from theresults of A. White and H. B. Lewis,s3 who find that the increasedurinary nitrogen elimination on 8, low cystine diet following theadministration of bromobenzene is prevented by the addition ofeither Z-cystine or dl-methionine. The further results of B. W. Chaseand H. B. Lewis 64 show that in rats the absorption coefficient ofdl-methionine from the gastro-intestinal tract is slightly greater thanthat of cystine.They also find that the urines of rats receivingmethionine give the cyanide-nitroprusside test for the -S-S-linking.L. W. Butz and V. du Vigneaud 65 have recorded the formationof an interesting higher homologue of cystine by the decompositionof methionine with sulphuric acid. To this homocystine theyascribed the structureH02C*CH( NH,) *CH,*CH,*S*S*CH,*CH,*CH( NH,)*CO,Hand this has been confirmed by V. du Vigneaud, H. M. Dyer, andJ. Harmon 66 by the conversion of homocystine into homocysteine,by reduction with sodium in liquid ammonia, and subsequentmethylation to yield methionine. The same workers confirm thegrowth-stimulating action of methionine on low cystine diets, butthe main interest of their results lies in the finding that dZ-homo-59 J .Biol. Chern., 1924, 58, 373; A., 1924, i, 438.6O LOC. cit.61 Science, 1931,74,414; A., 1932, 83; J . Biol. Chem., 1932, g8, 465; A.,6% Proc. SOC. Exp. Biol. &feu?., 1933, 30, 587; A., 975.63 J . Bwl. Chem., 1932,98,607; A., 89.64 Ibid., 1933, 101, 735; A., 1075.6 5 Ibid., 1932, 99, 136; A., 151.6 6 Ibid., 1933, 101, 719; A,, 1074.89POLLARD AND PRYDE. 335cystine likewise produces growth in rats in lieu of methionine orcystine. It may be that the action of homocystine is non-specificin simply supplying utilisable sulphur to the organism, but, as wasshown by B. D. Westerman and W. C. Rose,67 other disulphide acids,e. g., dithiodiglycollic, p-dithiodipropionic, and a-dihydroxy- p-di-thiodipropionic, are unable to support growth on cystine-deficientdiets in spite of their oxidation in the body.The results ofduvigneaud and his colleagues are therefore in harmony with the viewthat demethylation may occur in the intermediary metabolism ofmethionine with subsequent formation of homocysteine.Vitamin A .The impetus which from time to time organic chemistry gains frombiology has been demonstrated in several fields in recent years, butnowhere more strikingly than in the attempts to elucidate thestructures of vitamins A and C and cognate problems. In previousyears the relationship of vitamin A to natural polyene pigments ofthe carotene and related groups has been dealt with in this sectionof these Reports,68 but the interest has now widened considerablyand purely chemical problems of outstanding interest have arisen.Although these problems, and also those centring round the natureof vitamin C, may justly be regarded as biochemical in their incep-tion, they are perhaps now more appropriately dealt with in theorganic chemistry sections of these Reports. Thus the importantand voluminous structural work of R.Kuhn and P. Karrer and theirassociates on the polyene group is described elsewhere in thisvolume.69 The reader is referred to this account for details of therecent structural work on vitamin A and on carotene and relatedpolyene pigments. It will be seen that the C,, formula tentativelyadvanced for the vitamin by Karrer in 1931 'O still remains apossibility, although the C,,H,,O structure is now preferred.71According to the latter, vitamin A is derived from exactly one halfof the carotene molecule.The three a-, p-, and y-carotenes so fardescribed all possess growth-promoting action and it now appearsthat this action is dependent not on the number of double bondspresent in the parent polyene hydrocarbon, but on the presence of67 J . Biol. Chem., 1927, 75, 533; 1928, 79,413, 423; A., 1928, 87; 1928,1396.68 Ann. Reports, 1929, 26, 245; 1930, 27, 276; 1931, 28, 219; 1932, 29,248.8s P. 145.70 Ann. Reports, 1931, 28, 221.7 1 p. Karrer, 0. Walker, K. Schopp, and R. Morf, Nature, 1933, 132, 26;A., 805; see also F. H. Carr and W. Jewell, ibid., 131, 92; A,, 323336 BIOCHEMISTRY.at least one unmodified p-ionone ring.72 T.Moore 73 has publisheddata showing that P-carotene and a vitamin A concentrate are utilisedequally efficiently by the rat when administered at levels approach-ing the minimum dose, and it is concluded that P-carotene possessesa biological activity equal to that of about the same weight of“ pure ” vitamin A. It would be interesting, as Moore suggests, tocompare the activities of a- and y-carotene with that of @-caroteneat levels approaching the minimum dose. It is to be noted that,owing to the failure to secure crystalline preparations or derivatives ofvitamin A, absolute criteria of its purity are still lacking. The pre-paration of 3’. H. Carr and W. Jewel1 74 when administered to ratsin daily doses of 0.006 mg.gave slightly better growth than 0.001mg. (or 1 unit) per day of International Standard carotene. Thiswould seem to be the purest preparation of vitamin A so farobtained.Vitamin C.Last year an account was given of the progress made in the studyof vitamin C and the steps which had led to a tentative identificationof the vitamin with ascorbic a ~ i d . 7 ~ Later work has all tended tocodirm this identification, but the outstanding achievement in thisfield is the elucidation of the structure of ascorbic acid by E. L.E r s t and his collaborator^,^^ and the synthesis of the d- and E-forms(the latter is the natural form) of this acid and of structurally analog-ous substances by W. N. Haworth, E. L. Hirst, and their colla-borators 77 in the Birmingham laboratories.A detailed account ofthese brilliantly successful investigations i s given elsewhere in theseR e p ~ r t ~ s . ~ ~ It is sufficient to state here that the structurer-----o--~CH2( OH)*CH( OH)*CH--C==~--C:Odeduced by Hirst and his co-workers from degradation and otherexperiments has been fully substantiated by the later syntheses.Although it seems extremely probable that ascorbic acid is vitaminOH OH72 R. Kuhn and H. Brochann, Natumoiss., 1933, 21, 44; A., 278; Ber.,73 Biochem. J., 1933, 27, 898; A . , 871.74 L O C . cit.7 5 Ann. Reports, 1932, 29, 252.78 R. W. Herbert, E. L. Hirst, E. G. V. Percival, It. J. W. Reynolds, andF. Smith, J., 1933, 1270; A., 1143.77 R. G. Ault, I).K. Baird, H. C. Carrington, W. N. Haworth, R. Herbert,E. L. Hirst, E. G. V. Porcival, F. Smith, and M. Stacey, ibid., p. 1149; A.,1933,66, [B], 407; A., 431.275.7 8 P. 167POLLARD AND PRYDE. 337C, and numerous publications have appeared asserting or stronglysuggesting that this is it cannot yet be said that this is a cer-tainty. It is clear, however, that the biological investigation of thesynthetic material will place the matter beyond a doubt. At thetime of writing data are not available.J. L. Svirbely and A. Szent-Gyorgyi 80 find that the isopropylidenederivative of ascorbic acid 81 is a moderate antiscorbutic, whilst theacid recovered from it is fully active. E. L. IIirst and S. S. Zilva 82have examined the vitamin C activity of various preparations ofascorbic acid, and although considerable variations in activity werenoted,S3 it is found that active preparations can be regenerated frominactive, or much less active, oxidised preparations.A similarclaim is made by V. Demole.84 The general conclusion of Hirst andZilva is that it is much more probable that ascorbic acid is activeper se, than that the vitamin is associated with ascorbic acid and,like it, is reversibly oxidised and regenerated quantitatively. It isinteresting to note that ascorbic acid immediately after oxidationwith iodine shows little loss of antiscorbutic activity. This observ-ation corroborates earlier statements of S. S. Zilva 85 and of J.Tillmans and his collaborators 86 as regards the vitamin present indecitrated lemon juice, and further confirmation is furnished byS.W. J~hnson.~' The explanation now favoured by Hirst andZilva is that originally advanced by Tillmans, namely, that thevitamin itself, whilst retaining most of its activity, may be reversiblyoxidised. It will be evident that the labile structure of ascorbic acidwell fits it for such a r81e.Vitamin B,.The identity of the various crystalline preparations 88 with whichvitamin B, activity is associated is still uncertain. B. C. P. Jansenand his collaborators 89 now state that the analytical data agree best79 See T. Mi'. Birch, L. J. Harris, and 8. N. Ray, Nature, 1933, 131, 273;A., 433; W. J . Dann, ibid., p. 274; A., 433; S. S. Zilva, ibid., p. 363; A.,433; A. L.Bacharach, ibid., p. 364; A., 433; L. J. Harris and S. N. Ray,Biochm. J., 1933,27, 680; A., 646.80 Biochem. J., 1933, 27, 279; A., 541.81 Ann. Reports, 1933, 29, 252.82 Biochem. J., 1933, 27, 1271; A., 1091.83 Compare L. J. Harris and S. N. Ray, Zoc. cit.84 2. physiol. Chem., 1933, 217, 83; A., 756.85 Biochem. J., 1927, 21, 689; A., 1927, 702.86 Ann. Reports, 1932, 29, 252.87 Biochem. J . , 1933, 27, 1287; A., 1090.88 Ann. Repwts, 1932, 29, 250.89 B. C. P. Jansen, J. P. Wibaut, Y. J. Hubers, and P. W. Wiardi, Rec.trav. chim., 1933, 52, 366; A., 645338 BIOCHEMISTRY.with the formula C12H,,02N,S,2HC1 for their air-dried vitaminhydrochloride.During the past few years a series of investigations of the physio-logical action of vitamin B, has been in progress in the Oxfordlaboratories.These are of considerable interest in view of the newfermentation schemes of Embden and Meyerhof. There has fre-quently been discussed in the literature a supposed associationbetween the antineuritic vitamin and certain aspects of carbohydratemetabolism, but i t is only recently that the work of R. A. Peters andhis co-workers has established a clear correlation. I n 1929 H. W.Kinnersley and R. A. Peters 90 found that the brains of polyneuriticpigeons have a high lactic acid content, and Peters with N. Gavri-lescu 91 showed a little later that the brain tissue from such pigeonshas in vitro a sub-normal oxygen uptake. The latter workers thenfound,92 on adding a concentrate of vitamin B, to the polyneuriticbrain tissue in witro, that the oxygen uptake was increased. Theeffect was obtained with the optic lobes and with lower parts of thebrain and later with the cerebral and higher parts.So far no othertissue is known to show this effect.N. Gavrilescu, A. P. Meiklejohn, R. Passmore, and R. A. Peters 93established the specificity of the effect in the presence of addedglucose or lactic acid. Experiments by A. P. Meiklejohn, R. Pass-more, and R. A. Peters 94 on birds recovering from polyneuritis aftertreatment with vitamin B, concentrate, showed that there was animprovement in the oxidative behaviour towards lactate of mincedbrain from such birds, corresponding to the disappearance of thenervous symptoms, and with this improvement the effect of addedvitamin B, concentrate in vitro diminished.It seemed, therefore,that vitamin B,, or a substance present in B, concentrates, wascapable of repairing the same defect both in the living bird and in theisolated brain.The behaviour with regard to pyruvic acid, a question of obviousinterest, differs from that shown towards lactic acid. Although thenormal pigeon's brain when minced gives a large oxygen uptake,and the avitaminous brain a low oxygen uptake, both in the presenceof pyruvic acid, the low uptake is not usually increased in this caseby vitamin B, concentrates in vitro, although results are somewhatvariable. The effect of vitamin B, seemed, therefore, to be related90 Biochem. J., 1929,23,1126; 1930,24,711; A., 1929, 1496; 1930, 963.9 1 Ibid., 1931, 25, 1397; A., 1931, 1338.92 Ibid., p.2150; A , , 1932, 200.93 Proc. Roy. SOC., 1932, [ B ] , 110, 431; A . , 1932, 644; Biochem.. J . , 1932,9d Proc. Roy. ij'oc., 1932, [B], 111, 391; A , , 1933, 1176.26, 1872; A., 195POLLARD AND PRYDE. 339to lactic acid formation, with the possibility that pyruvic acid playedsome secondary r81e. E. Boyland 95 has shown that the vitamin isnot a co-enzyme for lactic acid oxidation, and A. P. Meiklejohn 96has obtained no evidence of an increased disappearance of lactic acidcorresponding to the increased oxygen uptake.R. A. Peters and H. M. Sinclair 97 have continued these studies andfind that after previous washing of the avitaminous brain tissue itsoxygen uptake under the influence of vitamin B, is reduced.Thein vitro action of B, upon the avitaminous brain is abolished bycyanide and fluoride, whilst pyrophosphate interacts with B, andlactate to produce large rises in oxygen uptake over periods of from2 to 3 hours. On the other hand, hexosediphosphate and Robison’smonophosphate increase only the initial rate in avitaminous (as innormal) brains, but the effect is not sustained and there is no specificinteraction with the vitamin. Of particular interest is the observ-ation that a-glycerophosphate (but not the @-isomeride) increasesthe respiration largely, but the increase is not related directly to thespecific vitamin action. Freshly minced avitaminous brain givesno pyruvate reaction, but this appears strongly after shaking withlactate-Ringer solution a t pH 7.3.It is suggested that pyruvate isformed from phosphoglycerate, which would account for the observedready disappearance of the reaction in the presence of a-glycero-phosphate. Although, therefore, there is no evidence a t present tosuggest that the vitamin action is concerned with a-glycerophosphate,or directly with pyruvate, it is inferred that a-glycerophosphate isprobably one of the missing tissue substrates and that, since vitaminB,, lactic acid, and pyrophosphate appear to form a coupled oxidationsystem, the vitamin lack must affect more than one phase of cellularmetabolism.Carcinogenesis by Pure Hydrocarbons.In continuation of their work reported last year 98 J. W. Cookand his associates 99 have now isolated from a medium soft pitchan actively carcinogenic hydrocarbon in a state of purity.This is1 : 2-benzpyrene. It was obtained along with the inactive 4 : 5-benzpyrene, 1 : 2-benzanthracene, and perylene. The structuresof both benzpyrenes, and the physiological activity of the 1 : 2-isomeride, have been confirmed by synthesis: This new hydrocarbonis the most active carcinogenic substance now known and its iso-lation and synthesis constitute a remarkable advance in this field.95 Biochem. J . , 1933, 2’4, 786; A., 872. O6 Ibid., p. 1310; A., 1090.O 8 Ann. Reports, 1932, 28, 246.99 J. W. Cook, C. L. Hewett, and I. Hieger, Nature, 1932, 130, 926; A . ,Ibid., 1933, No. 6.86; J . , 1933, 395; A., 601340 BIOCHEMISTRY.Like the synthetic carcinogenic hydrocarbons previously described,1 : 2-benzpyrene contains the 1 : 2-benzanthracene nucleus.Itwill be remembered that 1 : 2-benzanthracene is itself inactive, orvery nearly so, but that it yields substances, sometimes of a veryhigh order of carcinogenic activity, by the substitution of alkylgroups at position 6 or of new rings in the 5 : 6-position. J. W.’Cook1 makes the interesting statement that a mouse tumourproduced by 1 : 2 : 5 : 6-dibenzanthracene has now reached the 67thtransplanted generation, and in rats the 40th generation has beenattained. Spindle-celled tumours, with metastases in the heartand other organs, have also been obtained by the injection of1 : 2 : 5 : 6-dibenzanthracene into fowls.Synthetic Gstrogenic Substances.It will be observed that the tricyclic phenanthrene ring systemis common to all the carcinogenic hydrocarbons so far described.This system is now known to be present in a considerable numberof naturally occurring substances of great physiological interest.Amongst these are the bile acids and sterols, vitamin D (calciferol),the ovarian hormones (cestriol and cestrone),2 the cardiac-stimulat-ing glucosides (strophanthin and digitoxin): and certain alkaloidssuch as morphine and codeine of the opium group, and the corydalisalkaloids and colchicine.It is for tohis reason that the results ofJ. W. Cook, E. C. Dodds, and C. L. Hewett are of great interest.These workers found that the synthetic substance l-keto-1 : 2 : 3 : 4-tetrahydrophenmthrene could induce estrous when injected intocastrated animals, and Cook and Dodds found that similar effectswere obtained with 1 : 2 : 5 : 6-dibenz-9 : 10-di-n-butylanthraquinoland with the carcinogenic substances 5.: 6-cyclopenteno-1 : 2-benzanthracene and 1 : 2-benzpyrene. More detailed results havebeen presented by J. W. Cook, E. C. Dodds, C. L. Hewett, and W.Lawson,G who investigated a series of diols derived from 1 : 2 : 5 : 6-1 Contribution to “ Discussion on Experimental Production of MalignantTurnours,” PYOC. Roy. SOC., 1933, [B], 113, 275.This vol., p. 216.3 W. A. Jacobs and E. E. Fleck, J . Biol. Chem., 1932, 97, 57; A . , 1932,$48; see also W. A. Jacobs and N. M. Bigelow, ibid., 1033, 99, 521 ; A,, 278.4 Nature, 1933, 131, 56; A., 323.6 Communicated at the Meeting of the Royal Society, Nov.16tJh, 1933.Jbid., p. 205; A., 323341 POLLARD AND PRYDE.dibenzanthracene. These have the general formula shown. Ofthese the dimethyl, di-n-amyl, and di-n-hexyl compounds amH 2 5 & 2 1 : 2 : 3 : 4 - l-Keto-/v\ tetrahydro- I 11 f phenanthrene\A/inactive, whilst the intermediate diethyl, di-n-propyl, and di-n-butyl compounds are all highly active, the propyl derivative show-ing the maximum activity. I n addition to the compounds alreadymentioned, neoergosterol, calciferol, and ergosterol also exhibitsome estrogenic action, diminishing in the order cited. Thehydrocarbons are the least active of the estrogenic substances sofar investigated and it appears that the presence of oxygen-con-taining groups greatly increases the potency.I n all cases the activesubstances contain the phenanthrene ring system. S. Aschheimand W. Hohlweg,’ who record the presence of cestrogenic substlancesin extracts of bituminous materials, have no doubt encounteredthe same, or similar, hydrocarbons or derivatives of these. Thereis so far no evidence that carcinogenic substances are formed fromcestrin in the animal body, but, none the less, B. P. Wiesner andA. Haddow8 find that rats treated with naturally occurring ma-trogenic hormone show, in contrast to non-treated rats, a markedincrease in the rate of growth of implants of Jensen sarcoma.The OZstrin Group.I n a praiseworthy attempt to systematise nomenclature in themtrin group it has been suggested that the follicular hormonehydrate (trihydroxyestrin, theelol, emmenin) first characterisedby Marrian should be designated “ estriol,” and the follicularhormone (ketohydroxycestrin, theelin) of Butenandt similarlydesignated “ estrone.”It is satisfactory to record results which place beyond doubtthe relationship of members of the oestrin to the cholane group, forwhich assumed relationship much circumstantial evidence ad-mittedly existed.A. Butenandt, H. A. Weidlich, and H. Thomp-son 10 have converted cestriol (hormone hydrate) by fusion with7 Deut. rned. Woch.. 1933, 59, 12: A.. 870.8 Nature, 1933, 132, 97 ; A., 852.9 N. K. Adam, J. F. Dztniolli, E. C. Dodds, H. King, G. F. Marrim, A. S.Parkes, and 0. Rosenheim, ibia., p. 205.10 Ber., 1933, 66, [R], GO1 ; A.., 540342 BIOCHEMISTRY.potassium hydroxide into a phenoldicarboxylic acid ( C1,H,,O,)formed by fission of the five-membered ring between the twosecondary hydroxyl groups. Dehydrogenation of this dicarboxylicacid with selenium yielded hydroxy-1 : 2-dimethylphenanthrene,which was converted by distillation with zinc dust into 1 : 2-di-methylphenant hrene. The last -mentioned compound was alsoobtained by similar means from ztiobilianic acid of the cholane series.OH(Estriol.1 ; 2-Dimethylphenanthrene.A. Girard, G. Sandulesco, A. Fridenson, and J. J. Rutgers,llcontinuing their investigations of the sex hormones of pregnantmare’s urine,l2 have characterised equilenin l3 as C1,H1,02. Thishas a higher acidity than oestrone and apparently differs from itin having two aromatic rings in place of one. It would thereforehave the constitution shown and it represents a natural dehydrogen-ation product of oestrone. It is of interest to note that the reductionQH3 CHEquilenin. 3-Methyl- 1 : 2-cyclopentenophenenthrene.of the keto- and hydroxyl groups and the aromatisation of ring I11would yield a methylcyclopentenophenanthrene. Such a compound,C18H16, has been obtained from sterols and bile acids by 0. Diels,W. Gadke, and P. Kording l4 by dehydrogenation with selenium.0. Rosenheim and H. King l5 suggested that this hydrocarbon wasl1 Compt. rend., 1932, 195, 981; A., 98.la Ann. Reports, 1932,29, 242.l4 Annalen, 1927, 459, 1; A., 1928, 169.l6 J . SOC. Chem. Ind., 1933, 52, 299.l3 See A,, 1932, 433POLLARD AND PRYDE. 3433-methyl-1 : 2-cyclopentenophenanthrene and this has now beenconfirmed by its synthesis by G. A. R. Kon.l6 It is apparent,therefore, that a migration of the methyl group must occur duringselenium dehydrogenation, and the earlier observation of Dielsand Gadke 1 7 that more drastic dehydrogenation of cholesterolyielded the fully aromatic tetracyclic hydrocarbon chrysene,Cl8HI2’ must depend upon a further migration of carbon leading toring enlargement. A. Butenandt l8 has recorded the formation ofa hydrocarbon C18H14 by zinc dust distillation of cestrone. Thismay be impure chrysene.19 The hydrocarbon C17H14, 1 : 2-cyclo-pentenophenanthrene, has recently been synthesised by G. A. R.Kon 2o and independently by J. W. Cook and C. L. Hewett.,l Thisis Diels’s original hydrocarbon minus its methyl group. Thusremarkably interesting developments in the chemistry of thepolycyclic hydrocarbons have rapidly followed the biological prob-lems discussed in this and the preceding two sections of this Report.Flavins or Lyochromes ,The important newly-discovered group of flavin dyes or lyo-chromes is described elsewhere in this volume.22 The flavins arewater-soluble, nitrogenous substances which have been isolatedfrom human and canine liver and kidney (P. Ellinger and W..K0schara),~3 and from egg-white and milk whey (R. Kuhn and co-w o r k e r ~ ) . ~ ~ A flavin is also found associated with a protein in thenew oxidation enzyme isolated from yeast juice (0. Warburg andW. Christian),% and the co-enzyme (cytoflav) of I. Banga and A.Szent-Gyorgyi24 is stated by K. Laki25 to be identical with theflavin component of the Warburg oxidation enzyme. The pre-parations obtained by Kuhn and his co-workers possess an intensevitamin B, activity, and a preparation of lactoflavin (from whey)is claimed to have the highest B, activity so far recorded. TheAavins would seem to show every promise of a bright ‘future.A. G. POLLARD.J. PRYDE.16 J . SOC. Chern. I n d . , 1933, 52, 950.1 7 Ber., 1927, 60, 140; A., 1927, 241.1 8 Nature, 1932, 130, 238; A., 1932, 971.19 See J . SOC. Chm. Ind., 1933, 52, 268, 287.2o J . , 1933, 1081; A., 1153.22 P. 159.24 Biochem. Z., 1932, 246, 203; 247, 216; A., 1932, 537, 775.a5 Ibid., 1933, 266, 202 ; A., 1318.21 Ibid., p. 1098; A., 1299.23 This vol., p. 159