BIOCHEMISTRY.As in the past three years, this Report is written in two sections :plant, and animal biochemistry. The arrangement of the subjectmatter of the section dealing with the biochemistry of plantsfollows closely that adopted in the Report for 1926. Attentionhas been confined mainly to the biochemical and physiologicalaspects of the subject; the chemistry of soils and matters chieflyof agricultural chemical interest are discussed by the Reporteron " Soils and Fertilisers " in the Reports on Applied Chemistry.This arrangement brings these two Reports more or less into linewith the apportionment of papers by the Bureau of ChemicalAbstracts as between A and B Abstracts. There remain, how-ever, certain agricultural problems concerned with the biochemistryof soils and with plant nutrition which seem to find their placenaturally in this Report.It has been possible to deal only with a limited number of divisionsof the subject and those selected for treatment fall naturally intotwo main sections, one concerned with chemical changes accom-panying the activities of the lower forms of plant life and the otherwith the biochemistry of the higher plants.Consideration of workon the chemistry of the humic matter of the soil has been omitted,since, although a number of papers have appeared, no great advancehas been made and the subject has been very fully discussed inrecent Reports.I n the period under review, the publication by E. C. C. Balyand his co-workers of the results of further work on the photo-synthesis of naturally occurring compounds is of special importance.The announcement of their discovery of the mechanism of photo-synthesis in witro would appear to be a great step towards theunderstanding of this fundamental reaction as it occurs in natureand may be expected to lead to further rapid advances.The early work of Maze on the function of elements, other thanthe primary elements, in plant nutrition, and recent investigationson the importance of mineral elements in animal nutrition havestimulated the output of work on the inorganic constituents ofplants and considerable progress with this subject has been made.In considering the work of the year, a general impression is feltthat an increasing amount of interest is being taken in the bio-chemistry of plants.Apart from its fundamental importance froBIOCHEMISTRY. 219the purely scientific point of view, the subject is of the greatesteconomic consequence and its field includes investigations of valueto many industries besides the primary industry of agriculture.Broadly speaking, the general aim of a century’s work on plantnutrition has been the increase of the yield per acre-the quantity-of our agricultural crops, and, while that aim still remains, thereis now coupled with it an increasing tendency to investigate themore difficult and elusive problems connected with quality. Inthis connexion, research in this country has received welcome aidfrom the Empire Marketing Board, and it is to be expected thatfurther problems of this nature will be put forward for solution as anoutcome of the recent Imperial Conference on Agricultural Research.The writer of the section on plant biochemistry gratefully acknow-ledges the collaboration of Mr.H. J. G. Hines, B.Sc.In regard to the section of this Report which deals with thebiochemistry of animals the plan adopted is the same as that ofthe plant section, that is to say, only a limited number of subjectshave been dealt with and these comprise fields of research in whichnoteworthy and co-ordinated advances have been effected. Themain subjects reviewed are therefore : (1) the vitamins, in whichtheme special attention is directed to the differentiation of t’hecomponents of the group of water-soluble B-vitamins, and tothe formation of vitamin-l) by irradiation processes; (2) thechemistry of the hexose phosphates and the r6le of these com-pounds and other organic phosphates in muscle; (3) the work ofMeyerhof and his school on the lactic acid-forming enzymes isolatedfrom mammalian muscle; (4) the developments of the past twoyears in the investigation of hEmoglobin and related compounds.The Reporter feels that it is necessary to adopt some such schemeof restrictions as that just outlined in order to present a reasonablyhomogeneous and readable account within the space a t his disposal.At the same time, it is realised that consideration of many importantpapers pdblished during the past year has been omitted. That isinevitable and in no sense is the omission to be interpreted as ajudgment of inferiority in comparison with results which have beenincluded in the matter of this Report.Again no attempt hasbeen made to deal with isolated results in new or limited fields.It may be possible to deal with the latter category in future Reportswhen the lines of advance become more clearly demarcated.Micro-organisms.Decomposition of Organic Matter.-In last year’s Report mentionwas made of the work of Waksman and Skinner,l who showedAnn. Reports, 1926, 23, 213220 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.that both bacteria and fungi are concerned in the breakdown ofcelluloses in the .soil. The investigations of S. Winogradsky andof A. Kalnjns in this connexion have confirmed and extended theearlier work of Hutchinson and Clayton.* Both these workershave isolated new forms of bacteria from the soil capable of decom-posing cellulose aerobically ; the organisms studied by Winogradskyformed products resembling soil humus in being colloidal andnitrogenous, resistant to further bacterial attack, and soluble indilute alkalis.A careful study of the conditions attaching to the decompositionof cellulosic material has been made by R.D. Rege in continuationof the work of Hutchinson and Richards.6 The latter held theview that any cellulosic material containing 30% of pentosans anda relatively small amount of woody fibre would be readily decom-posable by soil micro-organisms provided that a supply of availablenitrogen and mineral nutrients was suitably incorporated with it.This has been confirmed by Rege's work, and it is shown that byusing suitable analytical methods, the " decomposability " of anycellulosic material can be predicted.A study of the agentsresponsible for decomposition showed that three common speciesof soil fungi were particularly active in the decomposition of ricestraw. Under aerobic conditions, these fungi, in combination,proved much more active than the soil bacteria alone. The optimumtemperature for the growth of one of them, a species of Acrirnoniella,lies between 40" and 50" and the maximum a t about 60°, and, underthe conditions obtaining in manure heaps, it is probable that thegreater part of the decomposition is performed by fungi. In experi-ments with poplar wood, attempts were made to hasten decom-position by increasing the supply of energy material, carbohydratesbeing added to the wood for that purpose ; the structural materialremained, however, unattacked until the easily available materialoutside was exhausted. It will be seen that, in the main, theresults of this investigation fall into line with the evidencemaccumul-ated in favour of the "lignin" hypothesis of the origin of humicmatter in soil and with the views of Waksman referred to in theReports for 1925 and 1926.7 It has been shown by A.C. Thaysenand W. E. Bakes,* in a study of the early stages of decompositionof oat-straw by micro-organisms, that the pentosans of the rawAnn. Report for 1925-26, Roth. Exp. Sta., 1927, p.37.J . Agric. Sci., 1919, 9, 143; Ann. Reports, 1919, 16, 174.Ann. Appl. Biol., 1927, 14, 1.J . Ministry Agric., 1921, 28, 398.0 Cornpt. rend., 1926, 183, 691; 1927, 184, 493.7 Ann. RepoTts, 1925, 22, 208; 1926, 23, 211.* Biochem. J., 1927, 21, 895BIOCHEMISTRY. 221material are a t least partly responsible for the appearance of thecarbohydrate fraction of the humus.C. Barthel and W. Bengtsson consider that, although in generalthe rate of decomposition of cellulose in plant material is directlyproportional to the content of nitrogen, the slower decompositionof leguminous plants in the soil as compared with straw crops maybe due to the higher content of non-cellulosic nitrogen-free materialin the former. This may be of interest in connexion with theresults obtained a t the Woburn Experimental Farm in the per-manent experiments on green manuring, where mustard has givenmuch better results than vetches.Our knowledge of the chemical and biological processes occurringin swamped and water-logged soils has hitherto been confined toscattered and isolated observations.In irrigated soils, moreparticularly those used for paddy rice, a water-logged conditionis normal and it is not to bc expected that the biochemical changeswill be the same as those occurring in well-aerated soils. V.Subrahmanyan 10 has published the first portion of a systematicinvestigation of this question. In his first paper he deals withthe influence of water-logging, under laboratory conditions, on thenitrogen compounds present, on the reaction, on gas production,and on bacterial numbers.The only prominent change in thenitrogen compounds is an increase in the amount of ammoniacalnitrogen, which results in a slightly more alkaline reaction. Theabsence of any appreciable production of carbon dioxide, and thelack of any marked increase in bacterial numbers, under aerobicor anaerobic conditions, suggested that the ammonia productionwas due to enzyme action. This hypothesis is confirmed by thework recorded in the second paper, in which it is shown that pro-duction of ammonia is not hindered by antiseptics and that anaqueous glycerol extract of the toluene-treated soil contains anagent which is able to produce ammonia from simple proteinderivatives and from which an active preparation of a deaminaseof a protein-like character was isolated.It is concluded thatthis enzymatic deamination may play an important part in plantnutrition in water-logged soils. It is perhaps worthy of notethat the rice plant seems to thrive better when supplied withnitrogen in the form of ammonium salts than when fertilised withnitrates.J. Konig l1 bas studied the decomposition of farmyard manureand its utilisation by plants and arrives a t conclusions which areKgl. Landtbruks. Ahad. Handl. Tid., 1927, 66, 306.lo J . Agric. Sci., 1927, 17, 429, 449.l1 Mitt. deut. Landw.-Qes., 1926, 662, 571 ; B., 1927, 198222 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.in close agreement with those of Bach referred to in last year’sReport.12 S.A. Waksman and F. G. Tenney13 have begun adetailed investigation of the composition of natural organic materialsand their decomposition in the soil.In dealing with the organic matter of soils and manures, thepublication of several papers on the action of hydrogen peroxideon organic matter should be mentioned. Following the methodproposed by G. W. Robinson and Jones l4 for the determinatioiiof the degree of humification of soil organic matter, G. H. G. Jones 15has attempted to determine the degree of humification of samplesof farmyard manure by means of hydrogen peroxide and findsgood correlation between the figures obtained and the degree ofdecomposition of the manure as judged by its appearance andhistory.W. 0. Robinson,16 in America, has also described amethod for the determination of the organic matter of soils bydigestion with hydrogen peroxide, but holds that hydrogen peroxidecannot be used to differentiate between humified and non-humifiedmaterial and that, in the presence of soil, it does not determine anyclearly defined type of organic matter. He further states that themethod is not in any case applicable to soils high in calcium carbon-ate, manganese dioxide, or chromium sesquioxide; and in thisconnexion, the work of K. Scharrer l7 on the catalytic decompositionof hydrogen peroxide by soils is of interest. He found that thepower to decompose hydrogen peroxide was much greater in neutraland alkaline soils than in acid soils, and that the greater the amountof manganese, iron or calcium in the soil the greater was its activity.Loss on ignition of soils involved loss of catalytic power only in sofar as it reduced the content of carbonate iind thereby the alkalinityof the soil.The soil containing the lowest number of bacteria hadalso the lowest catalytic power, but there was no direct relationshipbetween bacterial numbers and activity.Production of Acids by Micro-fungi.-The nutrition of micro-fungi and the chemical changes due to their activities have attracteda considerable amount of attention during the past two years, and,in the main, interest has centred in the production of acids, inparticular citric and oxalic acids, from glucose and sucrose. Theformation of citric and oxalic acids from sugars by Aspergillus nigerhas been confirmed by W.S. Butkewitsch,18 who showed thatl2 Ann. Repork?, 1926, 23, 213.l3 Soil Sci., 1927, 24, 275, 317.J . Agric. Sci., 1925, 15, 26; B., 1925, 140.Ibid., 1927, 17, 104; B., 232.l6 J . Agric. Rea., 1927, 34, 339; B., 535.Biochem. Z., 1927, 189, 125; B., 918.l8 Ibid., 1.927, 182, 99; A., 382BIOCHEMISTRY. 223gluconic acid also was produced.19 If calcium carbonate waspresent in the cultures or if nitrogenous compounds were absent,gluconic acid was formed in larger amounts than citric or oxalicacid. Under the same conditions, the mould Mucor 8tdoniferproduced fumaric and oxalic acids only. Working on the ferment-ation of various carbohydrates by two separate strains of A .niger,H. AmelungM observed that one of the strains gave rise to citricand gluconic acids only, whereas the other formed oxalic acid inaddition. Citric acid was obtained from compounds with three,five or six carbon atoms in the chain, but no acid was formedfrom four or seven carbon-atom chains (erythritol, glucoheptose).Gluconic acid was found only in cultures containing dextrose,sucrose, or maltose. It is considered doubtful whether gluconicacid is an intermediate stage in the fermentation of dextrose bythese moulds.3’. Challenger and his associates 21 have made a systematic studyof the mechanism of the formation of citric and oxalic acids fromrjugars by A . niger, investigating the fermentation of the variousbreakdown products in turn.When the mould is grown withcitric acid as the sole source of carbon, the formation of malonicand glyoxylic acids can be detected; acetone is also formed, thisbeing the first recorded instance of its production by a mould. Itwas suggested in the first paper by these authors that acetone-dicarboxylic acid was an intermediate stage in the formation ofacetone and the actual occurrence of this compound was demonstratedlater when ammonium citrate was employed instead of free citricacid as the source of carbon. Glyoxylic acid was obtained bothfrom malonic acid and from calcium acetate, in the latter casecalcium oxalate and glycollic acid also being formed. Franzenand Schmitt 22 have suggested that saccharic acid is an inter-mediate in the formation of citric acid in the higher plants; andthat this view holds for its formation from glucose by A .niger isshown by the isolation of potassium hydrogen saccharate fromcultures with glucose as the only source of carbon. Fermentationof calcium gluconate solutions gives rise to calcium saccharate andsome citrate and, further, potassium citrate is formed in consider-able amount when the mould is grown on potassium hydrogensaccharate solution. The formation of saccharic acid from glucoseThe formation of gluconic acid by other species of moulds has beeninvestigated by T. Takahashi and T. As&, Proc. Imp. Acad. Tokyo, 1927, 3,86; A., 696; 0. E. May, H. T. Herrick, C. Thom, and M. B. Church, J. Biol.Chem., 1927, 75, 417.2o 2. physiol. Chern., 1927, 166, 161; A., 703.*l F.Challenger, V. Subramaniam, and T. K. Walker, J., 1927, 200, 3044.23 Ber., 1926, 68, 222224 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.by certain yeasts23 appears to be the only other case known inwhich this compound has been shown to be formed by micro-organisms. The demonstration of the importance of this acid inthe mycological production of citric acid, and of acetonedicarboxylicacid in the further conversion of citric acid into oxalic acid, is ofconsiderable biochemical significance. The results obtained bythese authors lead to the suggestion that the mechanism of theformation of citric and oxalic acids by A . niger is as follows :-QH,*OH QH,*OH QO,H QH,*CO,HCHO C0,H C0,H CH,*CO,H(1) [QH*OH], + [$X*OH], + [QH*OH], --+ Q(OH)*CO,HGlucose.Gluconic acid. Saccharic acid. Citric acid.CH,*CO ,HQH,*CO,H QH,*CO,H f Acetic acid-(2) Q(OH)*CO,H -+ QO < QO2HCH,*CO,H CH,*CO,H h 7H2 4 CH,CO,H + CO,Citric acid. Acetonedicarboxylic C02H Acetic acid.acid. Malonic acid.(3) CH,*CO,H -+ QH,*OH --+ QHO + QO,HC0,H C0,H C0,HAcetic acid. Glycollic acid. Glyoxylic Oxalic acid.acid.AmelungZ4 states that oxalic acid appears to be the universalproduct of incomplete oxidation of organic substances and maybe produced by the breakdown of carbohydrates, proteins, fats,alcohols, and organic acids. Acid formation is unquestionablythe result of processes taking place in the living cell and does notoccur with dried preparations or with the expressed juice of fungi.The maximum action is attained a t the optimum temperature forgrowth.The fact, noted above, that different strains of A . nigergave rise to different end products may prove to be of importancein the detection and classification of strains of the same species offungi, a problem with which mycologists are much concerned a tthe present time.L. K. Pearson and H. S. Raper 25 have shown that the fatty acidsformed by A . niger and by Rhixopus nigricans vary with the tem-perature at which the organisms are grown. D. Chouchak26 dis-cusses the interesting question of the competition between themicro-organisms of the soil and higher plants for mineral nutrients ;23 See Griiss, Jahrb. wiss. Bot., 1926, 66, 156, 171, 177.24 LOG. cit., ref. 20.2b Biochem.J . , 1927, 21, 875; A., 906.26 Compt. rend., 1927, 185, 82BIOCHEMISTRY. 225and the calcium requirements of algze and fungi are dealt withby L ~ e w . ~ ' B. M. Bristol-Roach 2* has studied the carbon nutritionof some algae isolated from soil and finds that all the species testedwere capable of growth in complete darkness, provided that a suit-able organic compound was present, but the requirements ofindividual species and their responses to different conditions werewidely different. The respiratory and fermentative activities ofa number of species of green algae are discussed in an interestingpBper by L. Genev~is.~~ It is concluded that the " intramolecular "respiration of algae is essentially similar to yeast fermentation.Some other papers on a number of points relating to thenutrition of different species of fungi are noted below.30Higher Plants.Photosynthesis.-A distinct advance in our knowledge of themechanism of the photosynthesis of naturally occurring compoundsis marked by the appearance of three papers by E.C. C. Baly andhis collaborator^.^^ As the result of earlier work a t Liverpo01,~~the opinion was expressed that photosynthesis of carbohydratesby the action of ultra-violet light on carbonic acid took place intwo stages, involving, first, conversion of the carbonic acid moleculeinto activated formaldehyde and oxygen which then lost energyand appeared in their ordinary state, and, secondly, reactivationof the formaldehyde by light and its polymerisation to form reducingsugars.It is now held that it was unnecessary to have postulatedtwo separate stages and that the activated formaldehyde producedfrom the carbonic acid can itself polymerise to reducing sugarswithout loss of energy and subsequent re-activation. Accordingto this view, the small amounts of formaldehyde detected whenultra-violet light acts on aqueous solutions of carbonic acid are notdue to its direct formation in the first stage, but to the secondaryphotochemical decomposition of the photosynthesised carbohydrates.The earlier results obtained were criticised by C. W. Porter and27 Biol. Zentralbl., 1927, 17, 481.28 Ann. Bot., 1927, 41, 509; A., 994.29 Biochem. Z . , 1927, 186, 461; A., 905.30 M. Chikano and T.Kitano, 2. physiol. Chem., 1927,164, 217; A., 696;A. Rippel and H. Bortels, Biochem. Z . , 1927,184, 237; A., 597; H. Tamiya,Acta Phytochim., 1927, 3, 51; A., 906; Coupin, Compt. rend., 1927, 184,1575; Bach, ibid., p. 1578; A. HBe, Bull. SOC. Chim. biol., 1927, 9, 802;R. Meyer, 2. Pflanz. Diing., 1926, A , 8, 121; A., 1927, 280.s1 E. C. C. Baly, J. B. Davies, M. R. Johnson, and H. Shanassy, Proc. Roy.SOC., 1927, A , 116, 197; E. C. C. Baly, W. E. Stephen, and N. R. Hood,ibid., p. 212; E. C. C. Baly and J. B. Davies, ibid., p. 219; A,, 1040, 1041.See also E. C. C . Baly, Ind. Eng. Chem., 1924, 16, 1016.32 See Ann. Reports, 1922, 19, 220; 1923, 20, 220; 1924, 21, 184.REP.-VOL. XXIV. 226 -NU& REPORTS ON THE PROQRESS OF CHEMISTRY.H. C. Ramsperger,s who, taking extreme precautions in regard tothe purity of their materials, reached the conclusion that in com-plete absence of all impurities no trace of formaldehyde was formed.This divergence of opinion now appears to be explained, since Balyand his associates have shown that the action of ultra-violet lighton carbonic acid is to establish a photostationary state represented6H2C0, t C6HI2O, + 60,,the amount of carbohydrate present in this equilibrium being verysmall. This being so, the presence of oxidisable impurities wouldcause the reaction to proceed from left to right with the formationof a definite amount of carbohydrate which would be photochemic-ally decomposed to formaldehyde.The existence of this photo-stationary state was established by exposing carbonic acid toultra-violet light in the presence of Feder’s solution ; 34 definitereduction then took place, showing the presence of substances ofaldehydic nature.I n complete absence of carbon dioxide therewas no reduction. Hexoses were introduced as a component ofthe equilibrium on account of the work of J. C. Irvine and G. V.Pran~is,~5 who examined a photosynthesised sugar syrup obtainedby exposure of formaldehyde to ultra-violet light and found thatglucose is produced to the extent of about one-third of the totalreducing compounds formed.Attempts were then made to shift the equilibrium to the carbo-hydrate side by addition of a reducing agent to remove the oxygen,but without success, except in one case when rods of pure Swedishiron were used. The results obtained in these experiments, con-sidered in connexion with the work of Zenghelis,36 led to the investig-ation of the effect upon photosynthesis of carbohydrates of theintroduction of a surface capable of adsorbing carbonic acid.Brieflystated, it was found that, whereas no measurable reaction takesplace when pure carbonic acid in aqueous solution, free from allsuspended matter, is exposed to light, a very definite action occurswhen a surface which can adsorb the carbonic acid is present in thesolution. Complex organic compounds, and not formaldehyde, arephotosynthetically produced. These compounds char readily withsulphuric acid, and, after hydrolysis with hydrochloric acid, reduceBenedict’s solution.If ammonium carbonate or barium or potassiumnitrite was added, complex organic nitrogen compounds wereformed. The following materials proved effective in providingby38 J . Amer. Chem. SOC., 1925, 47, 79; A., 1925, ii, b73.a4 Arch. Phcsrm., 1907, 245, 25.Ind. Eng. Chem., 1924, 16, 1019; Ann. Reports, 1924, 21, 186.56 Cornpt. rend., 1920, 171, 167BIOCHEMISTRY. 227a suitable surface : aluminium powder, barium sulphate, freshlyprecipitated aluminium hydroxide, and the basic carbonates ofaluminium, zinc, and magnesium. Rigid precautions were takenand exhaustive tests carried out to ensure the complete absence ofall organic matter in the carbon dioxide and other materials used.It was noted that aluminium hydroxide loses its efficacy in pro-moting photosynthesis after being in contact with water for somehours, and since the experimental details were identical whennegative results were obtained with this material (the only variablefactor being the nature of the surface), this offers further proof thatthe possibility of the positive results being due to the presence oforganic impurities is excluded.A further important step towards an explanation of photo-synthesis of carbohydrates under natural conditions was achievedas the results of experiments with coloured powders in visible light.When the basic carbonate of nickel or of cobalt was used to providea surface capable of adsorbing carbon dioxide, photosynthesisedcompounds similar to those described above were produced byexposure of the solutions to visible light only.Formaldehyde couldnot be detected, i.e., the activated formaldehyde formed does notescape from the reaction sphere and become ordinary formaldehydeby loss of energy. Great care was again taken to ensure absenceof impurities. The yield of organic material obtained was greaterthan when white powders and ultra-violet light were employed.One a t least of the products was a carbohydrate which reducedBenedict’s solution, gave the Molisch and Rubner reactions, andformed a solid osazone. There were also more complex substanceswhich on hydrolysis reduced Benedict’s solution. If ammoniumbicarbonate was added, complex nitrogen compounds were formed,as in ultra-violet light. Photosynthesis of carbohydrates has thusbeen carried out in the laboratory using an exciting wave-lengthcharacteristic of natural photosynthesis.The oxygen set free during the photosynthesis tends to poisonthe surface.Nickel or cobalt sesquioxide is formed as a film onthe basic carbonate, and when the surface is completely poisoned,the carbohydrates previously formed tend to be photochemicallydecomposed under strong illumination. The surface slowly recoversitself under water.In the third paper of the series, Baly and Davies discuss thequestion as to how far the photosynthesis achieved in vitro is similarto the process as it occurs in the living leaf. Whilst some of theirsuggested explanations of details are admittedly speculative, thereis a close resemblance in many points.Ordinary formaldehydedoes not take part in either case. I n the laboratory, the proces228 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.has been realised by the action of light on carbonic acid adsorbedon a surface, and there is a considerable amount of evidence to showthat a limiting surface exists in the chloroplasts of plants and isnecessary for normal photosynthesis. Visible light and a visiblycoloured surface are concerned in both processes. Fatigue effectsare observed when the living leaf is exposed to too long and intenseillumination, and when, in the laboratory, the surf ace becomespoisoned by oxygen.37 There is a slow recovery in both cases andit appears that the photosynthesis must not proceed a t a morerapid rate than this recovery process.The actual nature of thecarbohydrates synthesised in the $laboratory still remains to beascertained.Numerous attempts have been made by various investigatorsto isolate simple aldehydes from the leaves of plants, with a viewto showing their existence as intermediate products in the processof as~imilation.~~ It would seem probable from the work justdiscussed that such substances, if present in the leaf, are likely tobe decomposition products rather than intermediates.Some other recent work bearing on this subject may be con-sidered in connexion with these interesting results. D. Burk39reports attempts to induce photochemical reactions betweenammonia and various carbon compounds, including carbondioxide, formic acid, formaldehyde, and dextrose.The solutionswere contained in thin glass vessels and exposed to sunlight, variouscoloured catalysts being used. In some experiments, sunlight wascondensed through lenses. No complex nitrogen compounds wereproduced from ammonia and carbonaceous substances, and withvery few exceptions, no action of any sort was observed. It issignificant that the exceptional cases when action was observedwere those in which insoluble oxides were used as catalysts. Whenmercuric oxide was employed, formates, carbonates, nitrates, andnitrites were produced photochemically and the amounts formedseemed t o be related to the extent of surface rather than to the bulkof the mercuric oxide used. Ammonia also gave rise to nitrites andnitrates in the presence of zinc oxide, but not when solutions ofzinc salts were used.It is clear from the Liverpool work that theessential surface capable of adsorbing carbonic acid was lacking inmost of Burk’s experiments, and, further, that the excessive illumin-ation employed, by inducing secondary photochemical decomposi-37 The probable formation of a peroxide is referred to; and it is of interestthat H. Gaffron (Bet-., 1927,60, [BJ, 2229; A,, 1225) has recently shown thatacceptor peroxides are produced during the photo-oxidation of aliphaticamines in the presence of chlorophyll.38 See Ann. Reports, 1926, 23, 225.3@ J . Phy~vicd Chem., 1927, 31, 1338; A., 1040BIOCHEMISTRY. 229tion, would not be favourable to the detection of any complexproducts formed.A. K. Bhattacharya and N. R. Dharm havefound that finely divided zinc oxide acts as a sensitiser for manyphotochemical reactions, including the formation of carbohydratesfrom formaldehyde.K. Noack 41 has studied the condition of chlorophyll in the livingplant. Chlorophyll adsorbed from solution in light petroleum bymeans of dry colloidal aluminium hydroxide or dry lipin-free globinshows the red fluorescence characteristic of the chloroplasts ofplants, and it appears that the red fluorescence is dependent on theexistence of chlorophyll in the molecular disperse condition. Inthe living plant, chlorophyll is probably adsorbed on the proteinsof the chloroplasts. The work of H. Gaffron 42 on oxygen transportby chlorophyll is also of interest.When chlorophyll is dissolvedin acetone and exposed to light, it undergoes gradual oxidation,but if an oxygen acceptor is present, the chlorophyll is unchanged.It was shown that the ratio photochemical action/radiant energyadsorbed, as determined experimentally, was substantially thesame as the value obtained from Einstein’s law of photochemicalequivalence on the assumption that one quantum of energy wasused up for every molecule of oxygen utilised. This held true overa considerable range of wave-lengths, but was found to be dependenton the concentration of the oxygen acceptor.Carbohydrate Production and Transport.E. J. Maskel143 has made observations on starch production inthe potato, using a technique that made it possible to carry out thework in the field. By employing Sach’s iodine test and a Ridgwaycolour scale, the net starch production was estimated as the differ-ence between the colour value developed by a leaflet exposed tolight on the plant for three hours and the colour value of theopposite leaflet which had remained covered.The observationswere made on plants growing on plots which had received respect-ively potassium chloride, potassium sulphate, low-grade potashsalts, and no potassium. Statistical analysis of the data showedthat the rate of starch production was appreciably increased bypotassium sulphate but not by the fertilisers containing chlorides.The rate of translocation of starch from the leaflets on the potassiumsulphatc plots was also increased, but this also varied significantlywith other factors, of which intensity of solar radiation and age4Q J .Indian Chern. SOC., 1927, 4, 298.41 Biochem. Z., 1927, 183, 135, 153; A., 595.42 Ber., 1927, 60, [B], 755; A., 428.43 Ann. Bot., 1927, 41, 237; A., 704230 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.were important. The results were in the main borne out bymanurial trials on the same land.The seasonal changes in starch content in one- to five-year-oldbranches of bush-type apple trees have been followed by T. Swar-Starch disappearance tended to lag behind cambial activityin vegetative shoots, whereas the reverse was the case in floweringshoots.It is maintained by Spengles and Wiedenhagen45 that sugaris transported from the leaves to the roots of sugar-beet in theform of hexoses and not as sucrose, the latter being synthesised inthe A similar view of the processes occurring in Cannaedulis is advanced by J.C. Rippert~n,~' who states that sucrose isformed in the leaves, and is transported through the stem in theform of invert sugar, resynthesis taking place in the root-stock withthe formation of sucrose and starch.Leaf Cytoplasm.A. C. Chibnall and H. J. Channon48 have made a study of theether-soluble substances of the leaf cytoplasm of cabbage. Havingworked out a method which enabled them to prepare these sub-stances in bulk, they showed that the fraction obtained by addingacetone to the ethereal solution contained no phospholipins butthat the main constituent was the calcium salt of a diglyceride-phosphoric acid to which the name of phosphatidic acid is assigned.The fraction not precipitated by acetone contained fatty acidswhich are not in combination with phosphorus compounds.Theunsaturated acids, linolenic and linoleic acids, predominated ;palmitic and stearic constituted the saturated acids ; oleic acid wasnot definitely identified ; arachidonic acid was absent. Hydrolysisof phosphatidic acid showed the presence of the same acids.The. Nitrogenous Metabolism and Constituents of Plants.A series of papers dealing with the nitrogenous metabolism ofapple trees has been published by W. Thomas.49 The distributionof nitrogen in the water-soluble fraction of leaves and shoots wasstudied a t intervals throughout a growing season, and in a subsequentseason comparison was made between an unfertilised tree and onereceiving a heavy dressing of sodium nitrate.Samples were4p J . Pornology, 1927,6, 137; A., 797. See also E. L. Proebsting, Hilgardia,1925, 1, 81; A., 1927, 488.4s 2. Verein Deu&. Zuckerind., 1926, Lief. 842, 767; Bied. Zentralbl., 1927,56, 459; compare Davis, Daish, and Sawyer, J. Agric. Sci., 1916, 7, 225.See also H. Colin and R. Franquet, Bull. SOC. Chirn. biol., 1927, 9, 114;A., 699; H. Colin, Cornpt. rend., 1927, 184, 835; A,, 696.47 Hawaii Exp. Sta. Bull. 56, 1927.4a Biochem. J., 1927, 21, 226, 233, 479, 1112; A., 386, 799, 1227.49 Plant Physiology, 1927, 2, 56, 67, 109, 246BIOCBIEMISTRY. 231desiccated a t 60" and, after suitable grinding, extracted with water.The separation of simple and conjugate proteins from their hydro-lytic products was effected by means of colloidal ferric hydroxide.An examination of the nitrogen distribution in several differentsamples of residual material after extraction with water showedvery consistent results, which in the opinion of T.B. Osborneindicated that a single protein is present, although the figuresdo not give absolute proof.The detailed results of the investigation of the changes occurringthroughout the year make interesting reading and work of this typewill undoubtedly lead to clearer views of the changes occurring duringthe various periods of growth. When growth is rapid, nitrogentends to migrate from the leaves to the shoots, where it is storedin the phloem.During bud formation, the reserve proteins aretransported to the actively growing parts in the form of amino-acids. The phenomenon of autumnal migration of nitrogen fromthe leaves to the branches, a point of controversy with earlierinvestigators, is established and storage takes place mainly in theone- and two-year growths. Although the quantities, not only ofsoluble proteins, but also of the total water-soluble nitrogenousproducts, are small in Pyrus malus and make this species anunsuitable plaht for investigation of the mechanism of protein syn-thesis, these results tend to confirm Chibnall's theory that amino-nitrogen is chiefly concerned in protein synthesis and " rest"nitrogen in protein degradati~n.~~ The results showed that nitrogenequilibrium of the whole tree would be just maintained by theapplication of 5 lb.of sodium nitrate.In the following year's work, distinct differences in conformitywith the fertiliser treatment were observed between two trees,one unmanured and the other receiving two heavy applicationsof sodium nitrate. The increased growth resulting from thetreatment was reflected in the analyses, and the possible applicationof the results to horticultural practice has been dealt with in anotherpaper.510. K. Stark 52 has carried out an investigation on the proteinmetabolism of the soya bean. Seedlings were grown under con-trolled conditioas in darkness and determinations of amino-nitrogenwere made at frequent intervals.In general, no correlation couldbe observed between period of growth and content of amino-nitrogen except in the very early periods; but since analyses werecarried out on the whole plants, and not on the separate parts ofAnn. Reports, 1924, 21, 192.6 1 PTOC. Amer. SOC. Hort. Sci., 1926, 73.63 Arne?. J . Rot., 1927, 14, 632232 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.the seedlings, this result is surely to be expected. The protein ofthe reserve material is broken down in one part and transferredto another ; hence analyses of the whole plant will presumably giveonly equilibrium values at any particular time, except in the veryearly stages when little growth has occurred.Among other papers on nitrogenous metabolism and nitrogencompounds in plants, the following may be briefly noted.C. 0.Appleman and E. V. Miller 53 have investigated the changes inthe nitrogen compounds in potatoes during growth and storage,the results failing to indicate any chemical or physiological basisfor the superiority of immature potatoes for “seed.” W. F.Gericke 54 has shown that the amount of nitrogen available a tdifferent stages of growth affects the protein content of wheat,the effects varying with different types of wheat. W. L. Davies 55has given an account of the proteins of forage plants of the ordersLeguminosce, Crucifer@, and Umbelliferce, with analytical details.S. L. Jodidi 56 has compared the proteins of rice with those of othercereals, and has isolated asparagine from etiolated maize seedlings.C .G. Vinson 57 has studied the nitrogenous compounds extractablefrom maize pollen by dilute sodium hydroxide solution.Storage of Fruit.In addition to its purely scientific interest, the Report of theFood Investigation Board for the years 1925-26 58 illustrates thegrowing connexion that is being established between industry andbiochemistry. The fruit trade suffers perhaps more than othersfrom loss by wastage, and the preservation of perishable goodsoffers many interesting problems.In this Report, investigations are described into the variousfactors favouring or preventing deterioration of fruit on storage,the work including both chemical studies on apples and pears ofdifferent varieties during ripening and storage and investigationsinto the suitability of various kinds of store.The best-keeping varieties of apples are found to contain the leastnitrogen and the most sugar and exhibit the lowest respiratoryactivity.Death ensues when the sugar is exhausted and this occursthe earlier if a large amount of protoplasm is present. Hence by asimple chemical determination the expectation of life of an appleduring storage can easily be found.The nature of the soil upon which the apples were grown had a53 J. Agm’c. Res., 1926, 33, 569; B., 1927, 22.54 Ibid., 1927, 35, 133; B., 826.55 J. Agric. Sci., 1926,16, 280; A., 1926, 761; ibid., 1927,17,33,41; B., 232.56 J. Agm’c. Res., 1927, 34, 309; A,, 800; ibid., 1927, 34, 649.6 7 Ibid., 1927, 36, 261; A., 1227.68 D.S.I.R., H.M. Stat. Office, 1927BIOCHEMISTRY. 233marked effect on the nitrogen content and hence on their keepingqualities. In general, specimens of the same variety from silt soilsurvived longer than those grown on gravel or fen soil at storagetemperatures of 1" and 8". At about the latter temperature, itwas found possible to double the storage life by keeping the fruitin an atmosphere containing 9.2% of carbon dioxide and 11.8%of oxygen instead of air. Death of the fruit stored at 1" is accom-panied by a browning of the tissue, a condition known as internalbreakdown ; in storage at 8" wastage is caused by a disease, " fungalrot," and not by internal breakdown. Slightly different resultswere obtained with pears, and indeed optimum storage conditionsvaried considerably amongst the several varieties.Differences in the rates of respiration of different varieties of appleshave been noted by B.D. Drain.59 F. Gerhardt 60 also has investig-ated some of the changes involved ikl the ripening and storage ofapples. He finds that the ripening process is accompanied by lossof moisture, acidity, dextrins, starch and acid-hydrolysable material,together with an increase in specific gravity, sugars, and solublepectin. Only very slight chemical differences between normaltissues and those showing internal breakdown could be detected.Work on the pectic substances of fruits has received attention inrecent ReportsJG1 and the subject continues to prove of interest.M. H. Carre and A.S. Horne 62 confirm earlier chemical investig-ations by a microscopical study of the tissues at various stages,and according to C. 0. Appleman and C. M. Conrad 63 the trans-formation of protopectin into pectin appears to be the only pecticchange associated with the ripening and softening of peaches.Other work on the chemistry of pectic substances is described byE. K. Nelson,64 A. M. Emmett,65 and by F. R. Davidson andJ. J. Williamson.66F. E. Denny6' has published an interesting summary of hisinvestigations on the curious effect of ethylene and other unsaturatedhydrocarbons in producing colour changes and a break in the restperiod of stored fruits and tubers. Other papers dealing with thissubject are noted below.68K g Bot. Gaz., 1926, 82, 183.6o Plant Physiology, 1926, 1, 251.61 Ann. Reports, 1925, 22, 213; 1926, 23, 228.62 Ann. Bot., 1927, 41, 193.64 J . Amer. Chem. Soc., 1926, 48, 2412, 2945.6 5 Biochem. J . , 1926, 20, 564; A,, 1926, 872.6 6 Bot. Gaz., 1927, 83, 329. 6 7 Proc. Nat. Acad. Sci., 1927, 13, 355.6* E. M. Chace and C. G. Church, Ind. Eng. Chem., 1927, 19, 1135; L. 0.Regeimbal and R. B. Harvey, J . Amer. Chem. SOC., 1927, 49, 1117; A., 599;L. 0. Regeimbal, G. A. Vacha, and R. B. Harvey, Plant Physiology, 1927, 2,357; G. A. Vacha and R. B. Harvey, ibid., p. 187.Maryland Agr. Exp. Sta. Bull. 283, 1926.H 234 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.Structural Constituents of Plants.In last year’s Report mention was made of current theories withregard to the origin of lignin and the work of M.H. O’Dwyer onthe hemicelluloses of beechwood was referred E. Schmidtand his collaborators 70 have been working for some years on theconstitution of the structural components of plants and they con-sider that the cell-membrane of both archegoniates and phanerogamsis made up of cellulose, hemicellulose, and incrustation. A cellulose-hemicellulose complex termed the ‘‘ skeletal substance ’’ is obtainedby repeated treatment with chlorine dioxide and sodium sulphitealternately, incrustants and part of the hemicellulose being removed.The existence of two types of hemicellulose (compare O’Dwyer) istherefore postulated. Glycuronic acid occurs in the hydrolyticproducts of the hemicellulose pf a number of plants differing widelyin nature, including both archegoniates and phanerogams, thusindicating that, although the free acid is not present, a carboxylatedpolysaccharide, as its precursor, is common to both groups.Anester-like union of cellulose, glycuronic acid and hemicellulose isassumed to be present. Trustworthy figures for the determinationof polyglycuronic acids were obtained by treatment of the skeletalsubstance with alkali hydroxide, followed by conductimetric titr-ation with hydrochloric acid. W. Fuchs and E. Honsig 71 havecriticised Schmidt’s views on the ground that lignin obtained bythe above treatment does not resemble lignins prepared in otherways. This would seem to be a fair criticism because of the drasticnature of the reagents employed.It is very improbable, however,that lignin has ever been isolated in the condition in which it existsin the plant, and, in this connexion, it may be noted that C. Dor6eand E. C. Barton-Wright 72 have obtained a new type of alkalilignin by treating spruce dust with sodium hydroxide underpressure. This has been termed meta-lignin and is stated to agreein composition with the a-lignin of Klason. A useful summaryof present-day views on the origin and formation of plant-cellmembranes from both the botanical and the chemical aspect of thesubject has been published by van Iter~on.?~Absorption of Ions by Plants.In 1923, Robbins 74 advanced a theory explaining the differentialabsorption of ions by plants which was based on the isoelectric6B Ann.Reports, 1926, 23, 231.‘O E. Schmidt, F. Trefz, and H. Schnegg, Ber., 1926, 59, [B], 2635; A.,1927,80; E. Schmidt, K. Meinl, and E. Zintl, ibid., 1927,60, [ B ] , 503 ; A,, 383.71 Ibid., 1926, 59, [B], 2850. 72 Biochem. J . , 1927, 21, 290; A,, 597.73 Chem. Weekblad, 1927, 24, 166.74 Amer. J . Bot., 1923, 10, 412; Ann. Reports, 1923, 20, 226BIOCHEMISTRY. 235relations of the components of the living cells, particularly theproteins. If the acidity of the medium does not increase beyondthe pH represented by the isoelectric point of the cell colloids, mostof the latter are on the electronegative side of their isoelectricpoints and therefore combine with an excess of basic over acidradicals. The medium increases in acidity, and hence some colloidspass over to the electropositive side, an absorption of anionsresulting. J.Davidson 75 makes use of this hypothesis to explainthe fact that relatively more potassium than phosphorus wasabsorbed by wheat seedlings grown in potassium phosphate solu-tions, irrespective of the initial hydrogen-ion concentration of themedium. Owing to buffer action, this preferential absorption didnot result in any marked increase of acidity when the initial reactionwas pE 6 or 7, but, with initial reactions of p,5 or less, increasedacidity was observed. At this lower pB relatively more phosphoruswas absorbed than a t pH 6 or 7. It would thus appear that thephysiological availability of phosphorus depends on the px value ofthe medium. In explaining absorption phenomena of this type,the author assumes that there is a relatively wide range in theisoelectric points of the individual protoplasmic ampholytes of thecells.K. Lemanczyk 76 considers that absorption of potassium fromnutrient solutions by the roots of barley consists of two phases,viz., equivalent absorption, including absorption of salt moleculesas such, and ionic absorption. In the latter phase, to which thechanges in the reaction of the solutions are due, potassium ionsand anions in the solutions are exchanged respectively for calciumand magnesium ions and hydrogen carbonate ions in the root-cells.The results of the experiments of A.R. C. Haas and M. S. Reed 77on the absorption of ions by citrus and walnut seedlings are moredifficult to understand. Citrus seedlings removed relatively morepotassium than calcium from solut'ions containing approximatelyequivalent amounts of these ions.A n interchange of ions wasobserved between the solutions and roots which resulted in anincreased excretion of potassium in the solutions when the originalconcentration was low. Calcium ions were readily absorbed whensodium and potassium were absent or low in amount. With walnutseedlings, presence of excess of sodium chloride hindered absorp-tion of calcium. More kations than anions were taken up from thesolutions of single calcium salts by both citrus and walnut seedlings,causing an increase in acidity. The changes in reaction of the7 5 J. Agric. Res., 1927, 35, 335; B., 950.Bull.Acad. Polonaise, 1926, B, 1109; A., 1927, 1228.7 7 Hilgardia, 1926, 2, 67; A., 1927, 907236 ANNUAL REPORTS ON THE PROGRESS OB CHEMISTRY.culture solutions are attributed directly to differential absorptionof ions, together with an excretion of certain ions. It appears thatin complete nutrient solutions, citrus seedlings may in a compara-tively short period bring about so great a concentration of hydrogenions aa to be injurious to the roots. In the view of these workers,“ the absorption of ions is veiled by a host of factors, few of whichare as yet understood,” but they are of the general opinion thatabsorption is related to some chemical or physical property of theprotoplasm. A purely physicochemical explanation is apt, however,to take little notice of chemical change and growth within theplant.Absorption is taking place, not in one cell only, but inchains of connected cells, and it is here perhaps that the explanationof some of the difficulties may be found.In connexion with penetration into and absorption by livingplant cells, Osterhout and his associates ‘8 have investigated theprotoplasmic surfaces in the alga, Valonia rnacrophysa. Theprotoplasm forms a delicate layer, only a few microns in thickness,the two surfaces being alike as far as microscopic observation goes.By measurements of potential differences the conclusion is reached,however, that the protoplasm actually consists of three layers.Electrometric determinations on the chain sap-protoplasm-sapshowed a potential difference of about 14.5 millivolts, the innersurface being positive with respect to the outer.The chain isassumed to bewhere X is an outer, non-aqueous layer, W a middle aqueous layer,and Y an inner non-aqueous layer. The work of M. Irwin hasbeen referred to in a previous Report.79 She has continued herstudies of the penetration of dyes into the vacuole of living cells ofNitella and VuZoniu,*O investigating particularly the effects of acids,salts, and buffer mixtures on such penetration. The assumptionof the existence of separate layers in the protoplasm proper isutilised to account for her results.also discusses the mechanism of the accumulationof dyes by living cells.SaplXIWIYlsap,G. W. ScarthInorganic Constituents of Plants.A considerable volume of work has appeared in the last few yearson the distribution and function of the inorganic constituents of78 W.J. V. Osterhout, E. B. Damon, and A. G. Jacques, J . Gen. Physiol.,1927, 11, 193.is Ann. Reports, 1926, 23, 224.81 Plant PFuysioEogy, 1926, 1, 215.J . Gen. Phyaiol., 1926,10,76,271; 1927,10,425,927; 1927,11, 111,123BIOCHEMISTRY. 237plants, the subject being of special interest on account of theimportance now attached by workers in animal nutrition to themineral constituents of food rations. There is a tendency to takeup problems of balance and correlation and emphasis is being laidon the relative quantities of the various elements present.Primary Nutrient Elements.-0. Arrhenius 82 has published twopapers dealing with experiments on the optimum concentrationsof the primary nutrients for plant growth.He reaches the con-clusion that in most soils the concentration of potassium is s~.&-cient, whereas the concentration of phosphoric acid is about halfthat required for a favourable crop. The. purpose of fertilishgshould be to alter the concentration of the nutritive substances tothe optimum, and not necessarily to satisfy the demands of the plant.Working with Helianthus, A. Rippel s3 has shown that the absorp-tion of elements which are readily mobile in the plant, such asnitrogen, potassium, and phosphorus, accelerates the formationof dry matter, whereas less mobile elements such as calcium,magnesium, sulphur, and silicon have little or no effect.It is suggested that there is a relatively greater uptake of mobile elementsduring the earlier stages of growth of the plant.84J. Davidson 85 has investigated the changes in nitrogen, potass-ium, and phosphorus during the germination and early stages ofgrowth of wheat seedlings and finds that they may either lose orgain potassium and nitrogen, according to age and conditions, butthat the content of phosphorus remains approximately constant.In an interesting paper, K. Maiwald86 discusses the influence oflarge amounts of potassium and chlorine on the growth and leafcolour of potatoes. He found that excess of potassium or sodiumions alone effected a reduction in leaf colour, as compared withnormal plants, of about 25%, chlorine ions alone about 70% andpotassium and chlorine ions together about 60%.It was clear thatwith calcium chloride the effects were due solely to the chlorine ionsand that with potassium and sodium sulphates the influence of thekations predominated. The author considers that, not only reduc-tion in chlorophyll content, but many other phenomena concernedwith plant metabolism, can be attributed to the alteration of theequilibrium between physiologically important ions.J. H. MacGilliaays7 has shown that in phosphorus-starved82 Medd. Centralanstalt f$rs@ksvasende€ jordbruks, 1927, Nos. 40, 41.83 Biochem. Z . , 1927, 187, 272; A., 1116.On the influence of fertilisers on absorption of plant nutrients and form-ation of dry matter, see W. Schleusener, Z.Pflanz. Dung., 1926, A , 7, 137;B., 1927, 55. 8s Bot. Qaz., 1926, 81, 87.86 Z . Pflanz. Diing., 1927, A , 9, 67; B., 565.a 7 J . Agm'c. RM., 1927, 34, 97; A., 699238 ANNUAL REPORTS ON THE PROGRESS OF HEMIS IS TRY.tomato plants there is a re-utilisation of the phosphorus present ;about half the total amount is found in the fruit, irrespective oftreatment, although, if there is a shortage of phosphorus, the sizeand number of the fruits are much decreased. There is an increasein the percentage of total nitrogen and of sugars present. Theeffects of deficient amounts of potassium, calcium, and magnesiumon various plants have been studied by R. C. Burre11.88The occurrence and distribution of sodium in plants and theratio of the amounts of sodium and potassium present have beenthe subjects of several papers.89 By comparing the compositionof the seed with that of barley plants grown in darkness, with andwithout sodium and potassium, t o the point of exhaustion of thereserve materials, A.Bobrownicka-Odrzywolska 90 has shownthat in presence of potassium a smaller amount of carbohydrate isrequired for the formation of a unit of cellulose. Sodium has asimilar effect if accompanied by other necessary mineral salts.Potassium also reduces the loss of organic matter and the per-centage of starch decomposed for respiration purposes. The youngplants made poorer growth in pure potassium or sodium chloridesolutions than in distilled water; none the less, a smaller per-centage of starch was decomposed for respiration.The translocationof potassium from leaves of ivy and poplar has been followed byT. Sabalitschka and A. Wei~e.9~Secondary Elements in Plant Nutrition.-The appearance offurther papers asserting the indispensability to plants of certainelements hitherto neglected in this connexion would seem to involvereconsideration of what were regarded as established facts in plantnutrition. The pioneering work of Mazk92 indicated the possi-bility that, by more refined methods, the ten elements postulatedby Knop and the older physiologists as satisfying all the require-ments of plant growth might be shown to be insufficient. Theseexperiments, coupled with the stimulus due to the brilliant workon deficiency diseases which has demonstrated the importance ofminute amounts of vitamins, hormones, and mineral elements inthe animal body, have led to a number of investigations on the r61eof secondary elements in plant growth.03Bot.Gaz., 1926, 82, 320; A., 1927, 596.G. Bertrand and J. Perietzeanu, Compt. rend., 1927, 184, 645, 1616;Bull. SOC. chim., 1927, 41, 709; A., 488, 704, 1116; G. Andre and E.Demoussy, Compt. rend., 1927, 184, 1501 ; A., 798.@O Bull. Acad. Polonaise, 1925, B , 801; A., 1927, 384.s1 2. Pfianz. Dung., 1926, A, 7, 166; B., 1927, 66.s2 Ann. Reports, 1916, 12, 231.Earlier papers are referred to in Ann. Reporb, 1922, 19, 225; 1923, 20,219; 1926, 22, 210BIOCHEMISTRY. 239The general line of the experiments now under notice is toattempt to grow plants in the usual nutrient solutions, preparedfrom highly purified chemicals, so that contamination with otherelements is reduced to a minimum.Under these conditions, manyplants fail to grow. The addition of very small traces of certainelements-boron, zinc, silicon, aluminium, manganese-has, in anumber of instances, secured normal growth of the plants and allthe above-named elements have been stated by different workersto be indispensable for proper growth.In regard to boron, the earlier work of Miss Warington hadshown that certain leguminous plants, but not barley and othercereals, could not be grown to maturity in solutions free from thiselement. Following up these observations, W. E. Brenchley andK. Warington 94 have attempted to replace boron for plants whichrequire it by other elements, particular attention being paid tomanganese; but, of 52 elements tested, none proved capable of sodoing.On the other hand, G . H. Collingsg5 states that, contraryt o Miss Warington’s findings, boron is not essential for the growthof the soya bean, although in water cultures a stimulating influencewas observed. A. L. Sommer and C. B. Li~man,~6 taking elaborateprecautions to purify all the materials used, have demonstratedthat both boron and zinc are indispensable to many leguminousand non-leguminous plants, barley (see above) requiring both theseelements. They are of the opinion that the explanation put for-ward by Brenchley andThornton in 1925,97 that the failure of legumesto grow in absence of boron is closely connected with injury to thenodule bacteria and consequent disturbance of the nitrogen meta-bolism of the plant, avoids the main issue.A.L. Sommer gs records experiments showing that small tracesof aluminium and silicon are also necessary for normal growth;and J. S. McHargue, from analyses of cotton99 and blue-grass,lconcludes thaC manganese, copper, zinc, nickel, and cobalt may allbe essential elements. Lack of manganese has been shown to bethe cause of chlorosis in one instance,2 and Bortels states that zincis necessary for the growth of the mould AspergiZZus niger.J. Stoklasa and his associates have for some time past concerned9 4 Ann. Bot., 1927, 41, 167; A., 385.s5 Soil Sci., 1927, 23, 83; B., 307.96 Plant Physiology, 1926, 1, 231.s7 Proc.Roy. SOC., 1925, B, 98, 373; Ann. Reports, 1925, 22, 210.s8 Univ. Calif. Pub. Agr. Sci., 1926, 5 , 57.J . Amer. SOC. Agron., 1926, 18, 1076; A., 1927, 599.Ind. Eng. Chem., 1927, 19, 274; B., 394.B. E. Gilbert, F. T. McLean, and L. J. Hardin, SoiE Sci., 1926, 22, 437;B., 1927, 171. Biochem. Z., 1927, 182, 301; A., 486240 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY,themselves with the occurrence of iodine in plants and the dis-tribution of this element in the earth's crust. Their views aresummarised in a recent paper,* in which it is pointed out that thepresence of iodine can be detected both in volcanic rocks and inmore recent rocks containing the fossilised remains of animal orplant life. They state that iodine promotes the growth of nitrifyingbacteria and that the simultaneous presence of iodine and iron inthe soil leads to a general enhancement of fertility; indeed, theyregard iodine as an essential biogenic element in the synthesisingprocesses of living cells.In this they are supported by K. Scharrerand his co-~orkers,~ who show that iodides, iodates, and periodatesmaterially increase the rate of reproduction of yeast, althoughwithout increasing the final maximum figure. Although these andother experiments make a strong case for the consideration ofiodine as an important element in the economy of plants, there isevidence that conflicts with this view and the claims put forwardcannot be completely accepted until further confirmation is forth-coming.It will be seen from this short summary that an interesting posi-tion has been reached.The work on '' secondary " elements isstill in an early stage and it is not surprising that somewhat con-flicting results have been obtained by investigators working underdifferent conditions. Experimental demonstration is difficult owingto the minute amounts involved, and conditions for growth indifferent parts of the world vary widely. Some plants may have asufficient reserve of " secondary " elements in their seeds to carrythem through a growing season, and therefore it is difficult to pro-duce rigid proof of the essential nature of these elements; but it isprobable that some, if not all, of the elements cited are necessaryfor the full and proper growth of different species of plants underdifferent conditions.Some clue as to the function of such elements is giqen by Brench-ley and Warington,' who observed that boron appears to beassociated with absorption or utilisation of calcium, possibly some-what as silicon appears to be associated with phosphorus nutrition.The inter-relations between silicon and phosphorus form the sub-ject of a paper by W.E. Brenchley, E. J. Maskell, and K. Waring-ton,s whose results are on the whole in agreement with thoseZ . angew. Chem., 1927, 40, 20; A., 171.K. Scharrer and J. Schwaibold, Biochem. Z . , 1927, 185, 405; A., 798;See also K. K. Scharrer and W. Schwartz, ibid., 1927, 187, 159; A., 903.Scharrer, Portschr. Landw., 1927, 2, 119, 249.6 See, e.g., W.E . Brenchley, Ann. Applied Biol., 1924, 11, 86.7 LOC. cit.Ann. Applied Biol., 1927, 14, 45BIOCHEMISTRY. 241reported previously.9 From statistical examination of the data,from pot experiments, they conclude that the effect of addedsilicate can be formulated in terms of an increase in the efficiencyof the phosphoric acid present.A valuable review of the literature regarding the effect on plantsof copper, zinc, arsenic, boron, and manganese is given by MissBrenchley in a recent publication.10General Changes with Growth.An investigation of agricultural and biochemical interest has beenpublished by H. E. Woodman, D. L. Blunt, and J. Stewart l1dealing with the seasonal variations in the productivity, botanicaland chemical cdmposition, and nutritive value of medium pasturage,both on light and on heavy soils.During late years, increasinginterest has been shown in our pasture lands, and although experi-ence and shrewd observation had led to the evolution of suchsystems of grazing as the “ Hohenheim system,” definite inform-ation of a chemical and botanical nature on the above factors waslacking. By far the greater part of the work done on the nutritivevalue of grassland has concerned itself with the hay crop, and littlewas known of the chemistry of the immature growth which obtainsunder grazing conditions. In the investigations now being con-sidered, an attempt has been made to imitate close grazing bycutting plots a t frequent intervals with a lawn mower.The pro-duce so obtained was subjected to botanical and chemical analysis,and, in addition, digestibility trials were carried out.It was found that, under these conditions, the grass contains avery high percentage of protein throughout the whole season, andthe percentage of fibre is much lower than in meadow hay. Theherbage in fact closely resembles a concentrated food, like linseedcake, that has been “watered down” and it has a much highernutritive value than had previously been supposed. Unlike manyfarm concentrates, pasture grass is well supplied with vitaminsand is also rich in bone-forming minerals. The experiments of1925 on light land were repeated on heavy land in 1926 with sub-stantially the same results, and the authors make the interestingsuggestion that a future development may be the production ofhome-grown concentrates for winter maintenance simply by dryingor ensiling short grass cuttings. E.J. Sheehy13 found that thebotanical composition and the nutritive value of the herbage ofSee Ann. Reports, 1925, 22, 210.10 “ Inorganic Plant Poisons and Stimulants,” Camb. Univ. Press, 1927.l1 J . Agric. Sci., 1926, 16, 205; 1927, 17, 209; B., 1926, 606; 1927, 688.l2 Ann. Reports Applied Chem., 1926, 11, 462.l3 Sci. Proc. Roy. Dub. SOC., 1927, 18, 389; B., 791242 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.two pastures of different reputation could be correlated with thedry matter content of the grass.Several investigations dealing with chemical changes during thegrowth of fruit may be noted here.R. H. Roberts l4 states thatblossom bud formation in apples accompanies a condition of balancebetween the nitrogen and carbohydrate content ; and G. T. Nightin-gale 15 has shown the importamce of the same balance in determiningthe growth of tomato plants under conditions of long and of shortillumination. The physical and chemical changes occurring duringthe ripening of grapes have been studied by P. R. V. D. R. Copemanand G. Frater.16B i o c h e m i s t r y of A n i m a Is.Vitamins.This year’s work in the field of vitamins has been characterisedby increasing activity and much important information has beenadded to that of preceding years. Although recent developmentshave not yet resulted in the isolation of any of the vitamins inwhat can be asserted with confidence to be a state of purity, thereare several indications that this advance cannot be long delayed.Vitamin-A.-Two publications have appeared from Japan bythe workers who are continuing there the investigations commencedby the late Dr.Takahashi, and these appear to the Reporter to callfor some comment. It was previously stated 1 that the prepar-ation made from the unsaponifiable fraction of cod-liver oil, towhich the formula C2,H4,(OH), was ascribed and which was called(‘ biosterin,” constituted essentially the pure vitamin. Verysimilar preparations had been obtained by Drummond and hisco-workers and an examination of these led to the conclusion thatthey were essentially unsaturated complex alcohols and hydro-carbons with which the vitamin was admixed in unknown butprobably small amount.Nakamiya and Kawakarni have nowmade a study of the hydrogenation products of “ biosterin,” andin a publication entitled (‘ Hydrogenation of Sterol-free Unsaponi-fiable Matters of Cod-Liver Oil ” they describe the isolation fromhydrogenated (‘ crude biosterin ” of nonacosane, batyl alcohol,myricyl alcohol, an unknown saturated alcohol of m. p. 89-91’,and octadecyl palmitate. Purther, cholesterol appears to haveIbid., Bull. 74, 1927; A,, 1225.Ann. Reports, 1925, 22, 219.Ibid., p. 219.14 Wisconsin Agr. E x p . Sta. Res. Bull. 68, 1926; A., 1927, 283.16 Dep. Agr. Union S. Afr. Sci. Bull. 50, 1926; A., 1927, 908.3 Sci. Papers, I m t .Phys. Chem. Rerrearch (Japan), 1927, 3, 62BIOCHEMISTRY. 243been isolated from the unhydrogenated “ crude biosterin ” whenthe latter was subjected to further purification. In a secondpublication, “ On the Hydrogenation of Biosterin ” 4 the isolationof the same products is described from a preparation called “ purifiedbiosterin.” In view of these results, it is a little difficult to under-stand why the terms ‘‘ unsaponsable ” and “ sterol-free ” are useda t all in the title of the first paper, and it would appear more thanprobable that in “ biosterin ” the Japanese workers are handlingthe same fraction of unsaturated alcohols and hydrocarbons asthat studied by Drummond and his associates. The Reporter hasthought it necessary to refer to these observations, since theywould seem to dispose of the view, advanced with much circum-stantial evidence, that “ biosterin ” really constituted vitamin-d ,and they illustrate afresh the great technical difficulties which besetattempts to separate the vitamin in a pure state.Nakamiya andKawakami confirmed the observation that the growth-promotingpower of their fractions was completely lost after hydrogenation.Rosenheim and Web~ter,~ as the result of a large series of bothcolorimetric and biological tests, have found that the amount ofvitamin-A present in liver fats other than that of the cod, in manycases far exceeds that present in the latter source. They statethat the liver oils of fishes such as the salmon and halibut are often100 times as rich in the vitamin as that of the cod.A discoveryof much greater potential industrial importance is that the liveroils of herbivorous mammals, such as the sheep, calf, and ox, usuallycontain some ten times the concentration of the vitamin found incod-liver oil. It is suggested that such mammalian oils, being freefrom the highly flavoured clupanodonic acid characteristic of fishoils, and from the chromogen responsible for the non-specificFearon colour reaction,6 are well suited for incorporation withmargarine and so constitute a ready means of raising the latter tothe same standard of biological efficiency as butter, so far as vita-min-A is concerned. There is, in fact, no reason why a higherstandard should not be attained. Using the antimony trichloridetest, Wilson has found that the human liver has the same highcontent of vitamin-A as the livers of other mammals.Althoughthe amount is rather variable, fatty extracts from human liver maycontain as much as 25 times the amount found in cod-liver oil.The mechanism of the arsenic or antimony trichloride colourreaction for vitamin-A still remains obscure. Rosenheim hasSci. Papers, Inst. Phys. Chern. Research (Japan), 1927, 7 , 121.ti Nature, 1927, 120, 440; Biochern. J . , 1927, 21, 111 ; A,, 271.Rosenheim and Webster, Biochern. J., 1926,20, 1342; A., 78; Willimott,Biochern. J . , 1927, 21, 1054; A., 1223. a Ibid., p. 386; A,, 486.Moore and Wokes, ibid., p. 1292; A., 78244 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.attempted to elucidate this question in a study of the chromogenicsubstance produced when a chloroform solution of cholesterol andbenzoyl peroxide is evaporated to dryness.The substance obtainedgives with arsenic trichloride a blue colour indistinguishable inappearance from that given by an oil containing vitamin-A. Butwhen the purified chromogen is added to vegetable oils which donot themselves give the arsenic chloride reaction, no colour is thenobtained. The colour developed by the artificial product does notfade so rapidly as that given by an oil containing the vitamin.Further, there are differences in the absorption spectra of the twopigments, and it is evident that this artificial chromogen, althoughit must bear some family resemblance to the natural chromogenor vitamin, is not identical with the latter.All the availableevidence still supports the view that the chromogen and vitamin-Aare one and the same substance.In a study of the determination of vitamin-A by the biologicalmethod, Steenbock and Coward 9 recommend supplying vitamin-l)in the form of an irradiated sterol, and state that the incidence ofophthalmia in experimental animals forms a better criterion of the .depletion of the animal’s store of vitamin-A than does cessation ofgrowth.Vitamin-€3.-The past year has seen the definite recognition ofwhat has been widely suspected by workers on this vitamin, namely,the existence of two distinct components of what has hitherto beencalled water-soluble vitamin-B. The question of nomenclature a tonce arises and the present occasion seems to be a suitable one forconsidering the general principles to be adopted in naming newvitamins-a problem which shows signs of becoming increasinglyacute.For the worker in fields other than that of the vitamins,and for the medical practitioner, it must be highly disconcertingto find a vitamin, familiar to him under the term, shall we say, X ,becoming fragmented into two or more vitamins X , Y , and 2,amongst which are distributed, in a manner quite mysterious tohim, the properties formerly exclusively assigned to the original X .With the view of preserving a greater degree of continuity in theliterature, it seems desirable to evolve a system which will obviatethis difficulty and a t the same time clearly differentiate the con-stituents of any complex vitamin group. For these reasons, itdoes not seem that the suggestions of Sherman and Axtmayer lC areto be recommended.These are that the term “vitamin-B” bereplaced by the terms “ vitamin-F ” (heat-labile, anti-neuriticcomponent) and ‘‘ vitamin4 ” (heat-stable, pellagra-preventiveJ . Biol. Chem., 1927, 72, 765; A,, 595.lo Ibid., 1927, 76, 207; A,, 1223BIOCHEMISTRY. 245component). A recent suggestion of the Accessory Food FactorsCommittee l1 in this country seems more serviceable, and is to thefollowing effect : (1) the term " vitamin3 " should be retained forthe group of water-soluble vitamins to which the term was firstapplied by McCollum and Davis in 1915 ; (2) the term " vitamin-B, "should be used for the more heat-labile, anti-neuritic vitamin(called " torulin " by Kinnersley and Peters) l2 required to preventpolyneuritis in birds, marasmus, with or without paralysis, inmammals, and beri-beri in man ; (3) the term " vitamin-B, " shouldbe given to the more heat-stable component (called P-P by Gold-berger and his associates in America) necessary for the maintenanceof growth and health, and for the prevention of characteristic skinlesions in rats and of pellagra in man.The committee also recom-mend that the term '' bios " be retained for the substance or sub-stances encouraging the rapid growth of yeast-cells. These sug-gestions are only tentative and have not as yet been officiallyadopted ; nevertheless for the sake of simplicity and clarity the useof the terms B, and B, will be adopted in this Report.Since the adoption of the view that the growth-promoting,water-soluble vitamin-B was identical with the anti-neuritic vitamincurative of polyneuritis in birds-a suggestion first made byMcCollum and Kennedy in 1916-much evidence has accumulatedwhich is slightly but definitely a t variance with that view, and itis the steady accretion and strengthening of this evidence which hasled to the recommendations mentioned above.The evidenceagainst the identity of water-soluble-B (in the original strict sense)with the anti-neuritic vitamin may be grouped under three heads : 13(1) distribution in nature ; (2) differences in heat stability ; (3) differ-ences in solubility and other physical properties.As regards (l),wheat embryo is rich in B, but poor in B,, whereas the reverse istrue of milk, meat, green leaves, roots and tubers. Many yeasts ofequal B, content vary considerably in regard to B,. Under (2)come the observations that a t 120" B, is much more sensitive toinactivation than is B,, so that on autoclaving yeast for four orfive hours there is obtained a preparation devoid of B, but stillpotent as regards B,. (3) The physical differences are shown by thegreater solubility of B, in alcohol, acetone, and benzene, andfurther by the greater tendency of B, to be adsorbed by charcoalor fuller's earth.Institute of Preventive Medicine.904.Axtmayer, loc. cit.; Salmon, J .Biol. Chem., 1927, 73, 483; A., 796.l1 Appointed jointly by the Medical Research Council and the Listerl2 Biochem. J , 1925,19, 820; 1927, 21, 777; A., 1925, i, 1616; A., 1927,l3 Chick and Roscoe, Biochem. J . , 1927, 21, 698; A., 702; Sherman an246 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.The studies of human pellagra by Goldberger and his associates 14dating from 1924 have led to the abandonment of their earliertheory that this disorder was caused by the inferior biologicalvalue of the dietary proteins, and to the adoption of the view thatthere is present in water-soluble vitamin-B a pellagra-preventiveprinciple (which they call P-P), identical with the growth-promotingprinciple of McCollum and Davis. Chick and Roscoe l5 corroboratethe results of Goldberger in regard to pellagra.Important furthersupport of these views regarding the multiple nature and physio-logical r61e of the components of vitamin-B is forthcoming fromDrummond’s laboratory,l6 and it would appear that much con-flicting evidence regarding the physiological r61e of vitamin-Bwill become more easily interpreted on wider recognition of itscomposite nature. That the complexity of vitamin-B may nothave been completely unfolded by the recognition of vitamins-B,and -B, is suggested by the observation of Boas l7 that crude egg-white, boiled and supplemented by an adequate diet, is not capableof supporting growth and health in young rats if the egg-whitehas been dried before being boiled. On the other hand theefficiency of the egg-white is not impaired if it be coagulated previousto desiccation.The ill-effects resulting from the ingestion of driedegg-white are counteracted by raw potato, potato starch, arrowroot,dried yeast, fresh egg-white, egg-yolk, milk, commercial casein,crude lactalbumin, spinach, cabbage leaves, banana, and driedhorse serum. These substances are supposed to possess someprotective principle which, although similar in distribution to theB-vitamins, is not identified with either B, or 23,. It is furthersuggested that there is a balance between the amount of the driedegg-white ingested and the amount of the protective principlerequired. A somewhat similar problem was encountered byReader and Drummond,18 who found that a diet consisting largelyof casein became adequate when the ratio of yeast extract to proteinwas raised considerably, and related problems of balance of food-stuff by vitamin-B have been investigated by Plimmer, Rosedale,and Raym0nd.1~The isolation of vitamin-B, is claimed by Jansen and Donath, 20l4 Goldberger and Tanner, U.S.Pub. Health Rep., 1924, 39, 87; 1925, 40,54; Goldberger, Wheeler, Lillie, and Rogers, ibid., 1926, 41, 297 ; Goldbergerl6 Kon and Drummond, Biochem. J . , 1927, 21, 632; Hassan and Drum-l7 Ibid., 1927, 21, 712; A., 797.la Ibid., 1926, 20, 1256; A., 1927, 79.lo Ibid., 1927, 21, 913, 1141; A., 905, 1224.zo Proc, K. Akad. Wetensch. Arneterdam, 1926, 20, 1390; A., 1927, 382.and Lillie, ibid., p. 1025. 1 5 L O C . cit.mond, ibid., p.653; A., 702BIOCHEMISTRY. 247who describe the isolation of a residue weighing 1.4 g. from 100 kg.of rice polishings and containing about one-quarter of the amountof the vitamin originally present. The product is stated to be thehydrochloride of a base and to it the formula C,H,,ON,,HCl isascribed. Its chemical behaviour suggests the presence of a gly-oxaline nucleus. Eykman 21 states that this preparation curespolyneuritis in fowls.'Vitumin-C.-No very striking advance falls to be recorded inregard to the anti-scorbutic vitamin, but Bezssonoff 22 suggests thatthis vitamin too is a complex consisting of two substances, differingin their heat stabilities, one probably being derived from the other.Hoyle and Zilva 23 report that the concentrated anti-scorbuticfraction of lemon juice contains iron, phosphorus, and sulphur, andthat these elements dialyse along with the vitamin.On the otherhand, Vedder and Lawson 24 state that their concentrated prepar-ations, made by extraction with alcohol, could be freed from phos-phorus and sulphur without loss of activity. In an interestingquantitative study of the reducing power of the anti-scorbuticfraction of lemon juice towards phenolindophenol, Zilva 25 showsthat if sufficient of this indicator be added to destroy the reducingproperty of the solution, the reduced leuco-compound of theindicator is re-oxidised in the air and is then further reduced bythe solution. This alternate reduction and oxidation proceeds untilthe reducing power of the medium is destroyed.The reducingproperty of decitrated juice or of its active fractions is lost, like theanti-scorbutic activity, in an alkaline medium in the presence ofair, but on fractionation of the juice the substance responsible isfound in as high quantities in the inactive as in the active fractions.On adding the indicator to decitrated lemon juice until the formeris no longer reduced, and on testing the solution so treated immedi-ately, no very appreciable loss in the anti-scorbutic activity isobserved. Neither the reducing capacity nor the anti-scorbuticactivity undergoes any appreciable diminution when the decitratedjuice is kept for one hour in neutral or acid solution in an autoclavea t a pressure of one atmosphere.On storing, both propertiesdeteriorate very much more quickly than in untreated decitratedjuice. Zilva suggests that the stability of vitamin4 possiblydepends on a sequence of reactions which are normally kept inequilibrium in the living cell-a hypothesis of great interest in21 Proc. K . Acad. Wetensch. Amsterdam, 1927, 30, 376; A., 1224.22 Compt. rend., 1926, 183, 1309; A., 1927, 283.23 Biochem. J . , 1927, 21, 1121; A., 1224.J . Biol. Chern., 1927, 73, 215; A., 702.z6 Biochem. J., 1927, 21, 689; A., 702248 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.view of the suggestion of Bezssonoff, already referred to, regardingthe complex nature of this vitamin.Yitumin-D.-In order to present a more complete survey of thepresent position of the numerous investigations in progress withregard to the formation and properties of vitamin-D, it will beadvisable to include in the present Report some reference to mattersmentioned in the Report of last year.Early in 1927 there appearedthe detailed results of Heilbron, Kamm, and Morton,26 who wereable to show in a spectrographic study of cholesterol, before and afterirradiation with ultra-violet light, that purified cholesterol containsin small amount another substance which can be accumulated inthe least soluble fraction on crystallisation from ethyl acetate,that this substance possesses absorption bands in the ultra-violeta t 293 pp, 280 pp, and 269 pp, whereas cholesterol itself has onlygeneral absorption, and that these bands disappear on irradiationwith a concomitant appearance of anti-rachitic potency.It wassuggested that this unknown substance and not cholesterol itselfwas closely related to the precursor of vitamin-D. While this workwas in progress Rosenheim and Webster 27 in this country andWindaus and Hess 28 in Germany were making a, detailed examin-ation of the effects of irradiating a, large number of cholesterolderivatives and related compounds. In the course of this work itwas shown that the trebly unsaturated and highly labile ergosterol,C27H420, gave rise to a highly potent anti-rachitic substance. Itwas further shown that cholesterol and the phytosferols, stigma-sterol, C,,H,,O, and sitosterol, C2,H460, when brominated, andsubsequently reduced by the action of sodium amalgam and aceticacid in order to regenerate the original sterol, were quite unable,after irradiation, to prevent the development of rickets in rats.It was suggested that the labile provitamin had been destroyed inthe course of this treatment.In view of the high sensitivity ofergosterol to oxidative processes, of its ultra-violet absorptionspectrum, similar to, but more intense than, that of impure “activ-atable ” cholesterol, and of the very high degree of anti-rachiticpotency developed by ergosterol on irradiation (Rosenheim andWebster state that the curative dose for rickets, developed in rats,is of the order of 1/10,000 to 1/20,000 mg. per diem), both Rosen-heim and Webster and Windaus suggest that provitamin-D isergosterol or some closely related sterol.They suggest that it isthe presence of small amounts of the latter, in the proportion ofabout 1 part in 2000, in all specimens of cholesterol prepared from26 Biochem. J . , 1927, 21, 78; A., 381.e7 Ann. Reports, 1926,23,254; Biochern. J . , 1927,21,127,389; A., 381,487.OB Nachr. ges. Wise. Gcttingen, 1927, 175, 84BIOCHEMISTRY. 249natural sources that is responsible for the development of anti-rachitic properties. Irradiated ergosterol is certainly the mostpotent anti-rachitic substance known and it is estimated that about5 mg. are equivalent to about 1 litre of a good cod-liver oil. Dr.Katharine Coward is reported by Rosenheim and Webster to havedetected the calcifying effect of 1 /100,000 mg. of irradiated ergosterolby means of the “ line ” test.Rosenheim and Webster 29 have published a further study of themechanism underlying the conversion of ergosterol into vitamin-D,in the course of which it is shown that the maximum activity isattained within 30 minutes after exposure to the radiations of amercury vapour lamp, the usual precautions being adopted toexclude oxidative changes.Thereafter the activity does not increasepari passu with the disappearance of ergosterol, but remains con-stant up to 4 hours’ irradiation. It is suggested that after a shortinitial period the formation and destruction of the vitamin proceedat the same rate until the available supply of ergosterol is exhausted.It would in any case appear that the conversion of ergosterol intovitamin-D is not a simple unimolecular reaction. Rosenheim andWebster record the further important observation that the com-paratively long-wave radiations of solar ultra-violet light are capableof activating ergosterol.The high content of cholesterol present inhuman skin (13 to 24%), and the presence in this cholesterol ofsome substance possessing the same ultra-violet absorption asergosterol, being borne in mind, this observation is of the greatestinterest in relation to the curative effect of sunlight in rickets.In view of the results just described, it would seem that there isno serious obstacle to the belief that provitamin-D is identical withergosterol. Nevertheless, the observations of Jendrassik andKemdnyffi 30 lead these authors to suggest an alternative theory.In the first place, they confirm the statements of Rosenheim andWebster and of Windaus and Hess that irradiated ergosterol pro-vides a highly potent anti-rachitic preparation, but they have failedto confirm the observation that cholesterol, after bromination andsubsequent reduction, cannot be activated by irradiation. It isalso asserted that in a series of fractionation experiments inactivecholesterol, after removal of the active fraction by recrystallisationand washing, can be reactivated repeatedly.The period of irradi-ation used by these investigators is 1 hour and the cholesterol isirradiated in thin layers containing 0.01 g. per square cm., and itis apparently assumed that complete conversion of the provitamininto the vitamin occurs under these conditions.The successive29 Lancet, 1927, ii, 622; A., 1224.3o Biochem. Z., 1927, 189, 180; A., 1224250 ANNUAL REPORTS ON THE PROGRESS Or CHEMISTBY.development of anti-rachitic potency in inactive fractions, fromwhich the active substance developed in prsvious irradiations hasbeen removed, and especially their success in activating cholesterolafter bromination, lead Jendrassik and KemBnyfii to the remarkableconclusion that, although cholesterol itself is not the provitamin, itgives rise to the latter in the presence of water. They also statethat on withdrawal of the last traces of water from cholesterol theprovitamin is destroyed and the possibility of its re-formation islost together with a concomitant disappearance of the characteristicabsorption bands, unless it be again treated with water.Theseremarkable suggestions of Jendrassik and Kembnyfii merit attention,and it appears to the Reporter that, apart from the brominationexperiments, they are not incompatible with the results of Rosen-heim and Webster and of Windaus. It is therefore highly desirablethat the question of the activatable nature or otherwise of bromin-ated and reduced cholesterol should be subjected to rigorous testsunder widely varying conditions. It is to be noted that Jendrassikand Kern&@ subjected the cholesterol which they regeneratedfrom the dibromo-compound to two evaporations on the steam-bathwith wet alcohol. Hess and Anderson,3l who have separated sito-sterol from corn oil into a-, p-, and y-fractions, the first-mentionedbeing the most soluble and least stable of the three, confirm thefact that neither the p- nor the y-fraction could be activated byirradiation after purification by means of the respective bromo-compounds.The freshly prepared a-sitosterol could be activated(omitting the bromine treatment), but Hess and Anderson are notcertain of the degree of purity of their preparation.In view of the results described above, it is obvious that thenumerous observations published during the past year on theactivation and fractionation of cholesterol and of its derivativesmust be re-examined, but at the same time much valuable inform-ation has been accumulated which will no doubt be more readilyinterpreted when the chemical natures of vitamin-D and its pre-cursor have been elucidated, an achievement which cannot now belong delayed.Vitamin-E .-Little has been added to our information concerningthe anti-sterility vitamin.Sure 32 and Hartwell 33 both report thatfertility of rats is diminished, or complete sterility may be pro-duced, by diets in which cod-liver oil is the sole source of fat andfat-soluble vitamins. Sure was able to restore fertility by supple-menting the diet with 0.035% of the unsaponifiable matter from31 J . Biol. Chem., 1927, 74, 651; A., 1224.82 Ibid., pp. 37, 45, 71 ; A,, 905.83 Biochem. J . , 1927, 21, 1076; A., 1107BIOCHEMISTRY. 251cotton-seed oil, or by a large addition (10%) of butter.Hartwellconfirms the advantage of butter over cod-liver oil. Simmonds,Beckcr, and McCollum 34 state that the death of rat-fcetuses throughlack of vitamin-E is due to a crisis in their assimilation of iron, theadministration of ferric citrate or wheat oil being beneficial. Theyreport that liver oils are rich in vitamin-3, a result which seems tobe at variance with the results of other workers on cod-liver oil.Speci$c Carbohydrates from Bacteria.Professor Drummond dealt in the Report for last year with thehighly interesting advance in the chemistry of specific immunologicalreactions rendered possible by the work of Heidelberger, Avery, andtheir associates.35 Further progress in the study of the nature ofthe specific polysaccharides isolated by them has been made in thepast year.Heidelberger and Goebe136 have shown that the poly-saccharide of Type I11 pneurnococcus, on acid hydrolysis, yieldsglucose and an acid of the type of glycuronic acid having theformula Cl,H,,Ol,. It has one half of the reducing power of glucose,gives a positive reaction with naphtharesorcinol, contains analdehyde group, and on oxidation with nitric acid gives saccharicacid. On oxidation with barium hypoiodite 37 the original acidgives a dicarboxylic acid, C,,H,,O,(CO,H),, which still responds tothe naphtharesorcinol reaction and yields the same amount offurfuraldehyde as the original acid. It is therefore deduced thatthe latter is an aldobionic acid composed of one molecule of glucoseand one of glycuronic acid combined in glucosidic linking throughthe aldehyde group of the latter.The following formula is thereforeto be ascribed t o this substance :CO,H*CH*[CH*OH],*CH-O - CH,*CH*[CH*OH J,*CH*OHL - 0 2Glucose.-0-Glycuronic acid.The reducing group of the glycuronic acid residue may be attachedto the glucose residue in the position 6 shown, or to any one of thepositions 2, 3, and 4. The evidence does not yet permit of a choicebeing made between these possibilities.Goebel 38 has obtained from the specific polysaccharide of Fried-lander's type A bacillus an aldobionic acid which is composedlikewise of glycuronic acid and glucose apparently linked in thesame way as the components of the pneumococcus acid, with whichit is isomeric.One would suggest that the presence of one of the94 J . Amer. Med. ASSOC., 1927, 88, 1047; A., 1224.as Ann, Reports, 1926, 23, 248.as J . Biol. Chem., 1927, 70, 613; A., 77.ST Ibid., 74, 613; A., 1114. 8 8 Ibid., p. 619; A., 1114252 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.possible linkings just mentioned, other than that present in thepneumococcus acid, is responsible for this isomerism.The parent polysaccharides from which these interesting sugaracids are derived are regarded as bei,ng composed of units of thealdobionic acid in the case of the pneumococcus substance, and ofunits composed of two molecules of the aldobionic acid and onemolecule of glucose in the case of the Friedlander substance.To the list of organisms which yield specific carbohydrates withimmunological properties is to be added the cholera vibri0.3~Organic Phosphates and Lactacidogen.During the past year much attention has been directed to thechemistry of the known hexosephosphates and to the question ofthe presence and functions of organic phosphates in muscle tissue.The most noteworthy advances have been made in the chemistryof the first discovered hexosephosphate, the diphosphate of yeastfermentation, in the chemistry of lactacidogen, the hexosephosphateof muscle, and in the demonstration that there are present in musclecertain organic nitrogenous phosphates which appear to play ahighly important r6le in muscle function.The Hexosediphosphoric Acid of Yeast Fermentation.-Morgan 40has been able to prepare from this acid the a- and p-methylhexoside-diphosphoric acids by subjecting the parent substance to theFischer-Speyer acid-methyl alcohol process.The two stereoiso-merides were obtained in the form of their brucine and barium salts.The glucosidic methyl group of the a-acid is more readily removedby hydrolysis than that of the p-acid, but neither hexoside ishydrolysed by emulsin. On the other hand, invertase causes apartial removal of the methyl group of the a-acid, but it does notaffect the p-acid. Of great interest is the observation that the boneenzyme (phosphatase) rapidly removes the phosphoric acid groupsof the p-acid, leaving a strongly laevorotatory, non-reducing sub-stance possessing the properties of a methylfructoside. An extensionof these results recently described by Morgan and Robison41 hasled to the suggestion that the structure of hexosediphosphoric acidis that of y-fructose-1 : 6-diphosphoric acid :H H TH YHs9 Landsteiner and Levine, J.E x p . Med., 1927, 46, 213.40 Biochem. J . , 1927, 21, 675; A., 749.41 Report of the Meeting of the Biochemical Society (Dec. 9th), J. Soc.Chern. Ind., 1927, 46, 1183BIOCHEMISTRY. 253By the action of bone phosphatase on the a- and p-methylhexosidedi-phosphoric acids, the corresponding a- and p-methylhexosides wereobtained, and these underwent rapid hydrolysis at room tem-perature on being treated with 0-O1N-hydrochloric acid to yield alaevorotatory sugar corresponding to +fructose. Such behavioura t once suggests one of the "reactive" butylene-oxidic types ofsugar and this was confirmed by converting the 8-methylhexosideby methylat ion into the corresponding tetramet hyl p -me t hyl-hexoside.The latter had [a]i2, - 64", and on hydrolysis yieldedan ap-tetramethyl hexose having [a]:$, + 30" in close agreementwith the known rotation of ap-tetramethyl y-fructose, and widelydivergent from that of the corresponding normal (amylene-oxidic)ap-tetramethyl fructose, which has [.ID - 142". Morgan andRobison seem justified in inferring that the existence of theunstable butylene-oxidic ring in conjunction with an unsubstitutedreducing group makes it very probable that the second phosphoricacid group occupies position 6, although it is obvious that the proofis not absolute.This result should not be regarded as unexpectedin view of the known fact that all hitherto described naturallyoccurring compounds of fructose have been shown to exist in they- or butylene-oxidic form, so that hexosediphosphoric acid wouldappear to fall into line with other fructose compounds. Schlubachand Rauchenberger 42 have recorded the complete methylation ofhexosediphosphoric acid via the silver salt, which on treatmentwith methyl iodide yielded tetramethyl hexosediphosphate, andon further treatment with silver oxide and methyl iodide affordedtetramethyl trimethylhexosediphosphate, which is reported to have[.ID + 20-77" in chloroform solution. It would be of great interestto know if the latter were susceptible to the action of any phos-phatase preparation.The Hexosernonophosphoric Acid of Yeast Fermentation.-Neubergand Leibowitz 43 have made a study of the hexosemonophosphoricacid first isolated from yeast fermentations by Robison.They arriveat the conclusion that the discrepancies between the reducing powerafter hydrolysis of the ester with taka-diastase, as determined bycopper (Bertrand) and by hypoiodite (Willstatter-Schudel) methods,cannot be explained, in view of the polarimetric findings, by assum-ing the presence of a mixture of 85% of glucose with 15% of fructose.The hexosemonophosphoric acid is thought to consist of from 80 to90 yo of a homogeneous substance which suffers secondary changesin the sugar residue on being hydrolysed. Meyerhof and Loh-r n a n ~ ~ , ~ ~ also employing the method of Willstiitter and Schudel,44 Ibid., 1927,185, 13; A,, 697,42 Ber., 1927, 60, 1178; A., 644.A3 Biochem, Z., 1927, 184, 489; A,, 700254 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.conclude that Robison’s monophosphate has a higher aldose valuethan Neuberg’s, a result which is not surprising in view of Morganand Robison’s investigations of the yeast diphosphoric acid fromwhich Neuberg’s acid is derived.In a further publication, Neubergand Leibowitz 45 record the conversion of hexosediphosphoric acidof yeast into Neuberg’s monophosphate by the action of taka-diastase, and of Robison’s monophosphate into the diphosphate bythe action of bottom yeasts. This is the first record of the prepar-ation of the Neuberg monophosphate by strictly biological methods,and the conversion of Robison’s monophosphate into the typicalyeast diphosphate probably involves, according to our presentconception of the relationship of these two compounds, the pre-liminary removal of the phosphoric acid group.Euler and Myr-back 46 also state that when Robison’s hexosemonophosphate istreated with dried bottom yeast, one half is fermented and theother half is converted into the diphosphate.An interesting point in the biochemistry of the carbohydrates,involving the behaviour of sugar phosphates, has been raised byProfessor R0binson.4~ He points out that in the hydrolysis ofphosphoric esters there is much evidence to suggest that, if theoxygen atom be directly attached to an asymmetric carbon atom,optical inversion should in many cases be observed.It has alwaysbeen difficult to explain the formation of glucose from galactose,or the reverse change, without assuming a profound disruption ofthe carbohydrate molecule. Professor Robinson now suggests thatthe galactose configuration may be derived from that of a glucose-4-phosphoric acid by a dephosphorylation involving a, Waldeninversion. This hypothesis is highly suggestive in view of the wide-spread occurrence of sugar phosphates in nature. It is to be notedthat it would be excluded if glucose possessed a butylene-oxidicstructure. Levene 4* has criticised this hypothesis unfavourably inpointing out that the acid hydrolysis of glucose-3-phosphoric acid isnot accompanied by any optical inversion, but the original theorypresumably did not regard every or any method of hydrolysis asnecessarily involving the inversion.Professor Robinson has alsosuggested that the pentose of plant nucleic acid, which is isolatedin the form of the rare sugar d-ribose, may in situ be the muchcommoner d-xylose, the removal of the phosphoric acid group fromthe latter during hydrolysis inverting the codguration of (neces-sarily) the 3-carbon atom. This view also is criticised by Levene.4 5 Biochem. Z., 1927,187, 481; A., 993.46 Z. phpiol. Chem., 1927, 167, 236; A., 794.4 7 Nature, 1927, 120, 44; A., 960.4 8 Ibid., 1927, 120, 621; A., 1226BIOCHEMISTaY. 255Lactacidogen.-During the period covered by this Report thechemistry of lactacidogen has undergone a modification which willprobably re - orient many investigations of the chemical mechanismsunderlying muscle contraction.Embden and Zimmermann 49 in1924 identified muscle lactacidogen with the hexosediphosphoricacid of yeast fermentation, since the two substances were shown toform the same neutral brucine salt. The method of isolation oflactacidogen which these investigators used a t that time involvedthe treatment of the muscle press-juice with glycogen, sodiumfluoride, and sodium bicarbonate, with the view of removing, by aprocess of fermentative re-synthesis, the free phosphate present inthe press-juice. It was thought that this synthetic processinvolved merely the re-formation of lactacidogen which had suffereddegradation during the extractive manipulations.That thisprocedure yields a hexosediphosphoric acid identical with thatproduced during yeast fermentation has recently been confirmedby Pryde and Waters,5o who obtained a brucine hexosediphosphatefrom rabbit's muscle with a specific rotation, [a]:& - 30*7",identical with that of a carefully purified preparation of the brucinesalt of the yeast acid. During the past year Embden and Zimmer-mann 51 have isolated lactacidogen from rabbit's muscle by amodified process which omits the fermentative re-synthesis usingglycogen and fluoride, and they have obtained, instead of hexose-diphosphoric acid, a hexosemonophosphoric acid-an observationwhich has also been confirmed by Pryde and Waters.52 Embdenand Zimmermann state that the new monophosphate differs fromthe two previously known natural monophosphates (Robison's andNeuberg's), but Pryde and Waters have encountered anomalies inthe rotation and solubility of their various preparations which leadthem to suspect the homogeneity of the monophosphate obtainedby them. Embden and Zimmermann have shown that the newmuscle monophosphate is converted by muscle press-juice into lacticacid, and by muscle press-juice, glycogen, and fluoride, into hexose-diphosphoric acid.They are therefore disposed to identify lact-acidogen with the new monophosphate. In the absence of theartificial re-syn thesising solutions, neither Embden and Zimmer-mann nor Pryde and Waters were able to detect the presence ofany diphosphoric acid. It is none the less possible that thetemporary formation of the diphosphate may play some part inthe muscle process (compare p.261).49 Ann. Repow, 1925, 22, 224.6o Report of the Meeting of the Biochemical Society (Dec. 9th), J . SOC.O1 2. physiol. Chena., 1927,167, 114; A., 749.Chem. Ind., 1927, 46, 1182.s2 LOC. cit256 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.Adenylic Acid.1n the course of the extraction of the mono-phosphoric acid from rabbit's muscle, Embden and Zimmermann 53encountered a nitrogenous organic phosphate which they were ableto identify as adenylic acid-an observation which again has beenconfirmed by Pryde and Waters.54 It would appear highly probablethat adenylic acid is the parent substance of inosinic acid, longknown to be a constituent of muscle extractives, since a simpleprocess of deaminisation of the former would yield the latter.That such a process, involving release of nitrogen, may play somepart in the contractile mechanism is suggested by a growing bodyof evidence, which will be considered shortly.Meanwhile mentionmust be made of another nitrogenous organic phosphate for whicha place will probably have to be provided in muscle chemistry.Phosphugen.-Eggleton and Eggleton, 55 in determining theinorganic phosphate of muscle, found that methods such as thoseof Briggs or Embden, in which acid reagen$s are used, gave results,on the resting gastrocnemius of the frog, which were higher, bysome 70 mg.P per 100 g. of muscle, than the results obtained by amethod such as the Bell-Doisy, which is carried out in a mildlyalkaline solution and gave results of the order 20 to 25 mg. P per100 g. of tissue. They found that the discrepancy was due to thepresence in the muscle of a labile organic phosphate which wasrapidly broken down in acid solution to form inorganic phosphate.In a further study 56 it was shown that this labile organic phosphateparticipates in the chemical mechanism of contraction and is com-pletely broken down when the muscle is fatigued in tetanus. Norestitution of the organic phosphate was observed under anaerobicconditions, but in the presence of oxygen it is rapidly re-formed andan equivalent amount of inorganic phosphate is lost.This Grobicrestitution process is apparently more rapid than the removal oflactic acid which occurs during the recovery phase. Eggleton andEggleton suggested the name " phosphagen " for this unknownlabile phosphoric acid compound. Fiske and Subbarow 57 inAmerica encountered the same labile phosphorus compound andshowed that in the normal resting voluntary muscle of the cat,what had previously been regarded as " inorganic phosphate "consisted of some 60 to 75 mg. of labile organic phosphate and some20 to 25 mg. of true inorganic phosphate per 100 g. of tissue, figureswhich are remarkably close to those of Eggleton and Eggleton forthe frog. Fiske and Subbarow also made the highly interestingb3 2. physiol. Chern., 1927, 167, 137; A., 787.64 L O G . cit.5 6 Biochem. J . , 1927, 21, 190; A,, 271.b6 Eggleton and Eggleton, J . Physiol., 1927, 63, 155; A., 990.Science, 1927, 65, 401 ; A., 990BIOCHEMISTRY. 257observation that the labile phosphorus compound was composed ofcreatine and phosphoric acid, an observation which was shortlyafterwards confirmed by Eggleton and E g g l e t ~ n , ~ ~ one molecule ofcreatine being associated with each atom of phosphorus.These observations are obviously of the greatest importance inrelation to all previous determinations of the inorganic phosphoricacid of muscle, and a further point of interest emerges when it isseen that incubation of a chopped muscle in the presence of sodiumfluoride leads to the conversion of “ phosphagen ” into an acid-stable organic phosphate.It would appear possible that ‘‘ phos-phagen ” is the source of the second phosphoric acid residue whichis added on to Embden and Zimmermann’s muscle hexosemono-phosphate when the latter is treated with muscle press-juice,glycogen, and fluoride. The part played by “ phosphagen ” in thecontractile process is not yet clear. That most, or probably all, ofthe muscle creatine is present in the resting muscle in combination,with phosphoric acid is strongly suggested by the fact that creatine,like “ phosphagen,” is most abundant in voluntary muscle, less soin cardiac muscle, and present only in traces in involuntary muscle.Ammonia Formation i n Muscle.In the Report for last year the observations of Hoet and Marks 59on the precipitate rigor, which sets in immediately after death froman overdosage of insulin or from excessive thyroid feeding, in whichconditions the muscles contain no glycogen, little or no lactacidogen,and show no accumulation of lactic acid, suggested that a possibledetermining factor in this type of rigor was an alkaline phase.Atthe time little or nothing was known of any possible source ofalkalinity, but since then it has been shown that the formation ofammonia is probably an integral part of the muscle process, andthis suggestion gains strength in view of the occurrence in muscleof the nitrogenous compounds which we have just considered.Parnas and Mozolowski 6o have shown that the maceration ofvertebrate muscle in water or saline leads to the formation of about5 mg.of ammonia per 100 g. of muscle. This formation of ammoniais inhibited by a borate buffer of pH 9.3. It is suggestive that thistraumatic formation of ammonia is most marked in voluntarymuscle, less so in heart and smooth muscle, and absent in glandulartissues. The process of formation is a rapid one, being complete inthe case of the frog’s gastrocnemius in 90 seconds. On extractingmuscle with a borate solution the ammonia precursor is obtainedJ . SOC. Chem. Ind., 1927, 46, 485.68 Ann. Reports, 1926, 23, 242.6o Biochem. Z . , 1927, 184, 399; A., 694.REP.-VOL. XXIV. 268 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.together with an enzyme which acts upon it, and ammonia formationoccurs on simple neutralisation of this extract, although a t a muchslower rate than in the intact cellular tissue.The precursor isstable in both acid and alkaline solutions. Since the ammonia ofthe intact muscle is increased by electrical stimulation and bystrychnine convulsions, it is inferred that its release is definitelyrelated to contraction. Habs,61 in association with Embden, alsohas studied the occurrence of ammonia in muscle, and shows thatits formation in muscle pulp runs parallel with the liberation of freephosphate. It is an attractive hypothesis to regard adenylic acidas the precursor of the muscle ammonia, and Embden 62 has indeedadvanced this suggestion, but Parnas points out that there wouldbe required for the traumatic formation of 5 mg.yo of ammonia25 mg. % of purine nitrogen as adenylic acid. Now the total purinenitrogen of the frog's muscle, including that of nuclear substances,is only 35 mg. yo. For the muscles of other animals, too, almostthe whole of the purine nitrogen would have to be present in theform of adenylic acid should this substance be the sole precursor ofthe ammonia determined by Parnas and Mozolowski. It is difficultto see how creatine can be regarded as the ammonia precursor, noris there any evidence to suggest that it is. As Habs points out,adenylic acid is the only substance capable of forming ammonia sofar detected in the muscle and as such it merits further consideration.In a comparison of the chemical processes of " trained '' (subjectedto short, periodic, daily faradisation in the intact animal) as com-pared with " untrained " muscle, Embden and Habs 63 show thatthe " trained '' muscle shows a marked increase in glycogen contentand a small but definite increase in the residual nitrogen.They donot suggest what particular nitrogen compound or compounds areincreased, but do show that the creatine figures are not affected.It is obviously difficult a t the moment to correlate this newerwork on the chemistry of muscle with the older work on lactic acidformation, but it seems certain that a considerable widening of ourviews on the whole subject must soon result.Lactic Acid-forming Enzymes from Muscle.Much interesting work has been published on the processes oflactic acid formation in muscle, but in the opinion of the Reporterthe most significant advance is the isolation by Meyerhof of anactive lactic acid-forming enzyme from muscle.This was referredto in the Report of last year,64 but since that date Meyerhof has61 2. physiol. Chern., 1927, 171, 40.s2 Klin. Wochenschr., 1927, 6, 628.e8 2. phy8iol. Chem., 1927, 171, 16.64 Ann. Reports, 1926, 23, 242BIOCHEMISTRY. 269himself provided a useful summary,65 and many more recent resultshave been published which justify a detailed account in the presentyear's Report.Meyerhof 66 has shown that it is possible to separate completelythe lactic acid ferment from frog or rabbit muscle and to obtain it inaqueous solution free from the carbohydrates of the muscle. Follow-ing a method of Buchner, it is possible, by precipitation with acetone,to obtain a dry enzyme preparation which, when redissolved,possesses 40% of the activity of the original extract.For example,when glycogen is added to it, the enzyme preparation shows forseveral hours an activity about as great as that of the mincedmuscle a t the same temperature, when calculated against muscleweight. Calculated against the dry weight of the extract, itsactivity is a t least five times as great as that of the muscle pulp.A co-enzyme, which is dialysable and thermostable, can be separatedfrom the enzyme mixture. It has been shown that this water-soluble lactic acid ferment splits hexoses only under special con-ditions, which will be referred to later, but on the other hand, in thepresence of inorganic phosphate 67 it readily acts upon starch,glycogen, the starch components amylose and amylopectin, and thesimpler compounds derived from them, such as tri- and di-hexosans,splitting them all to lactic acid with about the same velocity.During hydrolysis of the polysaccharides, a phosphoric esteraccumulates a t first quickly, then more slowly, and this ester canbe completely decomposed into lactic acid and phosphate by warminga t 37".The muscle enzyme also acts, but rather more slowly,upon the hexosediphosphoric acid of yeast. It would, however,appear that different enzymes are concerned in these reactions.For instance, it is easy to separate from the enzyme complex theenzyme which attacks hexosediphosphoric acid.Heating a t 37 Ofor 15 minutes destroys the capacity of the extract to split glycogenand other polysaccharides, but scarcely affects its capacity to splitthe hexosediphosphoric acid to lactic acid. Removal of the co-ferment by dialysis also yields an enzyme solution which stillsplits hexosediphosphoric acid, but which is without action onglycogen or starch. This varying behaviour towards glycogen andhexosediphosphoric acid depends upon the fact that brief heatingat 37", or removal of the co-ferment, destroys the ability of theenzyme extract to esterify the cleavage products of the poly-saccharide with inorganic phosphate. Thus the labile hexose firstformed by the cleavage of the glycogen must first be esterified with6 5 J .Gen. Physiol., 1927, 8, 631.6 6 Biochem. Z., 1926, 178, 395, 462; A., 1927, 75.G 7 Mcyer, ibid., 1927, 183, 216; A., 6902f3) ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.phosphoric acid before splitting and lactic acid formation can occur.This is well illustrated by the delayed appearance of lactic acidwhen the muscle enzyme preparation acts upon glycogen or starch,as compared with its immediate appearance when the substrate ishexosediphosphoric acid.Meyerhof’s muscle enzyme, even in the presence of the co-enzyme, acts but slowly on the fermentable hexoses,68 but byextracting baker’s yeast with water and precipitating the extractwith alcohol, there is obtained a substance which, when added to themuscle extract, greatly accelerates the rate of formation of lacticacid from hexoses.In neutral solution a t O”, this activator can bepreserved indefinitely, but it is readily destroyed by heat and byacids and alkalis. Treatment of a h.exose with muscle extract inthe presence of the activator leads to a rapid formation of lactic acidand a, parallel disappearance of inorganic phosphate. At the pointwhen all the inorganic phosphate has been used up, the velocity offormation of lactic acid falls rapidly and subsequently runs parallelto the liberation of inorganic phosphate, that is to say, in thesecond stage lactic acid and phosphate are being formed in equi-molar proportion. If at the end of the first stage a further additionof inorganic phosphate be made, the original velocity of lactic acidformation is restored.Meyerhof and Lohmann 69 have investigated the action of themuscle extract on the hexosemonophosphates obtained from naturalsources, and upon certain synthetic hexosemonophosphates.It isan interesting fact that the muscle extract is almost without actionon the synthetic monophosphates, whereas the natural mono-phosphates undergo a transformation similar to that of the poly-saccharides, hexosediphosphoric acid, and the fermentable hexoses,there being a rapid formation of lactic acid and a disappearance ofinorganic phosphate, followed by a slower production of lactic acidrunning parallel with the reappearance of inorganic phosphate. Inthe case of the monophosphates, as with the polysaccharides, thepresence in the muscle extract of the co-ferment is necessary.Meyerhof and Meyer 70 have described a method of purifying thelactic acid enzyme of muscle and a final preparation is obtained, afteradsorption on aluminium hydroxide made according to Willstatter’smethod, which is capable of forming 1.0 to 1.5 mg.of lactic acid ineach hour per mg. of protein present in the purified enzyme extract.Since in this process of purification the co-enzyme is removed, boiledmuscle juice must be added before any action on polysaccharides and6 8 Biochem. Z., 1927, 183, 176; A,, 590.Ibid., 185, 113; A,, 697.70 J . Physiol., 1927, 64, XVI; A., 1112BIOCHEMISTRY. 261hexosemonophosphates is observed. Meyerhof and Meyer alsostate that in addition to enzyme and co-enzyme a hydrolysableester is necessary and that the latter can be recovered from thebaryta precipitate of fresh muscle extract or boiled juice.Thissubstance is not Embden’s lactacidogen (monophosphate), but isstated to be an ester which behaves as a diphosphoric ester. Itsr6le would therefore appear to correspond to that of hexose-diphosphoric acid in inducing alcoholic fermentation in yeast juice.These results, which have been described in some detail in viewof their importance, show many striking similarities to the processesof yeast fermentation and would appear strongly to suggest thatboth hexosemonophosphates and hexosediphosphates are inter-mediate stages in the enzymic degradation of carbohydrate both bymuscle extract and by yeast. In regard to this question it isinteresting to note that Harden and Henley 71 have re-examinedthe data upon which the equation of alcoholic fermentation wasoriginally based.They now find that the ratio CO,/total P esterifiedis on the average 0-9, indicating that about 10% of the phosphorusis esterified without equivalent evolution of carbon dioxide. Theysuggest that the product of this esterification is probably a mono-phosphate. The ratio CO,/diphosphate is on the average 2.38,but varies considerably in individual cases. The fact that it isalways somewhat greater than 2, as required by the original equationof Harden and Young, suggests that the diphosphate is originallyproduced in accordance with the equation, but that a portion of thisis subsequently hydrolysed with the formation of a monophosphate.Insdin and its R61e in Carbohydrate Metabolism.Crystalline Insulin.-In the Report of last year mention was madeof the claims of Punk and of Abel regarding the isolation of insulinin a pure state.72 During the past year both these workers havemade further publications of great interest.Abel and hisassociates 73 state that when pyridine is added to an acid solutionof insulin, containing brucine acetate in amount sufficient to bringthe pH to 5.55-5.65, the insulin separates in a crystalline condition.On applying this method to the purification of commercial insulinthere was obtained, by working up the pyridine precipitate and themother-liquors, 0.4548 g. of crystalline insulin from 2.001 g.of thecommercial insulin powder. Abel has naturally coxsidered thepossibility that the crystals obtained are not really insulin but thoseof an inactive compound containing a very small quantity of highly71 Biochem. J . , 1927, 21, 1216; A., 1113.72 Ann. Reports, 1926, 23, 238.73 Abel, Geiling, Rouiller, Bell, and Wintersteiner, J . Pham. Exp. Ther.,1927, 31, 66; A., 701262 ANNUAL REPORTS ON THE PROQRESS OF CHEMISTRY.active material adsorbed on their surfaces. This possibility isdismissed on what appears to be convincing evidence. Crystallineinsulin has an activity exceeding 40 international units per mg.,and it gives the biuret, Pauly, Millon, and ninhydrin reactions,Tryptophan is said to be absent. The simplest formula correspond-ing to the analyses is C4,H,,0,4NllS,3H,0, which should becompared with the earlier alternative suggestions of Funk,C69H1,2022Nl,S and C,,H, 14024N2,S.Crystallographic observ-ations suggest that insulin is dimorphous. This observation is ofinterest in view of Funk’s most recent claims.74 Funk states thatinsulin may be fractionated to yield insulin-A and insulin-B. Theformer, which is present in larger proportion, decreases the bloodsugar of normal rabbits and of those having high initial blood sugars.On the other hand insulin-B is said to increase the blood sugar andto cause dilution of the blood with large retention of water. DuVigneaud T 5 has recently published observations which corroboratein large measure the statements of Abel. For example, he has beenable to obtain a highly active crystalline preparation of insulin byfollowing the method of Abel.Du Vigneaud’s preparation con-tains labile sulphur which he states is present in a disulphide form.He regards insulin as most probably a derivative of cystine and pointsout that the behaviour of the sulphur in insulin is quite parallel tothat of the sulphur in amino-acid derivatives of cystine. It issuggested, in view of the probable presence of a disulphide grouping,that Abel’s formula should be doubled. On the basis of theseresults, it now appears highly probable that sulphur is an integralpart of the insulin molecule, a suggestion first made by Dudley 76in 1923, when he formed the impression that insulin was a complexprotein derivative.The R6Ze of Insulin.-Thaniihauser and Jenke ,7 recently reportedthat glucosone, the keto-aldehyde derivative common to glucose,mannose, and fructose, was utilised by diabetics.Hynd 78 nowreports that, unlike dihydroxyacetone, which is also utilised bydiabetics (but compare p. 263) and readily alleviates the hypo-glyczmic symptoms, glucosone produces no alleviation. On thecontrary, when it is injected into mice a condition is produced similarto that following insulin injection, resulting in convulsions, coma,and death. Lactosone and maltosone, the corresponding keto-aldehydes derived from lactose and maltose, are quite negative in74 Science, 1927, 65, 39; A., 594.7 5 J . Biol. Chem., 1927, 75, 393.78 Ann. Repow, 1923, 20, 178.7 7 Arch.exp. Path. Pharm., 1926, 110, 500; A., 1926, 317.Proc. Roy. Soc., 1927, B, 101, 244 ; A., 480BIOCHEMISTRY. 263their actions. The glucosone effect, like that produced by insulin, isslightly modified by administration of glucose, and appreciably SOby adrenaline and pituitrin. Despite the similarity between theactions of insulin and glucosone, the latter does not lower the bloodsugar; on the other hand, increases of from 0.16 to 0*24% havebeen observed in mice. Hynd interprets these observations bysuggesting that insulin is an oxidase which catalyses the conversionof glucose into glucosone, that glucosone is an obligate first step inthe oxidation of glucose, and that therefore utilisation of glucose isdiminished or lacking if insulin be deficient or absent, as it wouldbe in the blood of pancreatic diabetics.Should the concentrationof glucosone become too high, owing to excessive insulin adminis-tration, convulsions would occur just as they do when glucosone isinjected directly. On this view the convulsions of insulin hypo-glycamia are not to be ascribed to the lowered blood sugar per se,but to the conversion into glucosone of that part of the sugar whichdisappears and undergoes oxidation. The glucosone symptoms areinhibited by a previous injection, and markedly relieved by asubsequent injection of acetoacetic acid. Glucosone would appearto be functioning here as a ketolytic substance in Shaffer’s sense.These conclusions are in accord with the views of Thannhauser andJenke, since these workers formed the view that the disturbancein the diabetic is due, not to inability to convert glucose afterglycogenolysis into a utilisable form (e.g., y-glucose), but to theinability to convert glucose into glucosone, whicb.is then utilisedfor the synthesis of glycogen or oxidised. It would appear thatglucosone is capable of forming glycogen even more readily than isfructose. Lambie 79 is in essential agreement with the suggestionthat the diabetic primarily lacks the power of transforming glucoseinto some intermediate product which can be oxidised by thediabetic as well as by the normal subject. But in view of the earlierresults of Kermack, Lambie and Slater,so and of Lambie and Red-head,s1 he attaches great importance to dihydroxyacetone as theintermediate product.Markowitz and Campbell 82 have, however,arrived a t diametrically opposite views regarding the utilisabilityof dihydroxyacetone by the diabetic, stating that, when it isadministered to depancreatised dogs, its concentration in the bloodsteadily falls, while the concentration of glucose rises, andultimately the dihydroxyacetone is quantitatively excreted asglucose. They therefore do not regard dihydroxyacetone as an79 J . Sot. Chem. Ind., 1927, 46, 300; A., 989.82 Amer. J . Physiol., 1927, 80, 848, 661; A,, 693.Biochem. J . , 1926, 20, 486; 1927, 21, 40; A., 1926, 861; 1927, 282.Ibid., 1927, 21, 649; A., 693264 ANNUAL REPORTS ON TRE PROGRESS OF CHEMISTRY.intermediate in the catabolism of carbohydrate.It is, in their view,not oxidised and has no anti-ketogenic action.8ynthaZin.-Clinicians have recently devoted some attention to asynthetic preparation called “ synthalin,” which is a decamethylene-diguanidine derivative (om-diguanidyldecane), and was introducedby Frank, Nothmann, and Wagner 83 as a substitute for insulin,but having the advantage over the latter in that it could be giveneffectively by mouth. Simola 84 has published a detailed examin-ation of the physiological action of “synthalin” and finds veryconsiderable differences between the action of the natural hormoneand the artificial substitute. For example, the hypoglyczmia pro-duced by the latter is far less marked, appears only after some hours,and is more lasting than that produced by insulin. In addition toits hypoglycsmic action, “ synthalin,” like insulin, depresses theblood inorganic phosphate, but in an irregular manner.If apoisonous dose be administered, the inorganic phosphate risessharply, reaching twice its normal value after 24 hours. Lacticacid also is increased in the blood after (‘ synthalin ” administration.Toxic symptoms of guanidine poisoning often develop and it wouldappear from clinical experience on the human subject that the toxicnature of the artificial substitute is likely severely to restrict itsuse. There is, however, no doubt that it does produce someincrease of sugar utilisation in the diabetic ; for instance, Lublin 85reports that diabetics, treated with “ synthalin ” by mouth, showan increase in the respiratory quotient following the administrationof glucose.During the past year two studies have been published on theaction of galegine.Galegine is a guanidine derivative shown byBarger and White 86 to have the constitution :Simmonet and Tanret 87 have shown that subcutaneous injection ofgalegine sulphate in rabbits produces in most cases hypoglyczmia,but in some cases the condition is preceded or replaced by hyper-glycamia. The effect is very similar to that of insulin and may berelieved somewhat by an injection of glucose. Miiller and Rein-wein88 have published very similar findings. They state that,as Deut. med. Wochenschr., 1926, 52, 2067.EP 2. physiol. Chern., 1927, 168, 274; A., 900.85 Arch.exp. Path. Phawn., 1927, 124, 118; A., 896.86 Biochem. J . , 1923, 17, 827; A., 1924, i, 272.81 Compt. rend., 1927, 184, 1600; Bull. SOC. chim. biol., 1927, 9, 908;88 Arch. exp. Path. Pham., 1927, 125, 212; A., 1109.A., 991BIOCHEMISTRY. 265when administered to rabbits or when given in large doses to dogs,it raises the blood-sugar. When it is given in small doses to dogs,the blood-sugar is lowered, and in the case of depancreatised dogsboth blood and urinary sugars are lowered. The hyperglycaemicaction is antagonised by ergotamine, andwhen the two substances areadministered together hypoglycemic convulsions may be producedin both rabbit and dog.Hcemogtobin, Hcemochromogen, and Cytochrome.Although the subject of haemoglobin has been dealt with byProfessor Drummond in the Annual Reports for the two precedingyears,sg the many important advances in the study of this respiratorypigment and related substances have, until very recently, proveddifficult to correlate with one another, and it is felt that a surveyof the developments of the past two or three years is called for in thepresent Report. The Reporter is encouraged in undertaking thistask in virtue of a very helpful summary of the present positionpublished by KeiIin.goThe complex substituted tetrapyrrole compounds called porphyrinsform the basis of the respiratory pigments which we shall considerin the present section of this Report. A large series of porphyrinsof animal and vegetable origin is known, and some, notably aetio-porphyrin, have been synthesised.They form characteristiccompounds with metals for which Schummgl has suggested thegeneral name porphyratins. Hzematin or haem, the prostheticgroup of hzemoglobin, which is an iron porphyratin, exists in twoforms, oxyhzematin and reduced hzematin, the latter being thehzm of Anson and M i r ~ k y . ~ ~ These two hzematins, differing intheir state of oxidation, also differ in solubility, colour, absorptionspectrum, and gas-combining power. Furthermore, characteristicdifferences are shown by the two haematins according as they arepresent in alkaline, neutral, or acid solution. The differences in theabsorption spectra are slight in the case of reduced haematin underthese varying conditions of reaction, but, on the other hand, markeddifferences are shown by oxyhamatin under the same conditions.An important advance in the study of the relationship of these iron-porphyrin compounds to hzemoglobin and related pigments wasrendered possible by Hill and Holden93 when, in 1926, they suc-ceeded in separating from hzemoglobin the natural globin withwhich the prosthetic group is associated, and they were able to show89 Ann.Reports, 1925, 22, 237; 1926, 23, 249.@O SOC. Biol., Rdunion PldniBre, May 1927 (Reprint).91 2. physiol. Chem., 1926, 152, 147; A,, 1926, 537.O2 Ann. Reports, 1925, 22, 237.93 Biochem. J . , 1926, 20, 1326; A., 1927, 67.I 266 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.that their natural globin combined with neutral oxyhaematin, overthe range pE 5 to 10, to yield methaemoglobin.Since methzemo-globin may be readily converted into reduced haemoglobin and sincethe latter, shaken with air, forms oxyhaemoglobin, the naturalrespiratory pigment may be synthesised in this way from its com-ponents. If in place of oxyhaematin reduced haematin (i.e., haem)be used, it also combines with natural globin (at pPH 9.0) to yieldreduced haemoglobin directly. Hill and Holden were able to showthat natural globin may also combine with porphyrins other thmthat of hEmoglobin, and even with porphyrins containing, not iron,but other metals such as manganese, cobalt, nickel, copper, zinc,and tin. The absorption bands of the compounds so obtained allshow a displacement towards the blue end of the spectrum whencompared with the bands of haemoglobin.Furthermore, naturalproteins other than globin do not combine with oxyhaematin or withreduced haematin to give compounds comparable with those of thehaemoglobin series. The only known example of a natural pig-ment parallel to haemoglobin is Munro Fox’s chlorocr~orin.~~ Inthis pigment the prosthetic group contains iron, but the porphyrindiffers from that of haemoglobin. Neutral oxyhaematin combineswith a number of nitrogenous derivatives, including denaturedglobin, nicotine, pyridine, and histidine, to give a series of com-pounds which Keilin has called parahaernatin~.~5 It would appearthat helicorubin, the pigment found in the liver and gut of pul-monate molluscs and in the liver of the crayfish, belongs to thisgroup of substances.Alkaline oxyhaematin does not combine withnitrogenous substances.Hcemochrornogen.-A highly important group of compounds isencountered when we consider those substances formed by reducedhaematin interacting with nitrogenous compounds. We havealready seen that when the nitrogenous substance is natural globin,reduced haemoglobin is obtained, but with denatured globin a sub-stance of a much lower degree of molecular complexity, namelyhzemochromogen, results. Many other nitrogenous compounds inaddition to globin may enter into combination with reducedhaematin : amongst them are to be numbered other proteins suchas denatured albumin and globulin, glycine, nicotine, pyridine,piperidine, hydrazine, and ammonia.We owe to Anson andMirsky 96 the general conceptions of the relationships of these com-pounds to haemoglobin, and of the molecular structure of thehzemochromogens. The latter all differ markedly from reduced94 Proc. Roy. Soc., 1926, B, 99, 199; A., 1926, 313.95 Ibid., 1926, B, 100, 129; A,, 1926, 857.me Ann. Reporb, 1925, 22, 237BIOCHEMISTRY. 267haematin in solubility, colour, absorption spectrum , and in the com-pounds which they form with carbon monoxide. The haemo-chromogens oxidise readily in the air, and in an alkaline mediumthey dissociate into their nitrogen compound and haematin, or, asAnson and Mirsky called it, haem. Keilin has shown that in aneutral solution they do not dissociate but are transformed intoparahzmatins, which we have seen to be compounds of oxyhaematin.Until the work of Anson and =sky was published, generalacceptance was accorded t o the idea that what were then calledhaematin and haemochromogen comprised the protein-free pros-thetic group of hzemoglobin in an oxidised and a reduced conditionrespectively.Anson and Mirsky, in the work which has just beenoutlined above, clearly established the true nature of hzemochrom-ogen as a compound of globin (or, in the case of artificial hzemo-chromogens, of some other nitrogenous substance), but they weremistaken in stating that haematin was also a globin compound.It was for these reasons, one correct, the other a mistake, that theterm hem was introduced by them to designate the free pros-thetic compound, and a good deal of confusion has since resulted.A&.HCI+OsL-- ReducedAlk. reduction ,7 hsmatin (hsm) HsIlliIl Alkaline A-oxyhamatin - 7Neutral z oxyhsmatindd \‘Acid HsmochromogenReduced hzmoglobin MethEmoglobinScheme showing relationship of the blood pigments. The expressions + N and- N are intended to indicate the addition or the removal of denatured globin orother nitrogenous compound.7reduc268 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.The reader will, however, find the position quite clear if he bears inmind the identity of Anson and Mirsky’s hem with reduced hzm-atin. The cause of the original mistake was cleared up by Keilinwhen he showed that the alkaline hzematins prepared from hzemo-globin and from hzemin crystals were identical and devoid of protein,whereas acid haematin prepared from haemoglobin is a colloidalsuspension of hzematin, which is not united with globin, but isprotected by the latter from precipitation.There is appended ascheme, modified slightly from one given by Keilin, which will befound helpful in tracing the relationships which we have discussed.The term hzemochromogen in its modern usage is really a verywide one, since one may vary, not only the nitrogenous compound,but also the iron-porphyrin. It has been shown that only slightalterations in the characterist,ic absorption bands are producedwhen reduced haematin is combined with a series of nitrogen com-pounds, but, on the other hand, more marked differences resultwhen the same nitrogen compound is combined with differentporphyrins.Another factor which influences greatly the positionsof the bands is the degree of dispersion of the hzemochromogen;with increasing molecular aggregation the bands become displacedtowards the red end of the spectrum. Thus when a haemochromogenis present both in solution and in a fine colloidal suspension, theliquid shows three absorption bands, formed of two superimposedspectra, comprising two a-bands and one fused p-band.Cytochrome.-In 1925 Keilin 97 showed that a very large numberof aerobic plant and animal tissues when examined with a micro-spectroscope showed a series of bands similar to those of thehzemochromogens, and he established the origin of these bands asbeing due to a widespread respiratory pigment which he calledcytochrome.These observations were confirmed and extended bySchumm 98 and by Euler, Fink and Hell~trOm.9~ It became clearthat cytochrome was in fact a porphyratin combined with a nitro-genous substance and showed a remarkable similarity to haemo-chromogen. Keilin has shown that in reality cytochrome is amixture of three haemochromogens, or rather of the same haemo-chromogen in three different physical states, each one of whichcontributes its characteristic a- and p-bands to the complex four-band spectrum of the complete substance, bands a, b, and c beingthe three separate a-bands, and band d the three fused p-bands.Under certain conditions these three hzmochromogens may become9 7 Ann.Reports, 1925, 22, 258.98 2. physiol. Chem., loc. cit. and p. 55; 1926, 154, 171, 314; 1927, 166, 1;99 Ibid., 1927, 169, 10; A., 993.A., 685BIOCHEMISTRY. 269oxidised (in which case the bands disappear) or reduced independentlyof one another, so that the particular bands shown by cytochromeand also their relative intensities may vary with the conditionsemployed. There is also present in all cells which contain cyto-chrome free haematin, and the latter, combining under variousconditions of oxidation and reduction with different nitrogen com-pounds, gives rise to the three haemochromogen components ofcytochrome, two differing in degree of dispersion and the thirdpartly modified by the active process of oxidation and reduction.The respiratory functions of cytochrome have been carefullystudied by Keilin.The fact that the pigment is confined to aErobicorganisms, and its behaviour in them, at once suggested that it isconcerned with the utilisation of oxygen, either directly or inconjunction with an oxydase system. It has been shown that alongwith cytochrome there is found an oxydase which can be detectedby its capacity to form indophenol from dimethyl-p-phenylene-diamine hydrochloride and a-naphthol. It seems probable thatthis oxydase system is identical with the respiratory fermentdescribed by Warburg,2 which is present in yeast and cocci cellsand is inhibited by carbon monoxide. Haldane3 has shown asimilar respiratory system to be present in the wax-moth and incress plants, so that its distribution is probably a very wide one.Keilin has brought forward evidence t o show that cytochrome, orat least two of its component hemochromogens, is oxidised bythis phenoloxydase and reduced by reductases or by other cellularconstituents which become oxidised irreversibly. Cytochrome maytherefore act as a “ Hilfkatalyst ” in the sense of Oppenheimer,*or as a respiratory chromogen acting either as a peroxydase or asa catalyst.It would therefore seem that in cytochrome there isrevealed a considerable part of the complex system of oxidativecellular catalysts.The Porphyrins.-There has been described a very large numberof closely similar porphyrins and their derivatives obtainable fromnatural sources directly or by simple chemical transformations.It would seem probable that the tetrapyrrole compounds fromwhich these are derived must be capable of existing in isomericforms and of giving rise to a large number of simple substitutedderivatives. The most striking recent advance in the study ofthese porphyrins, which fully justifies these suppositions, has been1 Nature, 1927, 119, 670; A., 592.2 Biochern.Z., 1926, 177, 471; 1927, 189, 354; A,, 1926, 1277; 1927,3 Nature, 1927,119, 352; A., 375; Biochern. J . , 1927, 21, 1068; A., 1110.4 ‘‘ Die Fermente und ihro Wirkungen,” Leipzig, 1926.1221270 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.the synthesis of aetioporphyrin, identical with Willstatter’s aetio-porphyrin from natural sources, by Fischer and Klarer,, and ofisoaetioporphyrin by Fischer and Halbigm6 As a result of thisachievement Fischer is able to make a more authoritative suggestionregarding the structure of aetioporphyrin, and therefore of porphyrinsrelated to it, than has hitherto been possible.The suggestedstructure, which forms the basis of these and other porphyrins, isas follows :The structure of aetioporphyrin as demonstrated by synthesis isobtained by substituting for R,, R,, R,, and R, the C,H, radical,and for R,, R,, R,, and R, the CH3 radical. On this basis, currentviews on the constitution of coproporphyrin are expressed bysubstituting for R,, R,, R,, and R, the group -CH,*CH,CO,H,and for R,, R,, R,, and R,, -CH,. The closely related uroporphyrin issimilarly formulated by replacing each of the groups -CH,*CH,*CO,Hin the coproporphyrin formula by the dicarboxylic acid groupIt is clear that from a tetrapyrrole nucleus of this type numerousisomeric and closely related porphyrins can be derived, and it istherefore not surprising that numerous representatives of the classare reported from time to time as occurring in nature. The subjectis still in too complex a state of development t o be suitable forreview, but as illustrative of the type of investigation in progressmay be cited the recent preparation of deuteroporphyrin by Fischerand Ljndner.7 This porphyrin has been obtained by fermentingfresh ox-blood spontaneously, or with yeast, an alkaline reactionbeing preserved throughout. Evidencs is adduced of its constitu-tion and this can be expressed by substituting as follows in theformula already given : for R,, R,, R,, and R,, -CH,, for R, andR,, -H, and for R, and R,, -CH,*CH,CO,H.-CH,*CH( COZH),.Annalen, 1926, 448, 178; A,, 1926, 9G2.Ibid., p. 193; A., 1926, 063.2. physiol, Ci~em., 1926, 161, 27: A,, 1927, 262BIOCHEMISTBY. 271All these porphyrine are capable of forming the correspondingiron porphyratins, and Fischer and his co-workers have preparedsynthetic aetiohamin and isozetiohaemin from the correspondingsynthetic porphyrins by treating the latter with ferric chloride andsodium acetate. The nature of these iron compounds has yet tobe elucidated, but a recent important publication by Hill* givessome indication of their probable nature. Haematoporphyrin wasprepared from pure hzemin and the former was then reconstitutedby artificial means. The corresponding nickel and copper por-phyratins were also prepared. Whereas the nickel and coppercompounds still showed their typical two-band spectra, the ironcompound resembled reduced haematin in showing only an ill-definedregion of absorption. The hzemochromogen type of spectrumshown by compounds of haematoporphyrin with metals other thaniron is shown by Hill not to be due to the presence of nitrogencompounds, and it is suggested that the property of forming haemo-chromogens is limited to the iron-porphyrin compounds. Onaddition of denatured globin, pyridine, or ammonia the iron com-pound gave haemochromogen. Thus the artificial iron-haemato-porphyrin showed the same behaviour as hamin prepared directlyfrom blood. Nickel and copper haematoporphyrins showed nochange on the addition of denatured globin. The iron compoundof hzmatoporphyrin thus behaved exactly as hzmatin and onreduction did not give the haemochromogen spectrum shown byother metallic derivatives of hamatoporphyrin, unless some nitrogencompound was present. It is deduced from these observationsthat haemochromogen is simply the ferrous compound of the por-phyrin, corresponding with the bivalent copper and nickel com-pounds, together with a nitrogenous substance. Since iron hzemato-porphyrin gives a different spectrum according to the reagentspresent when the pigment is reduced in alkaline solution, and sincethis property is not shared by any other porphyratin so far examined,it is inferred that the iron atom alone confers on the pigment theproperty of forming molecular compounds with a large variety ofsubstances when the pigment is in the reduced state.From quantitative measurements of the combination of pyridinewith reduced haematin, Hill has shown that in carbon monoxidehzemochromogen one nitrogen-containing molecule is replaced byCO, there being two such nitrogenous molecules in hzemochromogenitself. By adding the nitrogen compound t o CO-reduced haematin,one molecule of the former is taken up, and by adding excess ofthe nitrogen compound, the CO is displaced by the taking up ofanother molecule of the nitrogen compound and haemochromogen8 Proc, Roy. SQC., 1926, B, 100, 419; A,, 1927, 66272 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.is formed. These changes occur when any nitrogen compound isused which gives a typical haemochromogen, including denaturedglobin itself. Since iron is the only element in combination withthe pigment which causes it to have the property of forming haemo-chromogen, it is simplest to assume that the nitrogen and carbonmonoxide are directly co-ordinated with the iron, which has aco-ordination number of 6. The general formulz for a carbonmonoxide-hsmochromogen and for haemochromogen are thereforewritten thus (Hph being haematoporphyrin and N the nitrogenoussubstance) :HphEFe<N= co HphgFe<NE A'-- and -The chief objection to this view, as Hill himself points out, is thefact that the carbon monoxide compound of reduced hsmatin,without pyridine or other nitrogen compound, contains not twomolecules, but only one molecule of carbon monoxide. The formulacan, however, be written :Hph_FecX, cowhere X is either another molecule of the complex or a moleculeof solvent. It may be added that there is evidence that reducedhsmatin combines with itself to form large molecules in the absenceof the specific reagents mentioned, because of its insolubility andthe slowness with which it reacts with such reagents.C. T. GIMINGHAM.JOHN PRYDE