BIOCHEMISTRY.Metabolism of Moulds.THE marked increase in the amount of research devoted to a studyof the metabolic product of moulds since the war can be traced tothe curious fact that in the case of nearly all the chemical compoundsproduced by fermentation methods, there is no alternative, satis-factory, purely chemical method of preparation. The industrialimportance of such processes, therefore, cannot be over-emphasised,and it is noteworthy that the researches of Raistrick and his co-workers on the biochemistry of micro-organisms which are men-tioned later in this Report were carried out between the years 1922and 1928 in the laboratories of Nobel’s Explosives Company atArdeer, and are now published as 18 consecutive papers in a specialnumber of the Philosophical Transactions.lCarbon Balance 8heets.-Of the two classes of micro-organisms(bacteria and true fungi) which can be readily cultivated in artificialmedia, much attention has been paid in the past to the chemicalactivities of the bacteria,la and of the family of true fungi knownas the yeasts, but very little to the other classes of true fungi knownas the “ moulds.” In recent years an increasing amount of work onthe metabolism of the moulds, especially the production of acids,has been published,2 but in 1922 it seemed to Raistrick and his co-workers, who wished to make a general survey of the metabolicproducts of moulds, that it would be impossible to investigate indetail, in any reasonable period of time, the chemical compoundsformed by even a small proportion of the different known species offungi.For this reason, then, instead of attempting the isolationand identification of the compounds formed by any mould taken a trandom, it was decided to investigate quantitatively the types ofcompounds formed by each of a large number of fungi, so as toobtain a logical basis for the choice of any particular fungus for laterinvestigation. To this end a so-called “ carbon balance sheet ” wasprepared for every species that it was proposed to study : this wasfound to be of great utility, for it gave a clean-cut classification ofthe various types of product formed and of their quantitativerelationships. In order to reduce the scope of the work, and to1 PAX Tram., 1930, 220, 1.15 Compare Marjory Stephenson, “ Bacterial Metabolism,” London, 1930.a Compare Ann.Repvrte, 1927,24, 222230 CHIBNALL AND PRYDE :ensure the possibility of repetition, all experiments were carried outwith glucose as the sole source of carbon in a Czapek-Dox syntheticmedium. In this respect the work differs somewhat from that ofof her investigators.The balance sheets show the location, after the metabolism, of allthe carbon originally present as glucose in the culture solution. Forthe practical details of the method the reader must refer to theoriginal paper : it is possible here to mention but briefly the tentgpes of compound in which the carbon is estimated. These arc(1) evolved carbon dioxide, (2) gases other than carbon dioxide,(3) volatile products such as ethyl alcohol, etc., (4) the mycelium,(5) total carbon in the metabolism solution, which was furthersubdivided into ( a ) residual glucose, ( b ) dissolved carbon dioxide,(c) volatile acids, (d) non-volatile acids, ( e ) carbon compoundsprecipitable by iron, e.g., proteins, (f) carbon unaccounted for.By means of the carbon balance sheets it has been possible toeliminate from further investigation all those fungi-constitutingby far the greater number-which, under the conditions of theexperiment, produce practically nothing but carbon dioxide fromglucose.In all, some 96 species of Aspergill~s,~ 75 species ofPenicillium, 8 species of Citrmyces,5 23 species of Fusarium,6 and36 miscellaneous species of fungi have been examined, and fromthem a choice of species suitable for further intensive investigationhas been made. The general results obtained can be summarisedas follows.81.The carbon balance sheets may be used as a biochemicalmethod for the classification of different species in certain familiesof fungi. This is particularly so in the case of the Aspergilli, inwhich a classification based on biochemical characteristics followsclosely that founded on morphological observation. With speciesof Fusarium, however, there is little hope of a biochemical classi-fication, for each of the species tried gave rise principally to ethylalcohol and showed close alliance with the yeasts.2. Some light is thrown on the biochemistry of the initial stagesof the breakdown of the glucose molecule by fungi. It appears that3 J.H. Birkinshaw and H. Raistrick, Phil. Trans., 1930, 220, Pt. 2.J. H. Birkinshaw, J. H. V. Charles, H. Raistrick, and J. A. R. Stoyle,5 J. H. Birkinshaw, J. H. V. Charles, A. C. Hetherington, H. Raistrick, andJ. H. Birkinshaw, J. H. V. Charles, H. Raistrick, and J. A. R. Stoyle,J. H. Birkinshaw, J. H. V. Charles, A. C. Hetherington, and H. Raistrick,H. Raistrick and W. Rintoul, ibid., Pt. 1.ibid., Pt. 3.C. Thorn, ibid., P t . 4.ibid., Pt. 6.ibid., Pt. 6BIOCHEMISTRY. 23 1the first step is a Cannizzaro reaction involving the formation fromtwo molecules of glucose of one molecule of mannitol and onemolecule of gluconic acid. Depending then on whether the mouldin question prefers for growth an acid or a neutral medium, eithermannitol or gluconic acid (of course, in some cases both) is destroyed.Thus a certain species of white Aspergillus which gives rise to yieldsof mannitol approximating to, but never exceeding, 50% of theglucose metabolised produces very little if any gluconic acid, refusesto grow in an acid medium, and even when cultivated on a mediumwith an initial pH of 4.6 changes this during the course of its growthto 66-7.On the other hand, species such as Aspergillus Wentiiand Penicillium chrysogenum, which produce large quantities ofgluconic acid, give little or no mannitol, on which material they growquite well, but in all cases bring the pH of the medium down to about1-2. Other species again produce moderate amounts of both thesesubstances.The production of these two materiaIs from glucose raises aninteresting stereochemical point.Glucose on reduction should givesorbitol, and on oxidation, gluconic acid; mannose on similartreatment, mannitol and mannonic acid. Yet in spite of frequentsearch for sorbitol and mannonic acid as metabolic products of fungino trace of them has been found.3. Perhaps the most striking fact is the extraordinary specificityof some of the mould products. It would appear that certaingeneral biochemical reactions such as the production from glucoseof mannitol or gluconic acid, of ethyl alcohol, of citric and oxalicacids may be regarded as common to many species belonging tomany different families of fungi, but at the same time there arecertain highly specific substances which are produced, in some casesby a single species, and in others by a very small sub-group contain-ing a very few species.Citrinin, for instance, is specific for Penicill-ium citrinum Thom, and may be used as a test for this species by thepurely chemical test of adding ferric chloride to the metabolismsolution, whereupon a characteristic iodine brown colour is produced.All these specific products, some of which are referred to briefly later,are more complex than glucose and are good illustrations of theamazing synthetic powers of these organisms.Metabolic Products of Moulds.Oxalic, Citric, and Gluconic Acids.--It is now generally recognisedthat these three acids are produced by most moulds, and that thesecond two are not as specific as was a t first suspected.have found that Succinic Acid.-Raistrick and his co-workers' J.H. Birkinshaw and H. Raistrick, Phil. Trans., 1930, 220, Pt. 17232 CHIBNALL AND PRYDE :this acid is formed in small amounts from sugar by several speciesof Aspergillus and Fumccgo vagans. It was isolated as the ester,and identified as the free acid and anhydride. Its production byRhixopus species growing on a medium containing acetates has beenshown by T. Takahashi and K. Asai 10 and W. S. Butkevitsch andM. W. Federov.ll Using the calcium salt of n-butyric acid as asource of carbon for the growth of A . niger, H. B. Stent, V. Subra-maniam, and T. K. Walker l2 have isolated, with other products,succinic acid, which was identified by its m.p. and conversion intodi-p-nitrobenzyl succinate. They consider that the butyric acidfirst undergoes P-oxidation with the production of acetone. Furtheroxidation yields acetic acid, which then undergoes dehydrogenationaccording to the Thunberg-Wieland hypothesis :3CH,*CO*CH, -+ CH,*CO*CO,H + 2CH,*CO,H -+CH,*CH( OH)*CO,H + CO,H*CH,CH,*CO,HFumaric Acid.-This was first observed by F. Ehrlich l3 in 1911to be a product of Mucor stolonifer. Recently W. S. Butkevitschand M. W. Federov l4 claim to have obtained good yields of this acidwhen the organism is grown on solutions of glycerol, acetic acid andsugar. C. Wehmer l5 in 1918 obtained good yields from sugar by aspecies of Aspergillus which he named A.fumaricus. In a culturereported to be this species Raistrick and his co-workers failed toobtain fumaric acid, and it is worthy of note that in a recent paperWehmer 16 states that his culture, which originally gave largeamounts of this acid, now produces only traces of this substance, andthat gluconic acid is now formed instead. A similar experience isrelated by R. Schreyer.17 His culture of A. fumaricus has in thecourse of time lost its power of producing fumaric acid, and nowgives rise to citric and gluconic acids. It would appear that we havein this an interesting case of evolutionary change of metabolism.The claim of A. Gottschalk l8 that Rhixopus nigrz'cuns whengrown on a solution of pyruvic acid gave rise to fumaric acid wasdisputed by F.Ehrlich and I. Bender,19 who state that no growthtakes place on such a medium. This has now been admitted by10 Bull. Agric. Chem. SOC. Japan, 1928, 113.11 Biochem. Z . , 1930,219, 87; A., 643.l2 J . , 1929, 1987, 2485; A., 1929, 1271; 1930,66.13 Ber., 1911, 44, 3737.l4 Biochem. Z . , 1929,207,302; 1930,219, 87; A., 1929, 724; 1930, 643.15 Ber., 1918, 51, 1663.Is Biochem. Z., 1928,197,418; A., 1928, 1164.Ibid., 1928, 202, 131; A., 1929, 217.2. physiol. Chem., 1926,152, 136; A., 1926, 546.Is Ibid., 1927,170,118; 172,314; A . , 1928,95: 1928. 804BIOCHEMISTRY. 233Gottschalk and confirmed by W. S. Butkevitsch and M. W. FederovY2Owho state that in the presence of calcium carbonate, which was aconstituent of the nutrient solution, pyruvic acid undergoes a changeto a monobasic acid of empirical formula C,H604, which bears norelationship to the activity of the mould.Malic Acid.-Casual references to the production of this acid bymoulds occur in the earlier literarure. C.Wehmer l6 has now showndefinitely that it is produced in small amounts from sugar byAspergillus fumricus. Raistrick and his co-workers 21 also haveisolated the acid as its ester from the products of a white species ofAspergillus, and also from A. Wentii growing on glucose.The biochemical infer-relationship between succinic, fumaric andmalic acids was referred to in a previous Report.21a It would appearthat similar inter-conversion can be brought about mycologically.T. Takahashi and K. Sakaguchi22 have shown that a Rhixopusspecies can convert fumaric into 2-malic acid, and can also bringabout the reverse change.3'. Challenger and L. Klein 23 have shownthat a strain of A . niger affords excellent yields exclusively ofZ-malic acid when grown upon a fumarate medium. They thereforeconclude that the enzyme fumarase is secreted by the organism.Using the same strain of A . niger and a succinate medium, €3. B.Stent, V. Subramaniam, and T. K. Walker 24 obtained a mixture ofdl-malic and Z-malic acids. As oxidation of auccinic acid by hydrogenperoxide in the presence of ferrous sulphate yields as a first stagemalic acid, they consider that thisdl-malic acid 25 owes its formationto the direct hydroxylation of the succinic acid, a process which theythink could be effected by an aerobic oxydase system.The Z-malicacid, however, they consider has arisen through a preliminarydehydrogenation of the succinic acid to fumaric acid, which is thenacted on by the fumarase, and not to any preferential utilisation ofthe d-isomeride by the mould growing on the dl-malic acid. It isinteresting to note that Ruhland and Wetzel (p. 246) find anaccumulation of both dl-malic and Z-malic acid in developing rhubarbstalks.MannitoL-Although the presence of mannitol in the myceliumof fungi has long been recognised, it appears to have been con-sidered simply as a reserve food comparable with the glycogen ofyeast and not as a definite fermentation product. Raistrick and2o Biochem. Z., 1929,206,440; A., 1929, 607.*1 J.H. Birkinshaw and H. Raistrick, Phil. TTUW., 1930, 230, Pt. 17.21a Ann. Reprte, 1928,25, 226.22 Bull. Agric. Chem. SOC. Japan, 1927,3, 59.23 J., 1929, 1644; A., 1929, 1166.24 Ibid., p. 1987; A., 1929, 1371. 26 Ibid., p. 2486; A., 1930, 66.H 234 CHIBNALL AND PRYDE :his co-workers26 have now shown that it is produced by manymoulds, and by certain species of white Aspergillus in particular, inyields as high as 50% of the sugar metabolised. The alcohol 27 wasestimated in presence of glucose by a method depending on theincreased rotation observed when borax was added to the solution,allowance being made for the fact, hitherto unrecorded, that glucosein 0.9% concentration in 6% borax solution is optically inactive andat concentrations up to 0.9% is lzvorotatory.It was found thatculture solutions in which the amount of aeration was unrestrictedgave only small yields, and the high values quoted above were onlyobtained when the amount of aeration was strictly limited.Po1ysaccharides.-A few polysaccharides from the lower fungihave been previously recorded, and given appropriate names, butattempts to fmd out their constitution have been limited to showingthe reaction, if any, to iodine and the sugar given on hydrolysis.Raistrick and his co-workers 28 have encountered several suchsubstances in the course of their work. From a species of Asper-gillus they have isolated what appears to be glycogen.27From Fumago vagansZ1 they have isolated a new substance,which separates from water as a white amorphous precipitate, andfrom the mycelium of Penicillium digitatum, S a ~ c a r d o , ~ ~ a similarsubstance which is insoluble in cold, but moderately soluble in hotwater.These two substances give no colour with iodine, have anempirical formula CeHlo05, and in aqueous solution are neutraland strongly dextrorotatory . They give rise quantitatively toglucose on hydrolysis with dilute acids, and are unaffected byinvertase or diastase.The most interesting product, however, is that formed fromYenicillium luteurn, Zukal, a mucilage which the authors havenamed “ luteic ’’ acid.30 The crude product as first isolated is themagnesium salt of a complex organic acid, and from this the pureacid, a white amorphous mass resembling starch, was eventuallyprepared.It gives a gelatinous solution in water and does notreact with alkaline iodine, reduce Benedict’s solution or form anosazone, thereby showing that it contains no free aldehyde groups.It is lzvorotatory and on hydrolysis with dilute acids it gives riseexclusively to glucose and malonic acid; a t the same time theacidity of the material is doubled. From these and other con-siderations, it is concluded that the substance is a complex built upz6 J. H. Birkinshaw, A. C. Hetherington, and H. Raistrick, Phil. Trans.,1930, 220, Pt. 9.27 H. Raistrick and W. Young, ibid., Pt. 10.28 Idem, &id.29 J. H . Birkinshaw, J. H. V. Charles, and H. Raistrick, ibid., Pt. 18.30 H. Rnistrick and M. Rintoul, ibid., P t . 13BIOCHEMISTRY.235of units, each of which is a condensation product of two moleculesof glucose with one molecule of malonic acid with the loss of twomolecules of water, in which one carboxyl is free and the otherin combination, and two aldehyde groups are linked in such away as to destroy their aldehydic properties. Polysaccharides ofthis type, in which part of the simple hexose units is replaced bya simple organic acid, are very rare, and the only other well-authen-ticated example is, curiously enough, also of microbiological origin.This is the “ soluble specific substance ” from young cultures ofpneumococcus which has been recently isolated by Heidelberger andGoebel3l and shown to give rise to glucose and glucuronic acid onhydrolysis. The natural gums, e.g., gum arabic, are of course ofsimilar type, but in these cases the nature of the complex constituentacid has not been worked out.Kojic Acid.-This acid was shown by T.Yabuta in 1924 32 to be5-hydroxy-2-hydroxymethyl-y-pyrone, and is characterised by theintense wine-red colour it gives with ferric chloride in dilutions ofeven 1 : 200,000. In ignorance of earlier work Raistrick and hisco-workers 33 in 1923 found that the balance sheet of A . parasiticusshowed that more than 19% of the sugar consumed was unaccountedfor, and from this investigation rediscovered this acid, noted inYabuta’s paper. A . Jlarvus, e$fwus, and tamarii also give this acid,which appears to be of diagnostic value, in that it justifies one inplacing in the flavus-oryzce-tamarii group of Aspergilli any speciesof this genus which, if cultured under certain specified conditions,gives rise to the typical kojic acid reaction, without, however,necessarily excluding from this group any Aspergiltus which gives anegative reaction.F.Traetta-Mosca34 showed that the acid is produced fromglycerol, sucrose, glucose and laevulose by a mould which he calledA . ghww, but which Raistrick and his co-workers believe to be amember of the Jlavus-oryxce group. F. Challenger, L. Klein, andT. K. Walker 35 showed that it was produced by a mould they callA . oryzce diastccse from arabinose and xylose, and the list of pre-cursors given by Raistrick a.nd his co-workers includes all thesesubstances together with lactose, galactose and mannitol.Thelatter workers point out that the idea originally held, that kojic acid(C,H,O,) arises from glucose (C,H&,) by a simple oxidation anddehydration, offers no explanation of the formation of the acid from31 Ann. Reports, 1927, 24, 251.33 J. H. Birkinshaw, J. H. V. Charles, C. H. Lilly, and H. Raistrick, Phil.34 Buzzetta, 1921, 51, ii, 269; A., 1922, i, 91.35 J . , 1929,1498; A., 1929, 1042.32 J., 1924,125,575.Trans., 1930, 220, Pt. 7 ; J. H. Birlrinshaw and H. Raistrick, ibid., Pt. 8236 CHIBNALL AND PRYDEcompounds containing less than six carbon atoms in the molecule,e.g., xylose, arabinose, and glycerol. They think that either ofthe following explanations seems to offer a solution of the difficulty,but consider that there is at present no conclusive experimentalevidence as to whether either is correct :-(1) I n common with some other types of fermentation, as shownfor glycerol by Neuberg and his co-workers, acetaldehyde may beproduced by the fungus from the carbon source supplied, whetherthis be a poly-, di-, or mono-saccharide, a pentose or a polyhydricalcohol.The acetaldehyde may then be condensed by a series ofreactions to kojic acid. This explanation is apparently supportedby the fact that it has been shown that all those fungi which producekojic acid also produce at the same time ethyl alcohol, and hence,in passing, acetaldehyde.(2) The source of carbon supplied, whatever its nature, may befirst metabolised by the fungus into a reserve carbohydrate, which islater hydrolysed by the micro-organism, as occasion arises, into amono-saccharide which in its turn gives rise to kojic acid.Theclose similarity between the amylene-oxide form of a 6-carbon sugarand kojic acid renders this a probable explanation, and it is furtherQH,*OH QH,*OHsupported by the fact that various fungi are known to store reservecarbohydrates, and to utilise them later as occasion demands, e.g.,glycogen in yeast, trehalose in A . niger.The view that the immediate precursor of the kojic acid is a3-carbon compound is favoured by F. Challenger, L. Klein, andT. K. Walker, who cite as a parallel the production of citric acidErom pentoses, and by A. Corbellini and B. Greg~rini,~~ who con-sider that the pyrone nucleus is formed by synthesis from 3-carbonoxidation products of glycerol by a reaction analogous to thatoccurring with aldehydes under the action of carboligase.New Phenolic 8ubstctnces.-Using glucose as source of carbon andthe carbon balance sheet as a guide to indicate the formation of" unaccounted carbon," Raistrick and his co-workers have isolatedthree new highly-coloured phenolic substances.From certain species of Citrolnyces 37 they have obtained yields36 Gazzetta, 1930, 60, 244 ; A., 959.37 A.C. Hetherington and H. Raistrick, Phil. Tram., 1930, 220, Pt. 11BIOCHEMISTRY. 237as high as 25% of the sugar fermented of a new yellow benzopyronederivative having the formula C,,Hlo0,,2H,0, which they havenamed citromycetin. It is a dihydroxy-carboxylic acid containinga benzopyrone nucleus, and from a study of its decompositionproducts it is considered to be (I).HO,C CO Et(1.1 (11.)0From 1'.citrinum, T h ~ m , ~ * and from no other species they haveisolated a new yellow crystalline colouring matter having theempirical formula C,,H1,O, which they have named citrinin. Froma study of its decomposition products F. P. Coyne, H. Raistrick, andR. Robinson39 tentatively assign to this compound the formula (11).They call attention to the fact that this carbon skeleton containstwo straight chains of 6 carbon atoms each, joined at their y-positionsby a thirteenth carbon atom.A third coloured substance, which separates as purplish-blackpermanganate-like crystals of the formula C8H805, was produced,together with much citric acid, by a strain of Penicillium spinulosum,It is a dihydroxymethoxytoluquinone, and is the firstrecorded instance of the production from glucose by fungi of aquinone derivative.It appears fairly certain that the substance isa p-quinone, but the relative positions of the methyl, methoxy, andtwo hydroxyl groups in the quinonoid nucleus can only be finallysettled by synthesis, as compounds of this type have not yet beendescribed in the literature.Perhaps the most interest'ing of the new products obtained fromglucose is the new polybasic fatty acid given by P. spiculisporumLehman.41 It is the lactone of y-hydroxy- pa-dicarboxypentadecoicacid (111), and its constitution has been proved in the following way.The substance is a dibasic acid and is stable towards acid.Onhydrolysis with caustic soda it yields a tribasic acid (IV), whichforms a monoacetyl derivative. The ease with which this tribasicacid reverts to the parent substance with loss of water on heatingsuggests that it is a y-hydroxy-tricarboxylic acid. On fusion withpotash the tribasic acid yields lauric acid (VI), succinic acid (VII)and carbon dioxide, and on oxidation with permanganate in acetone38 A. C. Hetherington and H. Raistrick, Phil. Trans., 1930, 220, Pt. 14.3Q Ibid., Pt. 15.4O J. H. Birkinshaw and H. ftaistrick, ibid., Pt. 12.4 1 P. W. Clutterbuck, H. Raistrick, and M. Rintoul, iW., Pt. 16238 CHIBNALL AND PRYDE :solution it gives a theoretical yield of a keto-acid shown by synthesisto be y-ketopentadecoic acid (V).CH3rp21tlQH3 QH3IQH219 LCH219CiH- 1+-YH*OH YoFH*CO,H 0 YH2FH*CO,H _j YH*CO,H -+ CH2CH2*C0--A/ CH,C 0 ,H(ITI.) (IV.) (V.1Y+ ?H2*Co2H + co, + H,O (iH3[ ~ H & CH,*CO,H CH,*CO,H(VI.) (VII.)These results show that the tribasic acid (IV) is y-hydroxy-@-di-carboxypentadecoic acid, and that the parent substance (111) isits y-lactone.It is interesting to note that y-ketopentadecoic aciditself was isolated from the metabolism solution, so that we havehere an undoubted instance of the production by a living organismof a fatty acid containing an odd number of carbon atoms. It isto be remembered, however, that this C,, acid and its dicarboxy-derivatives are not constituents of the mould " fat " but are excretedinto the fermentation solution, so their discovery does not necessarilyupset the view, now generally accepted, that natural fats containonly even-numbered carbon fatty acids.The degradation of the lower fatty acids themselves by fungi hasbeen recently investigated by T.K. Walker and his co-workers.A . niger was grown on an aqueous solution of the calcium salt of thefatty acid together with the requisite inorganic salts.42 Calciumpropionate was oxidised a t the cc-carbon atom, and lactic acid wasalways detected (thiophen test) before pyruvic acid (coloration givenwith benzenediazonium chloride in the presence of sodium acetate).Calcium n-butyrate, n-valerate and isovalerate gave the respectivep-hydroxy-acid and p-keto-acid and methyl ketone successively,showing that (3-oxidation had occurred in these cases.43The precise mechanism of p-oxidation of the normal fatty acidsin vivo is not yet known with certainty, previous experimental workindicating that the first product may be either a p-keto-acid, aP-hydroxy-acid or an ap-unsaturated acid.A11 of these are equallyeasily oxidised by the liver and are interconvertible.**42 T. K. Walker and P. D. Coppock, J., 1928,803; A., 1928,804.43 P. D. Coppock, V. Subrammiam, and T. K. Walker, ibid., p. 1422; A.,1928, 804. 44 H. D. Dakin, J . Biol. Chem., 1923,56,43BIOCHEMISTRY. 239When A . niger was grown on calcium n-butyrate Walker and hisco-workers cpuld not detect crotonic acid in the culture medium,nor would the mould grow on calcium crotonate as sole source ofcarbon.They consider that their evidence points to the initialformation of a p-hydroxy-acid and its subsequent oxidation to thecorresponding p-keto-acid. They therefore differ from W. N.St0koe,~5 who considers that the p-keto-acid is the initial product.The Chemistry of Plant Pathology.Pressing problems of economic importance have led during recentyears to a great increase in the amount of research devoted to plantpathology, but the mode of attack has been almost entirely bio-logical and research on strict chemical lines has been rarely initiatedand still more rarely successful. It is therefore noteworthy that thedifferences in susceptibility to a parasite of closely related plantshave been definitely connected with the presence or absence of achemical entity in the host.46 Red and yellow varieties of thecommon onion (Alliurn c e p ) are in general resistant to the diseasescaused by the fungus Colletotrichum circinans (Berk), whereas thewhite varieties are susceptible.Investigations revealed that anaqueous extract of the dry outer pigmented scales causes rupturingor abnormal germination of the spores and retards the growth ofthe mycelium of the fungus, whereas a similar extract from the dryouter white scales is not endowed with this property. Fractionationof the former extract led to the isolation of protocatechuic acid,which was found to be as toxic in dilutions of 1 part to 3000 parts ofwater as the original extract itself.The mode of preparation of theextract (digestion with 20 parts of water at 30" for 2 hours) and thechemical methods used in the fractionation exclude the possibilityof the acid having arisen by decomposition from quercetin, whichPerkin 47 showed was present in pigmented onion scales.The spike disease of Sandal (Santalum album, Linn.) has beeninvestigated by M. Sreenivasaya48 and his colleagues. They findthat the diseased leaves have a higher content of reducing sugars,total carbohydrates, and both total and soluble nitrogen than healthyones, and the sap has a definitely higher dinstatic activity. Further-46 Ann. Reports, 1929,26,210.4 6 K. P. Link, H. R. Angel, and J. C. Walker, J . Biol. Chem., 1929,84,719;A., 1929, 122; J .C. Walker, K. P. Link, and 13. R. Angel, Proc. Nat. Acud.Sci., 1929,15,845; A., 1929,262; H. R. Angel, J. C. Walker, and K . P. Link,Phytopath., 1930, 20, 431 ; A., 1224.4 7 A. G. Perkin and J. Hummel, J., 1896,69, 1295.48 J . I W n Imt. Sci., 1928,11A, 23, 97; 1929, 12A, 163; A., 1928, 804,1291; 1930, 385240 CHIBNALL AND PRYDE :more they state that mannitol can be isolated from diseased leaves,whereas it appears t o be absent in healthy ones.49A. A. Dunlap 5* has studied the virus diseases of plants, andsuggests that they may be divided into two classes, mosaic diseasesand yellows diseases, according to the effect of the disease upon thetotal nitrogen and total carbohydrate contents of the leaves of thehost plants. Mosaic disease caused an increase of nitrogen and itdecrease of carbohydrate, whereas yellows disease brought aboutthe reverse effect.His results are in general agreement with thoseof earlier workers; E. G. Campbell,51 for instance, found that leaf-roll disease of potato is accompanied by increase of carbohydrate,and R. H. True and L. A. Hawkins 52 found an increase of carbo-hydrate, and S. L. Jodidi 53 a decrease of total nitrogen, in blightdisease of spinach. Dunlap considers that the latter should beclassified as a yellows and not as a mosaic disease. Again, in fruits,A. S. Morne and F. G. Gregory 54 find that resistance to disease inapples is associated with high acidity, high potassium and lownitrogen, although the converse may also be true.Turning to the question of inorganic deficiency and plant diseases,it is of course well known that lack of certain essential elements suchas iron, potassium, and calcium brings about leaf chlorosis.Butduring recent years it has become increasingly evident, chieflythrough the work of J. B. Orr and his school a t Aberdeen,55 thatinorganic elements play a fundamental part in determining thenutritive value of pasture grasses. Deficiencies may occur which,though not so marked as to cause gross signs of disease, may yetlimit the rate of growth and the rate of productivity. Furthermorethese smaller deficiencies may adversely affect the “ constitution ”of the animal, rendering it more susceptible to some disease ofbacterial origin.R. Adam 56 and his colleagues have carried out an interestinginvestigation into the bactericidal action of a number of cyclic andstraight-chain fatty acids, which they have synthesised fromappropriate alkyl halides by the malonic ester condensation.The six different isomeric series of acids containing cyclohexylgroups,Nature, 1930,126,438 ; A ., 1483.50 Amer. J . Rot., 1930,17, 348.61 Phytopath., 1925,15, 427.52 J . Agric. Res.. 1918, 15, 381.63 J . Arne?. Chem. SOC., 1920, 42, 1061, 1885; A., 1920, i, 586.64 Proc. Roy. SOC., 1928, [B], 102, 444.6 5 Reviewed by J. B. Orr, “ Minerals in Pasture,” London, 1929.66 See W. M. Stanley, M. S. Jay, and R. Adams, J . Amer. Chem. SOC., 1929,1,1261 ; A., 1929, 676, in which reference is given to t% series of 15 papersBIOCHEMISTRY.241C,H,,*[CH,],*CO,H, C,H11*CHR*C02H, C6H,,*CH,*CHR*C02H,C,H 11-[ CH,],*CHR*CO,H, C6H, ,*[CH2],*CHR*CO2H, andC,H1,*[CH,],*CHR*CO,H, were found to possess high bactericidalaction in &TO to B. Zeprce. The effect increased with increase ofmolecular weight of the alkyl group, and was most potent when theacids had 16-18 carbon atoms. Further, those acids with thecarboxyl group a t the end of the chain were not nearly as effectiveas the isomerides with the carboxyl near the ring. As chaulmoogricand hydnocarpic acids both contain the A2-cyclopentenyl group, itseemed possible that a ring was necessary for bactericidal action;accordingly a series of acids of the general formula RCH(CO,H)R',in which R is a cyclopentyl, cyclopentenyl or cyclopropyl group,or one of these groups substituted in the w-position of theulkyl group, and R' is an alkyl group, was prepared and studied.l'he results indicated that there was no very marked differencebetween the acids containing the 3-, 5- or 6-membered rings,and that those with 16-18 carbon atoms were again the mosteffective. Finally a number of isomeric octadecoic and hexadecoicacids, which included a complete series of compounds with chainsof seventeen and fifteen carbon atoms, and a carboxyl group sub-stituted in every possible position, were prepared.The resultswith these acids showed conclusively that no ring was necessary forbactericidal action, and again it was found that the terminal carboxylgroup was the least effective.Adams and his co-workers concludefrom their studies that the effect of these acids towards B. Zeprceand other acid-fast bacteria can hardly be attributed to thechemical specificity of the individual acids, and is probably dueto a combination of physical properties common to many ofthem.J. M. Schaeffer and F. W. Tilley 57 have investigated manyisomeric alcohols in a similar way and find that those with thelongest straight chains or phenols having the longest straight chainin the para-position are the most efficacious germicides. Thecoefficients of cyclohexanol and of methylcyclohexanols are abouthalf as great as those of phenol and the corresponding cresols.The efficacy as contact insecticides of the n-fatty acids fromformic to stearic, and of their sodium and ammonium salts andmethyl esters, has been investigated by F.Tattersfield and C. T.Girni~~gham.~* The toxicity to Aphis rumicis L. of the acids fromacetic to undecoic rises with increase of molecular weight. Thesalts and esters were in general less toxic than the acids, but showeda similar rise with increase of molecular weight. The authors suggest5 7 J . Bact., 1927,14, 259; A . , 1928, 795.5 8 Ann. Applied Biol., 1927,14, 331242 CHIBNALL AND PRYDE:that the toxicity of these acids is bound up in some way withwater-solubility relationships.Nitrogenous Metabolism in the Plant.During the past year several interesting papers dealing with thenitrogen question in plants have been published. Many of theseemanate from Agricultural Stations, and much of the matter con-tained therein, although of great agricultural or horticulturalinterest, does not find a place in a Report devoted chiefly to progresson the chemical side. In addition, certain papers of fundamentalimportance published in a new journal, Phnta, during the pastfour years-to which the attention of the Reporter has only recentlybeen directed-fall to ’be discussed this year.Nitrogen Fixation by Bacteria.-Recent work by D.Burk on themetabolism of Axotobacter has led him to question the validity ofthe theory, now generally held, that the first stage in the fixation ofatmospheric nitrogen is the production of ammonia. He has usedthe manometric micro-methods for the study of cell-metabolismdevised by 0.W a r b ~ r g . ~ ~ He has shown-partly in collaborationwith 0. Meyerhof 60-that Axotobacter behaves uniquely towardsoxygen gas. Its rate of oxygen consumption and its efficiency ofnitrogen fixation are markedly conditioned by oxygen pressure ina manner characteristic of no other living organism. (1) The rateof respiration (measured directly by oxygen consumption) attainsa maximum at 0-15 atmosphere, diminishing rapidly at both higherand lower pressures, being only one-third as great at 0.005 and 1.0atmosphere. (2) The decrease in rate of respiration between 0.2and 1.0 atmosphere of oxygen is linear; this is true whether thecomplementary gas making the total pressure up to one atmosphere isnitrogen or hydrogen. (3) In the absence of free or fixed nitrogen,which would permit growth, the rates of respiration are independentof time at any given oxygen pressure, except in the high region of1 atmosphere, when they fall off with time.(4) In a young culturethe rate of respiration is enormously high, 2000 c. mm. of oxygenper mg. of dry matter per hour, or about three times its own dryweight of glucose. In contrast it may be noted that 0. Warburg’svalue for baker’s yeast is only 75 c . mm. and that the rate of respir-ation is independent of oxygen pressure between 0.03 and 0.97atmosphere. (5) The rate of nitrogen fixation attains a maximumat 0.4 atmosphere of oxygen, and is only one-third to one-sixth asgreat at 0.008 and 0.21 atmosphere. (6) The most important59 “ Uber den Stoffwechsel der Tumaren,” 1926, p.1-11.6o 2. physiiol. Chem., 1928, 139, 117; A,, 1929, 473; J . Physical C‘hem.,1930,34,1174,1195; A., 1068BIOCHEMISTRY. 243influence of oxygen pressure, however, is upon the efficiency ratio,nitrogen fixed/oxygen consumed, which increases some ten- totwenty-fold between 0.21 and 0.01 atmosphere.Burk admits that it is logical to suppose that these unusual meta-bolic properties are either the cause or the result of the similarlyexceptional ability of Azotobacter to fix nitrogen, and that anytheory of the chemical or catalytic mechanism of nitrogen fixationmust provide an explanation of them. This type of reasoning, forinstance, has been applied by certain workers recently to thebehaviour of certain strains of Axotobacter; 61 because ammonia wasfound in the culture fluid, it was considered to prove that ammoniawas concerned in nitrogen fixation.He does not think that suchreasoning is valid and recalls Winogradsky’s 62 original &dings thatthe ordinary metabolism and nitrogen fixation of these organismsare two distinct phenomena. The strains of Axotobacter used bythese workers produce ammonia extracellularly whether growingin either nitrogen gas or nitrate, and therefore there is no proof thatammonia is involved in their mechanism of fixation ; indeed, sincenitrates and nitrites are reduced vigorously to ammonia, it is equallylikely that the mechanism involves, rather than precludes, theformation of nitrogen-oxygen compounds such as nitrates.Burk’sstrains of Azotobacter produce neither ammonia nor nitrates extra-cellularly during fixation, nor ammonia when growing on nitrates,and he does not think that they can possess a different method offixation.Burk has so far established certain facts concerning the mechanismof fixation which are free from the above logical inconsistency.(1) At 0.2 atmosphere of oxygen, nitrogen is fixed at an appreciablerate only above 0-05 atmosphere, and tends to reach a maximumvalue a t about 5 to 10 atmospheres. (2) The efficiency of nitrogenfixation increases markedly with the rate of fixation. (3) All theunique oxygen pressure functions described above obtain in culturesgrowing on fixed nitrogen, and therefore offer no indication as to thenature of the chemical mechanism of fixation.63 (4) Humic acid,which greatly accelerates nitrogen fixation, is not directly concernedwith the mechanism of fixation but is a growth stimulant.( 5 ) Thefailure of legume bacteria t o fix nitrogen in the absence of the hostplant has been confirmed by gasometric studies under a variety ofpressures of nitrogen, hydrogen or oxygen between 0 and 1 atmos-6 1 S. Kostychew, A. Ryskaltschuk, and 0. Schwezowa, 2. physiol. Chem.,62 Compt. rend., 1893, 116, 1385; Arch. Sci. Biol. St. Petemburg, 1894-5,63 D. Burk and H. Lineweaver, J . Bact., 1930,19,389; A,, 1219.1926,134, 1.3, 297244 CHIBNALL AND PRYDE :phere. ( 6 ) The view of previous workers is supported and enlargedupon, that fixation is a function resorted to only in the absence ofsufficiently available fixed nitrogen.D.W. Cutler 64 records a new group of organisms distinct fromNitrosomoms and Nitrocrococcus, which produce nitrite from ammonia.Nitrate Reduction in Plant Roots.-The presence of small amountsof nitrate in green leaves has led to the view that the synthesis oforganic from inorganic nitrogen generally takes place in these organs.An interesting paper by G. T. Nightingale and L. G. Schermerhornshows that active reduction of nitrate to ammonia may take placcin the root system. When the asparagus plant is in a condition ofactive vegetative growth of tops, nitrates can be found only in thcfibrous roots. I n a plant lacking nitrates in its tissue and nutrientmedium, but containing reserve carbohydrate within its roots, thcexternal supply of nitrate leads to the production of nitrites andammonia in the fibrous roots only and not in the storage roots.Atthe same time asparagine and amino-acids appear in these organsin considerable quantities and the amount of reserve carbohydrateis reduced. The transformation of nitrates is most rapid from 20"to 30°, and is very slow at 10". The storage roots and activelygrowing tops of the asparagus plant apparently may assimilatenitrates) but rather seldom have an opportunity to do so becausenitrates are transformed to organic nitrogen in the fibrous rootsbefore reaching other organs of the plant. If, however, the tem-perature is 10" or lower, nitrates may be translocated to other partsof the plant.Later, with a rise of temperature, nitrates are rapidlyassimilated by and disappear from both the storage roots and theactively growing tops and are then found again only in the fibrousroots.A similar transformation of nitrate to organic nitrogen wasobserved by G. T. Nightingale and W. R. Robbins66 in the finefibrous roots of the paper-white narcissus (Polyanthus narcissus),and W. Thomas 67 concludes from his research on the nitrogen meta-bolism in Pyrens malus, L., that the transformation takes place forthe most part in the roots.Nitrogenous Metabolism in Underground Storage Organs.-1%.Gruntuch 68 has reviewed what little earlier work there is on thissubject and has carried out a long series of experiments with theunderground storage organs of many plants, chiefly with XohnurnNature, 1930,125, 168 ; A ., 376.65 N . J . Agric. Exp. Sta. Bull., 1928, No. 476 ; A., 607.66 Ibid., 1928, No. 472; A., 1929, 612.6 7 Science, 1927,66, 116; compare Ann. Reports, 1927, 24, 230.68 Planta, 1929, 7, 382BIOCHEMISTRY. 245luberosum, Helianthus tuberosus, Dahlia variabilis, and Asparagusoficinalis. He finds, as one would expect, that the total nitrogenin these organs varies greatly, not only in different plants, but alsoin the same plant a t different stages of growth. In spite of this theratio of protein/soluble nitrogen remains fairly constant. Griintuchconcludes that the metabolism of these storage organs remainsobscure, and is not to be compared with that of the ripening seed.69If nitrates (which Griintuch has not determined) are transformed inthe fibrous root, as discussed above, and do not accumulate in thestorage organs, then the soluble nitrogen is presumably all organicnitrogen, and it is possible that we are dealing here with some“ mass-action ” relation between protein and soluble nitrogensimilar to that postulated by L.R. Bishop 70 in the development ofthe reserve proteins in the barley grain.The R61e of Ammonia in Plant Metabolism.-In a series of paperspublished during the last thirty years D. Prianischnikov has deve-loped a theory, now generally accepted, that ammonia is the “ alphaand omega ” of nitrogen metabolism in the plant. Ammonia, how-ever, was never stored as such in the plant but was metabolisedto “ amides ” (asparagine and glutamine). If the carbohydratereserve in the plant was insufficient for this purpose, the plant rapidlydied.He considered, therefore, that ammonia was a plant poison,and that r‘ amides ” were an innocuous form of ammonia storage.’lIn a brilliant series of papers published during the last three yearsW. Ruhland and K. Wetzel have shown that in certain types ofplant-those with a very acid sap-these conditions do not hold,and that an accumulation of ammonia (as salts, not free) can occurto an extent hitherto considered improbable. Within the limits ofthis Report it is possible to give only a brief outline of the moreimportant of their results in as far as they relate to the metabolismof nitrogen.The high sap acidity (pE 1-54-1-56) of leaves of Begonia semper-$orens suggested that they might be used for investigations on themetabolism of organic acids.72 The following differences betweenthese leaves and those of normal acidity (pE 5-6.5) were observed.(1) If the leaves were kept in the dark, the respiratory quotientrose from an initial 1.1 to 1.47-1-85 at the end of 3 days. Normalleaves showed a fall*belowil.(2) They contained 20% of their dry weight as oxalic acid.E9 F. Czapek, ‘‘ Biochemie der Pflanzen,” 11,279.70 Ann. Reports, 1929, 26, 219.71 Biochem. Z., 1928,193,211; 1929, 207,341; A., 1998, 662; 1929, 728;72 W. Ruhland and K. Wetzel, Planta, 1926,1,658; H. Ullrich,ibid., p. 565.compare M. E. Robinson, New Phyt., 1929,88,11724G CHIBNALL AND PRYDE:(3) They contained 5-10 times the normal preformed ammonia,and the value increased threefold during the day.Whereas innormal leaves the value (soluble N - ammonia N)/amrnonia N wasusually about 50, in begonia it was only 2-3. If the leaves werekept in the dark a t 28-35' to bring about protein decomposition,the ammonia content rose enormously-in 106 hours to 30% of thetotal nitrogen, while the small initial amount of " amide " nitrogendisappeared. The deamination was accompanied by a parallelincrease in oxalic acid (the pH of the sap decreased to 1.3) so that theammonia was always present as ammonium oxalate, and at no timewas free ammonia poisoning possible. In normal plants, of course,the " amide " nitrogen increases in the dark, the ammonia remainingmore or less stationary.Ruhland and Wetzel therefore considerthat plants can be separated into two physiological types-" amide "plants and " ammonia " (or " acid ") plants.In further illustration of this new important observation may becited certain of their results with rhubarb (Rheum hybridurn h o ~ t ) . ~ ~The rhizome, which has only a slight acid reaction, contains " amide "nitrogen (20% of the total soluble nitrogen), much amino-nitrogen(50% of the total), and only traces of ammonia. The stalks of the veryyoung leaves synthesise protein rapidly from the amino-acids andamides translocated from the rhizome. This ceases as the leavesopen, and a little later, when the stalks are 15 cm.long, strongdeamination takes place. The fully grown stalks contain some 62%of their total nitrogen as ammonia, and if they are kept in the darkthis value may rise to 72%. Parallel with the production ofammonia during growth is the production of (chiefly) malic andsuccinic acids, which give place slowly, as the stalk ages, to oxalicacid; so that free ammonia poisoning is again prevented. BothZ-malic and dZ-malic acids are found and it is considered t h a t theinactive acid has been translocated from the rhizome, in which thismodification only is found, and that the active acid has arisen bydeamination of amino-acids, since the relative concentrations ofammonia and this active acid are nearly I : 1. It is interesting t onote that the leaf lamin=, which are much less acid than the stalks,contain the usual amount of protein (88-9170 of the total nitrogen)and only traces of ammonia.That " amide " plants can, in anemergency, mobilise ammonia is shown by some results of D.Prianischnikov, 74 in which the roots of certain embryos, after beingdipped in acid, gave off ammonia and, to a certain extent, neutralisedthe acid.Ruhland and Wetzel call attention to the close similarity between73 W. Ruhland and K. Wetzel, Planta, 1927, 3, 765; K. Wetzel, ibid.,74 Biochem. Z., 1928, 193, 211 ; A., 1928, 562. 1927, 4, 476BIOCHEMISTRY. 247the metabolism of these “ acid ” plants and that of Aspergillus nigergrowing on p e p t ~ n e , ~ ~ in which 78y0 of the soluble nitrogen is con-verted into ammonia and there is an equivalent production of oxalicacid.Another interesting pwallel is the production of dl-malic andZ-malic acid during the metabolism of rhubarb stalks and the pro-duction of both these acids by A . niger (p. 233).8’ynthesis of Protein in G e e n Leaves.-J. Bjorksth 7G has putforward some interesting views on the intermediary products ofsynthesis in leaves which are based on an entirely new method ofexperimentation. The intercellular spaces of leaves of wheatseedlings suffering from nitrogen starvation were injected withvarious nutrient solutions by the evacuation method. The proteinnitrogen (in mg. per 1 g. of dry leaf) was then compared every hourfor 6 hours with that of control leaves. As an efficient control sourceof nitrogen, 0-O1M-urea was used. Injection with hydrogenperoxide greatly increased the production of carbon dioxide, butprotein synthesis went on normally.Changing the acidity of thesolution between pH 4 and pH 8, or the osmotic pressure by addingpotassium, sodium, or chlorine ions, again had no effect on proteinsynthesis. As sources of carbon for protein synthesis, none of thesimple aliphatic or hydroxy-acids was of use. Pyruvic acid (butnone of its homologues) was, however, readily utilised, and as therewas no increase in carbon dioxide formation it would appear that theacid did not undergo decarboxylation. Of the sugars tried,only glucose was utilised. Aliphatic amines, amino-acids, andammonium salts of aliphatic organic acids were good sources ofnitrogen supply, nitrates less so, and cyclic products were notutilised at all.Hydrogen cyanide and nitrites, which Traub 77considered might be precursors of amino-acids, were not used assources of nitrogen.Bjorksthn discusses the various hypotheses that have beenadvanced to account for the synthesis of amino-acids in the light ofhis own results, and puts forward a new suggestion that the simplestbuilding stone for protein synthesis is a-aminoacrylic acid, formedby the condensation of enolic pyruvic acid and ammonia.In a second paper J. Bjorksthn and I. Himberg 78 discuss certainaspects of the rble of ammonia in protein synthesis. Injection ofetiolated wheat leaves with urea, acetamide, and butyramide leadsto protein synthesis but no increase in free ammonia.Under ethernarcosis, injection of urea leads to an increase of free ammonia, but76 W. Butkevitsch, Biochern. Z., 1922,129,445.7 6 Ibid., 1930,225,l; A., 1482.7 7 Compare M. E. Robinson, Biol. Reviews, 1930, 5, 126.78 Ibid., 1930, 225, 441248 CHIBNALL AND PRYDE:no such increase is produced by acetamide and butyramide. As faras is known at present, there is no enzyme which will hydrolyse theseamides, whereas, of course, urea is readily broken down by urease.They conclude, therefore, that ammonia plays no direct r6le inprotein synthesis from these amides, and postulate a mechanism bymeans of which the amides are condensed directly with pynivic acidto give a-aminoacrylic acid.They consider that urea can also condense directly with enolisedpyruvic acid to form a-aminoacrylic acid, which is of interestbecause G.Klein 79 has recently asserted that the major part of theurea in plants-and its wide distribution in small amounts is wellknown-is not free, but bound with formaldehyde or acetaldehydeas a ureide.Xitrogen Exchange in Phcrnts.-Certain aspects of this problemwere discussed in last year’s Report. During this year severalpapers have appeared, some of which favour the view that amidesare concerned chiefly with storage, and that organic nitrogen istransported as amino-acids. incontinuation of their researches on the cotton plant have traced themovements of nitrogen in the boll, and conclude from a study of thenitrogen gradients into the boll during growth that the organicnitrogen enters from the sieve-tubes as “ residual ” rather than as“ amide ” nitrogen.H. Engel 82 has made some interesting observ-ations on plants suffering from nitrogen starvation. Cut shootswere kept for some weeks with their ends in distilled water, and theflow of nitrogen between the older fully grown and the youngdeveloping leaves was observed. The older leaves suffered muchprotein decomposition, but the soluble nitrogen was decreased also,showing that the products of decomposition had been translocated.Engel discusses the variations found in the ammonia-, amide-, andwhat he refers to as amino-nitrogen, and concludes that the nitrogenfrom the older leaves has been translocated to the growing parts asamino-nitrogen.Single leaves were used in some experiments, so thematerial available for micro-chemical analysis was necessarily limited.His value for amino-nitrogen (total soluble N-ammonia N - twicethe amide N) is open t o criticism.83K. Mothes 8* has made somewhat similar experiments. Cuttobacco plants were kept for 5-6 weeks in air that was 70q4 satur-ated. The older leaves withered and lost the major part of their79 2. Pflanz. Dung., 1928, 12A, 390; Abstracts of Communications, 5thInternational Congress of Botany, Cambridge, 1930.E. J. Maskell and T. G. MasonAnn. Reports, 1929, 28, 216.81 Ann. Bot., 1930, 46, 657; A., 1323.*3 Compare H. R. Vickery, Ann. Reporfs, 1929,26, 21 2.84 Rer. deut. Rot. Qe.?., 1928, 46, 591.82 Plantn, 1929,7, 133BIOCHEMISTRY.249protein, while the young growing leaves were still green and turgidand were actively synthesising protein. His analyses led him tothe conclusion that the nitrogen was transported as amides from theold leaves to the younger. That the protein of old leaves-even inthe presence of abundant carbohydrate-is more labile than that ofyoung leaves was shown by K. Mothes in an earlier paper,85 so thatH. Engel's assumption that the nitrogen starvation leads to theprotein breakdown in his older leaves does not necessarily follow.H. L. Newby and W. H. Pearsall86 show that as the leaves of thevine and rhubarb become old the ratio of the protein to non-proteinnitrogen decreases, as also does the acidity of the sap.A study of the soluble nitrogen in the leaves of the soya beanduring development has been made by J.E. WebsterY8' and of theseasonal variations in the protein and soluble nitrogen in the matureleaves of three evergreen plants by H. Sattler.88Phnt Bases and Alkaloids.-Very little information on themetabolism of these substances in the plant is available, and therehas been during the past two years a welcome increase of researchdevoted to the subject. G. Klein and M. Steiner 89 have investigatedabout 100 species of plant. All of them contained ammonia, andabout 40 contained volatile arnines. Methyl-, dimethyl-, trimethyl-,isoamyl-, and isobrityl-amine were identified by microscopic methodsbased on experiments with synthetic products. The amountspresent ranged from 0.0005 mg.--0.2 mg.per 100 g. of fresh leaves.The authors consider that these volatile products are used to attractinsects, and that they may be used as a basis for the systematicclassification of plants. R. Kapeller-Adler and T. Csaf6 findmethylamine and trimethylamine in s e a - ~ e e d . ~ ~T. Weevers 91 has investigated the metabolism of caffeine andtheobromine of several plants. They are found in the wood, under-ground organs, and especially in leaves. The amount in the lastdecreases as the leaf ages and disappears before it dies. Weeversconsiders that when this happens the bases are not translocated assuch, but undergo degradation in the same way as protein. Excisedleaves of Ilex paragwriensis 92 St. Hill show an increase of caffeinein daylight and a decrease if kept in the dark.Excised leaves of thetea plant, when kept in water, accumulate xanthine equivalent to30% of the decomposed protein, an amount too large to permit theassumption that it has its origin in the leaf nucleo-protein. He con-8 6 Proc. Led8 Phil. Lit. Soc., 1930, Section 2, 81.89 Jahrb. wiss. Bot., 1928, 68, 602.8 5 Planta, 1926,1,472.87 Plant Physiol., 1928,3,31; A., 1929,612.Planta, 1929, 9, 315.90 Biochem. Z., 1930,224,378; A., 1484.91 Arch. nkerhnd. Sci., 1930, VIIB, 5,111.92 Proc. Acad. Sci. Amsterdam, 1929, 32,281250 CHIBNALL AND PRYDE :siders that xanthine and its derivatives are storage products, andthat their nitrogen can be utilised when occasion arises for proteinsynthesis.The position with regard to alkaloids is not yet so clearly defined.It will be recalled that Pictet considered them to be secondaryproducts formed by the plant to remove poisonous primary productsof metabolism.T. Weevers and H. D. van Oort 93 draw no definiteconclusions from their experiments with the leaves of Cinchonusuccirubia, Pavon. K. Mothes 94 shows that there is a small gradualincrease in nicotine as the leaves of the tobacco plant develop, butthe connexion, if any, between the synthesis of nicotine and proteinmetabolism is not clear.K. Mothes 95 has also investigated the metabolism of arginine inPinus spinea. The base was estimated as flavianate after prc-liminary precipitation with phosphotungstic acid. In view ofVickery's 96 results on the composition of the basic fraction fromalfalfa-which contained but very small amounts of arginine andconsisted chiefly of basic compounds of undetermined composition(some of which may be precipitated by flavianic acid)-it seems tothe Reporter that this method of analysis is unsound.It is interesting to note that a new base closely allied to argininchas been isolated by M.Wade 97 from the press juice of the water-melon (Citrullus vulgaris). Its constitution, x-amino-6-carbamido-valeric acid, NH,*CO~NH*[CH,],*CH(NH2)*C0,H, has been con-firmed by synthesis from ornithine, by way of dibenzoylornithine,6-amino-a-benzamidovaleric acid, and 6-carbamido- ct-benzamido-valeric acid.Carbohydrate Constituents of Plant Tissue."The structure of pectic acid, which is generally accepted as thebasis of all pectic substances, still provokes some discussion. Theformula of Nanji, Paton, and Ling,98 in which pectic acid is regardedas composed of 4 molecules of galacturonic acid, 1 molecule of gal-actose, and l molecule of arabinose combined in a hexa-ring, has beenquestioned by S.T. HendersonJs9 who obtained from flax a producthaving the composition of a galactose-tetra,galacturonic acid (i.e., con-93 Proc. Acad. Sci. Amsterdam, 1929, 32, p . 1.n4 Planta, 1928, 5, 563; Apoth.-Ztg., 1930,13, 3.9 5 Ylanta, 1929,7,685. s6 Ann. Reports, 1929, 26, 212.9' Proc. Imp. Acad. Tokyo, 1930, 6, 15; Biochem. Z., 1930, 224, 420; A.,B8 J . SOC. Chem. Ind., 1926,44,253~.99 J . , 1928,2117; A., 1928,1119.* The Reporter gratefully acknowledges assistance from Dr.H. W. Buston1224.in the preparation of this sectionBIOCHEMISTRY. 251taining no arabinose) and possessing all the properties of pectic acid.F. W. Norris 1 considers that the pectic acid of flax is of the normaltype, as indicated by the furfural and " uronic anhydride " content.Ehrlich 2 still describes as " pectic acid " a water-soluble compoundhaving the formula C41H60036, formed by the loss of 9 molecules ofwater from 4 molecules of galacturonic acid, 1 molecule of galactose,1 molecule of arabinose, 2 molecules of acetic acid, and 2 moleculesof methyl alcohol. This product was obtained in the form of acalcium magnesium salt, in association with an araban, by hot-waterextraction of beetroot residues.The presence of methyl alcoholresidues, and the solubility of the substance in water, seem toindicate that it is actually an intermediate between the completelydemethoxylated, insoluble pectic acid (C35H50033) of other workersand " soluble pectin " (containing 4 methoxy-groups). The presenceof acetic acid in the molecule has not been accepted by other workers.J. R. Bowman and R. B. McKinnis3 obtained from oranges anarabinogalacturonic acid, and by a similar process from apples adigalacturonic acid. These they regard as the nuclear units of therespective pectins. They suggest that the digalacturonic acidundergoes transition in nature to arabinogalacturonic acid (andpossibly to arabinose), and that pectins contain these acids invaxying proportions.Although the ring formula for pectic acid is now widely accepted,the relation between this acid and " soluble pectin " still presentscertain unexplained aspects. The action of weak alkali on pectinconsists mainly in a removal of the methyl ester groups, the finalproduct being the insoluble, completely demethoxylated pectic acid.It has been pointed out, howeverY4 that pectic acid and methylalcohol are not the sole products of the reaction, small amounts of ahemicellulose being invariably produced, a fact not accounted forby a simple saponification.may in partexplain this.By the action of sodium hydroxide solution (0-5-4%) at temperatures between 37" and loo", pectic acid was shown toundergo rapid decarboxylation, yielding products of a low " uronicanhydride" content-20yo instead of 70% in the case of onionpectic acid.These products were shown to resemble the hemi-celluloses in many of their properties. A similar decarboxylationwas demonstrated by F. V. Linggood,(j by heating pectic acid withThe results of E. J. Candlin and S. B. Schryver1 Biochem. J., 1929, 23, 195 ; A., 1929, 729.2 F. Ehrlich and F. Schubert, Ber., 1929,62, [B], 1974 ; A., 1929,1273.3 J . Amer. Chern. Soc., 1930,52,1209; A., 746.4 F. W. Norris, Biochern. J., 1926,19,676; A., 1926, i, 1226.5 PTOC. ROY. SOC., 1928, [B], 108,365; A., 1928, 1162.6 Biochem. J., 1930,24,262; A., 824252 CHIBNALL AND PRYDE:water under pressure. About 12% of the pectic acid was accountedfor as hemicellulose, the remainder being present possibly asdegradation products of sugars.Since the pectins and the hemi-celluloses have been proved to be closely related, the term '' poly-uronide " has been proposed to include both classes.An investigation on the hydrolysis of pectin by alkali has beenmade by A. G. Norman and J. T. Martin,' in an attempt to determinet'he mode of linkage between the individual members of the pecticacid ring. They pointed out that the pectin ring was extremelysusceptible to attack by weak alkali (e.g., 0.2% solution a t lOO"),indicating that the linkages between the units are probably unlikethose found in other polysaccharides such as cellulose and starch.They found that rupture of the ring proceeded more rapidly thanapparent decarboxylation, and criticised the conclusions drawn byCandlin and Schryver, since they were able to show that substancesother than hemicelluloses and uronic acids were formed, and werecapable of yielding large amounts of carbon dioxide with boilinghydrochloric acid.Work on somewhat similar lines was carried out by A.G. Normanand F. W. Norris,* who studied the oxidation of pectic acid byFenton's reagent. Pectic acid they proved to be readily oxidised,giving complex mixtures containing galactose and galacturonic acidresidues. Their products strongly resembled the hemicelluloses,and the authors put forward the suggestion that, in nature, thelatter may arise from the pectins by prolonged mild oxidation, ratherthan by decarboxylation.Certain of the gums have been shown tocontain uronic acid residues, and may be formed ~imilarly.~Attention has often been directed to the fact that tissues rich inpectin are poor in lignin, and vice versa, and the suggestion has beenmade that pectin is the precursor of lignin. No practical evidencehas been put forward in support of this view.While evidence has been furnished that the hemicelluloses arerelated to the pectins in that they are based on conjugated sugarand sugar acid residues, their structure remains undecided. F. W.Norris and I. A. Preece lo have isolated five types of hemicellulosefrom non-lignified tissues (wheat bran, maize cobs). Of these,hemicellulose A was precipitated from the caustic soda extract of thet,issue on neutralisation with acetic acid; hemicellulose B, by thcsubsequent addition of a half-volume of acetone ; and hemicelluloseC by excess of acetone. B and C were subdivided into fractionsI31 and C1 by precipitation with Fehling's solution, B2 and C27 Biochem.J., 1930, 24, 649; A., 966.9 A. G. Norman, ibid., 1929, 23,524; A., 1929, 856.10 Ibid., 1930,24, 69; A., 383; I.A. Preece, ibid.,p. 973; A., 1326.Ibid., p. 402; A . , 824BIOCHEMISTRY. 253remaining in the respective filtrates. Of these products, A washydrolysed to xylose ; B2 to glucose only ; B1 and C1 gave varyingamounts of xylose, methylpentose, and a uronic acid; C2 gavearabinose, methylpentose, and uronic acid. The uronic acidpresent was probably glycuronic acid.From a consideration of theamounts of pentose, etc., obtained, the hemicellulose molecule isevidently one of considerable complexity, B1 having a minimummolecular weight of 6500.In the case of the hemicelluloses from lignified tissue, M. H.O'Dwyer l1 showed that the methoxyl groups were of two types, oneof which was extremely resistant to hydrolysis, and therefore wasnot an ester group. The' hemicelluloses derived from timber are byno means of the same structure as those from non-lignified tissues,although extracted by similar means. The products from wood have,in general, a much higber uronic anyhdride content and are appar-ently more complex in nature. Other tissues (e.g., flax) yieldh emicelluloses containing varying proportions of uronic anhydrideresidues .99Certain substances intermediate between the pectic substancesand the simple sugar acids have been isolated and studied, notablyby F.Ehrlich.2 By careful hydrolysis of pectic acid, three isomerictetragalacturonic acids have been isolated, differing in their opticalproperties, reducing power, etc. These acids have been shown topossess a cyclic structure, only tetragalacturonic acid B having a freealdehyde group. Ehrlich 12 has described a new pectic enzyme,pectolase, isolated from old cultures of Perisporaceae and shown tobe present also in taka-diastase, diastase, and emulsin, which wasable to hydrolyse these cyclic acids to (mono) galacturonic acid andwas also able to liberate soluble pectic compounds from the insolubleprotopectin.Ehrlich has suggested that the cyclic tetragalacturonicacids are normal intermediates in the enzymatic degradation ofpectin.Constitution of Long-chin Fatty Acids from Natural Sources.I?. Francis, S. H. Piper, and T. Malkin 13 have synthesised then-fatty acids from C,, to C,,, and have made a study of the meltingpoints and X-ray spacings of the acids, their ,ethyl esters, and ofequimolecular mixtures of the acids. The melting points of the oddand the even acids lie on two smooth curves. Two important typesof spacing are given by each acid, and when these are plotted againstthe number of carbon atoms four straight lines are obtained, twol1 Biochem. J., 1928,22, 381; A., 1928, 669.l2 Celluloeechem., 1930,11,140,161; A., 1163.l8 PTOC.RoY.SOC., 1930, [A), 128,214; A,, 1161254 CHIBNALL AND PRYDE :belonging to the even and two to the odd carbon acids. Theauthors call attention to the fact that reliance cannot be placed onmelting point or " mixed " melting point for purposes of identi-fication. For instance, acids of carbon content 20 and 21 atoms,and the following mixtures, 21 + 22, 22 + 23, 22 + 23 + 24, allmelt between the limits 74.9" and 75.2". The X-ray spacings andmelting points of mixtures of known composition convince theauthors that a n-fatty acid cannot be considered pure unless it hasthe correct melting point and correct acid value and gives bothX-ray spacings. They have analysed arachidic, lignoceric, cerotic,and montanic acids prepared from various natural sources, and theacids obtained by oxidation of the alcohols present in Chinese waxand carnauba wax.All were shown to be mixtures of n-fatty acidsand there was no indication of the presence of so-called iso-acids.The authors show that by intense fractionation it is possible toobtain pure acids from natural sources. Samples of lignoceric acidprepared by Dr. Brig1 from beechwood tar and of cerotic acidprepared by Professor Holde from Chinese wax were shown to bepure n-tetracosanoic acid and n-hexacosenoic acid respectively.Glutathione.Conclusive evidence has now been obtained that this tripeptide isy-glutamylcysteinylglycino,HO,C*CH( NH,) *CH,*CH,*CO*NH*CH( CH,*SH)*CO*NH*CH,*CO,H ,by E. C. Kendall, H. L. Mason, and B.F. McKenzie.l* If glutathioneis first oxidised to the corresponding sulphonic acid by means ofbromine, and then treated with sodium hypobromite, it yields onemolecule of carbon dioxide and a substance from which, before orafter further treatment with nitrous acid, succinic acid and glycineare liberated by hydrolysis with hydrochloric acid. If glutathioneis oxidised with hydrogen peroxide in the presence of ammonia,succinic acid is not liberated by the oxidation, but a significantpercentage of the total glutamic acid can be separated as succinicacid after hydrolysis. Also, if glutathione is treated with nitrousacid and then with alkaline hypobromite, succinic acid cannot beisolated until after hydrolysis. Finally, if glutathione is oxidisedwith chloramine-T, the mononitrile of succinic acid is not liberatedin significant amount, but succinic acid can be recovered afterhydrolysis. These reactions show that glutathione must be either( 1) glutamylglycylcysteine or (2) glutamylcysteinylglycine, the freeamino-group of the glutamic acid being in the y-position to thepeptide linkage.Since the product of interaction of glutathioneethyl ester hydrochloride with magnesium phenyl bromide yields,1 4 J . Biol. Chm., 1930,87,66; 88, 409; A,, 946, 1299BIOCHEMISTRY. 255on hydrolysis, diphenylacetaldehyde, it is probable that the carboxylgroup of the glycine is free, and constitution (2) the correct one.It will be recalled that (Sir) F. G. Hopkins l5 showed that whenglutathione was boiled in aqueous solution much decompositionoccurred, and together with unidentified products the diketo-piperazine of glycine and cysteine (or, in the case of the disulphideform, diglycylcystine dianhydride) was isolated.Kendall and hisco-workers find that, by heating glutathione in aqueous solution at62" for 120 hours, hydrolysis to pyrrolidonecarboxylic acid and adipeptide of glycine and cysteine occurs. On treatment of the latterwith sodium hypobromite or with nitrous acid, followed by acidhydrolysis, glycerol was isolated, though in poor yield. The dipep-tide was next oxidised to the disulphide, this condensed with2 : 3 : 4-trinitrotoluene, and the product hydrolysed with hydro-chloric acid. Glycine was again isolated, showing conclusivelythat the dipeptide was cysteinylglycine, and that glutathione mustbe y-glutamylc ysteinylglycine .Additional evidence that glycine occupies a terminal position inthe molecule has been furnished by B.H. Nicolet.ls Glutathionewas condensed directly with ammonium thiocyanate and aceticanhydride to give a compound C,,H,,O,N,S,. This was condensedwith benzaldehyde in the presence of acetic acid and sodium acetateto give a, compound which, when hydrolysed with sodium hydroxide,gave a 50% yield of benzylidenethiohydantoin. The compoundC1,Hl,O,N,S, is therefore regarded as a bisthiohydantoin, in theformation of one of the thiohydantoin groups of which glycine musttake part. It follows, then, that glycine occupies a terminal positionin the molecule.W.Grassmann, H. Dyckerhoff, and H. Eibeler l7 show thatglutathione is not hydrolysed by pepsin, pancreatic proteinase,papain, or the dipeptidase and aminopolypeptidase from yeast andintestine. It is, however, readily attacked by pancreatic carboxy-polypeptidase, which hydrolyses only one peptide linkage, yieldingthe total glycine in the free state and a peptide residue recovered in90% yield. They consider that this shows the glycine to be at theend of the chain and that the carboxyl group is free.This clarification of the chemical nature of glutathione has,unfortunately, introduced considerable uncertainty as to its actualphysiological r6le in the pure condition. The earlier preparationspossessed such characteristics as constituted glutathione a chemicalcatalyst in the interaction between atmospheric oxygen and fats,l6 Ann.Reporta, 1929,26, 222.1@ J . Biol. Chem., 1930, 88, 389; A., 1299.l7 2. physiol. Chem., 1930,189, 112; A., 1067256 CHIBNALL AND PRYDE :proteins, and the " insoluble, thermostable residue ? ' left afterexhaustive extraction of tissue.18 All of these substances areoxidised in the presence of impure glutathione, which in turn isre-oxidised by oxygen gas. N. U. Meldrum and M. Dixon,19 how-ever, now indicate that glutathione when pure possesses theseproperties only in respect of the fats, where its catalytic activity isknown to occur a t an acidity not found in animal tissues. Itappears that in all preparations of glutathione there exists in varyingdegree some hydrolytic product to which, in the presence of iron orcopper ions, the autoxidisability of reduced glutathione must beattributed.Pure glutathione even in the presence of iron is notautoxidisable. Neither are other cysteine peptides. Nevertheless,the evidence presented by these authors indicates that the fissionproduct is closely comparable in properties with cysteine itself.Thus, though existing evidence as to the function of the thiol groupin conjunction with iron in a respiratory capacity is in no wayaffected, the actual participation of the tripeptide, glutathione, inthis capacity has become much open to question.Recent attempts to show the presence of glutathione in planttissues are not very convincing.20Muscle Contraction.The investigation of the physical and chemical processes whichconstitute the mechanisms of muscle contraction continues toabsorb much of the energies of biochemists.In recent yearsconsiderable information concerning the chemical nature of thesubstances participating in the muscle process has accumulated,but the more purely physical side has been somewhat neglected.In a highly interesting series of studies of the physical chemistryof muscle globulin, J. T. Edsall 21 directs attention to this neglectwhen he says : " Knowledge of the energy-liberating reactions inmuscle has made enormous strides in the past 20 years, and farsurpasses our knowledge of the machinery which they set in motion.It seems beyond doubt that the proteins play a large-if not thelargest-part in this machinery. It is to be expected that thephysico-chemical properties of the isolated protein will be foundintimately related to its function within the muscle fibre." Edsalldescribes the preparation of muscle globulin, which is regarded asidentical with the myosin of Danilewsky, von Fiirth, and Weber,and with the paramyosinogen of Halliburton, although the terml 8 Ann.Reports, 1925,22, 225. lS Biochern. J., 1930, 24, 472; A., 803.2o W. H. Camp, Science, 1929, 69, 455; A., 1929, 1499; V. B. White, ibid.,z1 J . Biol. Chem., 1930,89,259.1930,71,74; A., 826BIOCHEMISTRY. 257myosin is reserved, in a later paper of the series, for the anisotropicprotein responsible for the double refraction of flow. Muscleglobulin, if kept in the cold, protected from bacteria, dissolved insalt, at pH 6.5 to 7-5 preserves its properties unchanged over aperiod of several months.The protein is insoluble in all salt con-centrations between pu 5 and 6, but it possesses an extraordinaryaffinity for water, from which it cannot be separated without someradical change in the protein itself. Even concentrated precipitatesof the protein contain 98% of water, and it remains undenaturedonly in the presence of a large amount of water. This phenomenonmay, as Edsall suggests, have a special physiological significance,since it has been recorded that no significant change occurs in thewater content of skeletal muscle, even in cases of profound dehydr-ation, when the fluid of the intercellular spaces is greatly depletedand the blood volume is diminished well below normal.Whenmuscle weight is lost, proteins and salts are lost along with thewater removed. In other words, the muscle appears to lose waterto any extreme degree only when the protein is broken up and lostas well. The behaviour of muscle globulin suggests that the wateris directly held by the protein, and that this water-holding poweris not lost when the protein is extracted from the muscle.In two further communications A. L. von Muralt and J. T.Edsall22 have published many interesting observations on thephysical properties of muscle globulin. It is found that the proteinshows double refraction of flow, which is ascribed primarily to theorientation of anisotropic particles and secondarily to photo-elastic phenomena. Apparatus is described for measuring theangle of isocline (the angle at which the arms of the black crossappear when polarised light is passed through a solution of theprotein in a rotating cylinder).The measurements of the angleof isocline are interpreted as indicating a monodisperse system,that is, the muscle globulin particles are of uniform shape and size.The angle of isocline of muscle globulin solutions is independent ofage. After repeated washing of the muscle globulin solution, thepreparation becomes practically salt-free and forms a clear geleven a t an extremely low protein concentration (about 0.3%).Merely by vigorous shaking, this gel is transformed to the fluidstate and sets again to a gel in a few minutes.It is therefore athixotropic gel.23The double refraction of flow shown by muscle globulin solutionsis intimately related to the chemical nature of the protein solution,22 J . Biol. Chem., 1930, 89, 315, 351.23 A. Szegvari and (Frl.) E. Schalek, KoZEoid-Z., 1923, 32, 318; 33, 326; A,,1923, ii, 423 ; 1924, ii, 116.REP.-VOL. XXVII. 258 CHIBNALL AND PRYDE :Thus it is found that typical denaturing agents produce rapid andcomplete destruction of the double refraction of flow, which appears,therefore, to be a property only of the undenatured protein. Fromthe work of H. StuebelZ4 it has been inferred that oriented rod-shaped particles, small compared with the wave-length of light,are present in the intact muscle and are responsible for its doublerefraction.The double refraction and other properties of myosinsolutions indicate that they may contain these rod-shaped particles,and also point to the probable location of the myosin in the aniso-tropic disc of the cross-striated muscle fibre. It seems highlyprobable that these remarkably interesting investigations mayelucidate new aspects of the functional activity of the muscle, andin any case they constitute a new and welcome attack, by estab-lished methods of physical science, on this very complex biologicalproblem. Similar remarks may be made concerning the investig-ations of G. Boehm and K. I?. S c h o t ~ k y , ~ ~ who have obtainedX-ray diagrams of living muscle in a state of rest and of excitation.Despite the wealth of new discoveries made in recent yearsconcerning the chemical processes of muscle contraction, somerecent observations of 0.Meyerhof 26 suggest that there are stillmaterial gaps in our knowledge. Meyerhof has employed a thermo-electric method for measuring the depression of freezing point ofmuscle in fatigue and in rigor. I n prolonged fatigue and rigor theobserved depression is some 30% greater than would be expectedon the basis of the known products of hydrolysis, Similar con-clusions are reached by A. V. Hill and P. s. Kupalov,27 who findthat the increase of osmotic pressure in the fluids of a stimulatedmuscle is about 2.8 times as great as would be exerted by thelactate ions produced, if dissolved in the “free” water of themuscle.It is 1.8 times as great as would correspond to the lactateions together with the creatine liberated by the breakdown ofphosphagen, and is appreciably greater than can be accounted forby all the chemical changes at present known, or suspected, tooccur in stimulated muscle. A. V. Hill 28 defines the “ free ” waterfraction as the weight of water in 1 gram of fluid or tissue whichcan dissolve substances added to it with a normal depression ofvapour pressure. The “ free ” water fraction of frog’s muscle,whether resting or in rigor, is about 0-77, or perhaps a little greater,the total water fraction being 0.80 or 0.81. On the other hand,0. Meyerhof and F. Lipmann 29 find that the pH change observed2 5 Naturwiss., 1930,18, 282; A,, 637. 24 Arch.ges.Physiol., 1923,201,629.26 Biochem. Z., 1930, 226, 1 ; A., 1614.2Q J . Physiol., 1930, 69, Proc. XXI; A., 1211; Naturwiss., 1930, 18, 330;Proc. Roy. SOC., 1930, [B], 106, 446. 28 Ibid., p. 477.A., 810BIOCHEMISTRY. 259in a muscle during a prolonged series of twitches agrees well withthat calculated from the lactic acid formed and the phosphagensplit. The muscle at first becomes more alkaline and only in anadvanced state of fatigue does it become more acid. The explan-ation of the phenomenon is found in the fact that the breakdownof creatinephosphoric acid, which in the first stages of fatigue islarge compared with the simultaneous formation of lactic acid,increases the amount of basic equivalents.Muscle Contraction without Production of Lactic Acid.-In thefield of muscle chemistry one of the most interesting and significantdiscoveries announced during the past year concerns the action ofiodoacetic acid on the lactic acid formation of contracting muscle.Early in the year it was announced by E.Lundsgaard30 that thepost-mortem formation of lactic acid in the muscles of rabbits andfrogs poisoned by sodium iodoacetate was completely inhibited.At the same time the contractile power of the muscle did not sufferdamage. Muscles treated with sodium iodoacetate will contractwith a hydrolytic cleavage of the phosphagen, which takes placeeven more rapidly than in normal muscle, and without lactic acidformation. After total breakdown of the phosphagen the muscleremains in a state of contraction.The phosphoric acid of thephosphagen is, under these conditions, rapidly and completely con-verted into hexosephosphoric acid. On the basis of these observ-ations it was suggested that phosphagen might be the energy-producing substance in muscle activity, and that the productionof lactic acid might cause progressive resynthesis of the hydrolysedphosphagen. Later, Lundsgaard showed that the anaerobic re-synthesis of phosphagen was abolished after the muscle had beentreated with iodoacetate, but on the other hand, in the presenceof oxygen, the poisoned muscle did more work than under anaerobicconditions and showed a smaller diminution in phosphagen content.This result is ascribed to oxidative resynthesis of phosphagen.Thechronaxie of the poisoned muscle is normal. 0. Meyerhof, E.Lundsgaard, and H. Blaschko31 have shown, by a comparison ofthe total anaerobic development of tension with the decompositionof phosphagen, that the whole of the energy for anaerobic con-traction with the poisoned muscle is derived from phosphagen.Reference has already been made to the observations of Meyerhofand Lipmann on the change of pH during muscle activity. Asmight be anticipated these observers 32 have demonstrated that inthe presence of iodoacetic acid only the alkaline phase of the change30 Biochem. Z., 1930, 217, 162; A,, 368.31 Naturwiss., 1930, 18, 787; A., 1312.Biochem. Z., 1930, 227, 84260 CHIBNALL AND PRYDE :is observed, and that when the muscle is arrested in contracturethe alkalinity is maximal.Two further communications by Lundsgaard 33 extend theseremarkably interesting observations.It is shown that iodoaceticacid completely inhibits alcoholic fermentation by living yeastand by zymase preparations, but in neither case is hexosephosphoricacid formation observed. The actions of invertase, ptyalin, andcatalase are not affected by iodoacetic acid. I n general, in thepresence of iodoacetic acid, oxidative processes involving carbo-hydrates are able to proceed normally after glycolysis is completelyinhibited.34I n addition to these observations concerning the energy sourcesof muscle contraction, a recent paper by G. Embden and E. Metz35directs attention to the fact that bromo- and iodo-acetic acidscause a marked diminution in the solubility of the muscle proteins,similar to that observed by H.J. Deuticke36 in fatigued muscles.It would appear to be highly probable that the halogenated aceticacids are destined to be of great service in elucidating, and possiblyeven in revolutionising, the complex chemistry of the muscleprocesses.It has been recognised for many years that fluorides inhibitfermentation and glycolysis, and F. Lipmann37 has pointed outthat, if muscles are poisoned with fluoride in such concentrationas to leave unimpaired their contractile power, such contractionshould occur without lactic acid formation. This he shows to bethe case, the contraction occurring with decomposition of phos-phagen, esterification of hexose with phosphoric acid, and hydrolysisof adenylpyrophosphoric acid.The mechanism of the action ofiodoacetic acid and of fluoride on the lactic acid-forming fermentcomplex is similar, save that the former acts slowly and is irre-versible, whereas the latter acts instantaneously and is reversible.Phosphagen.-Continuing his observations already summarisedand in support of his views 38 regarding the relation between phos-phagen and rate of excitation, D. Nachmansohn39 has shown thatthe increased extent of decomposition of phosphagen caused byveratrine is exactly proportional to the increased rate of excitationcaused by the drug. On the other hand, curare and the ammoniumbases generally produce a marked reduction in the extent of decom-position, the greatest effect in this respect being observed withtrimethyloctylammonium iodide.This base has been used by33 Biochem. Z., 1930, 220, 1, 8 ; A., 958, 954.3 5 2. physiol. Chem., 1930, 192, 233.3 7 Biochem. Z., 1930, 227, 110. ** Biochern, Z., 1929, 213, 262; A., 1929, 1484.34 Ibid., 1930, 227, 51.36 PJEiiger’s Arch., 1930, 224, 1.3a Ann. Reports, 1929, 26, 232BIOCHEMISTRY. 2610. Meyerhof and D. Nachmansohn 40 in investigating the synthesisof phosphagen in living muscle. It is found that the anaerobicresynthesis of phosphagen in living muscle after decompositionduring tetanus is as great, expressed absolutely, in muscle poisonedwith trimethyloctylammonium iodide as in the unpoisoned muscle,but is much greater when expressed as a percentage of the totaldecomposition.Aerobic resynthesis of phosphagen is observed tooccur both after previous decomposition on admission of oxygen,and without previous decomposition when the muscle is placedin Ringer's solution saturated with oxygen, especially after additionof phosphate. The aerobic resynthesis after previous decompositionproceeds more quickly and completely at lower temperatures andmost quickly at - 0.5" to -- 1". The molecular ratios, phosphatesynthesised : oxygen used, and phosphate synthesised : lactic aciddisappearing, both give values of 5.B. Kisch41 has published some interesting observations on theoccurrence of phosphagen in the electric organ of Torpedo. Theamount present is found to be about the same as that of the generalmusculature.In the fresh unstimulated organ 77% of the phos-phorus of the acid extract is present as phosphagen. Duringactivity of the organ and during asphyxiation the amount of phos-phagen rapidly diminishes, but resynthesis occurs during rest inthe presence of oxygen. Thus the analogies between the electricorgan and the skeletal muscles developed on morphological groundsare supported by these biochemical observations.The Adenylic Acid Complex of Muscle.-There is now a generalacceptance of the view that the adenine present in an aqueousextract of muscle occurs as a nucleotide. The observations ofK. P ~ h l e , ~ ~ A. Dmoch0wski,4~ and P. Osterna are in accord inthis respect, and the further association of the nucleotide (adenylicacid) with pyrophosphoric acid, an association first indicated byK.L0hrnann,4~ is supported by the data of C. H. Fiske and Y.S~bbarow.*~ The last-mentioned observers do not regard adenylicacid as such, as a normal constituent of muscle, but find it to be adecomposition product of a substance which is precipitated as thecalcium salt from a protein-free muscle filtrate. This substanceyields adenine, carbohydrate, and three molecules of phosphoricacid, two of which are readily removed by acid hydrolysis.Concerning the significance of adenylic acid as the source of40 Biochem. Z., 1930, 222, 1 ; A.. 1210.42 2. physiot. Chem., 1929, 185, ! f ; A., 1929, 1479.43 Biochem. J., 1929, 23, 1346; A., 1930, 238.44 Biochem. Z., 1930, 221, 64; A ., 945.*1 Ibid., 1930,225,183; A., 1464.46 Ann. Repwt-s, 1929, 26, 231.S&ence, 1929,70, 381 ; A., 1930, 492262 CHIBNALL AND PRYDE:muscle ammonia, G. Embden and G. Schmidt47 have publishedsome convincing data. The enzymes of fresh frog-muscle weredestroyed by acid treatment and the muscle was exposed to theaction of an enzyme capable of deaminising muscle adenylic acidonly.48 The ammonia eliminated by this procedure correspondswith that obtained by exposing the minced muscle for 3 to 4 hoursin a slightly alkaline solution. It is concluded, therefore, that theammonia formed in a short autolysis of frog’s muscle is derivedexclusively from the muscle adenylic acid, and that it is now possibleto exclude even adenosine as a possible source. Adenylic acid mayparticipate in reactions involving more profound degradation ofits molecule than this deamination.In some of the results pub-lished by G. Embden, J. Hefter, and M. Lehnartz49 there is asuggestion that a t the moment of contraction a fission of adenylicacid, or of its deamination product, inosinic acid, may occur withthe formation of orthophosphoric acid, and K. Pohle 50 has sug-gested adenylic acid as a possible precursor of endogenous uricacid.Adenylic Acid and the Kidney .-Considerable interest attachesto the occurrence and functions of adenylic acid in kidney tissue.G . Embden and H. J. Deuticke 51 have isolated the acid from thissource and shown it to be identical with that obtained from muscle.It is acted upon by the specific deaminase of rabbit’s muscle.B. E.Holmes and A. Patey 52 have studied the ammonia-forming systemswhich occur in washed kidney tissue. One of these acts aerobicallyand has an optimum of pIl 5.2 or lower. A second aerobic system,acting upon glycine, and an anaerobic system are also shown to bepresent in the kidney.53 It is suggested that the first-mentionedsystem is concerned in the normal production of ammonia by thekidney, as this is known to be greatest when the urine is acid. Itis not yet possible to state whether or not adenylic acid is theammonia-precursor in this system. There is, however, generalagreement that adenylic acid can be completely deaminated underanaerobic conditions. Therefore, although it may be the sourceof some of the ammonia in the urine, it cannot be the substrate ofthe particular aerobic system studied, and the pH optimum curvesdo not suggest that it is the substrate for extra ammonia formationin the case of an acid urine.47 2.physiol. Chem., 1930, 186, 205; A., 494.40 Ann. Reports, 1929, 26, 228.49 2. physiol. Chem., 1930,187, 53; A., 637.61 2. physiol. Chem., 1930, 190, 62; A., 1203.52 Biochem. J., 1930, 24, 1664; A., 1614.LOC. cit.A. Patey and B. E. Holmes, ibid., 1929,23, 760; A., 1929, 1194BIOCHEMISTRY. 263Enzymic Hydrolysis of Glycogen.A. D. Barbour 54 records the hydrolysis of glycogen by means ofa glycerol extract of fresh muscle or liver tissue. The hydrolysisis carried out for 5 hours at the optimum pH of 6-3.The soleproduct of the action of the muscle extract upon the glycogen is atrisaccharide, C,,H,,016, having + 181", and 30% of thereducing power of glucose. It is readily converted into the anhydro-trisaccharide, C18H3,-,01,, having + 187", and 8.5% of thereducing power of glucose. The digestion of glycogen by salivaryor pancreatic amylase appears to follow a course different from thatof the muscle and liver enzymes. Attempts to use the enzyme forthe synthesis of glycogen from the trisaccharide and its anhydro-derivative were not successful. If the trisaccharide can be obtainedin reasonable amount, it would become a matter of great interestand importance to subject it to the recognised methods of structuralinvestigation in the sugar group, more especially in view of theapparent chemical identity of trimethyl glycogen and trimethylstarch.55 It is of interest to recall here the preparation fromglycogen by H.Pringsheim and G. Will 56 of a trisaccharide deriv-ative which they called glycogesan. It was obtained by chemicalmeans. Under the influence of pancreatic amylase, glycogesanwas degraded to maltose, fission being practically quantitative ifyeast complement was added. Although glycogesan behaved as atrisaccharide on cryoscopic investigation, the facts that it formeda colloidal solution in boiling water and possessed the same opticalactivity and iodine colour reaction as glycogen suggest that it hasa greater molecular complexity than the trisaccharide described byBarbour.Alcoholic Fermentcttion.In an important paper by R.Robison and W. T. J. Morgan 57on the phosphoric esters of alcoholic fermentation a detailed de-scription is given of the met,hods of fermentation and separationemployed. The details will be of great service to other workers inthis field. The distribution of the total esterified phosphorusamongst the four known esters is given for a number of fermentationexperiments with yeast-juice, zymin, and dried yeast. There is54 J . Biol. Chem., 1929, 85, 29; A., 1930, 249.55 W. N. Haworth, E. L. Hirst, and J. I. Webb, J., 1929, 2479; A., 1930,72.Ber., 1928, 61, [B], 2011; A., 1928, 1225.57 Biochem. J., 1930, 24, 119; A., 374264 CHIBNALL AND PRYDE :appended a table, taken from this paper, which records analyticaldata of the barium salts of these esters :P, %.Ester.Barium fructosediphosphate 10.16phate ............... 7 .85,, glucosemonophos-,, fructosemonophos-,, trehalosemonophos-phate (Neuberg) ... 7-85phate ............... 5.57Reducing poweras glucose. SeliwanoffH. and Iodine, fructose,12 2 10 + 3.5"36 41 0 +19*5J., %. %. %-22 + 0.7 2 360 0 0 +132In the course of this work some indication has been obtained ofthe existence of a fifth ester, forming, at most, only a very smallproportion of the fermentation products. Following on the dis-covery of pyrophosphate as a constituent of muscle,58 E. Boyland 59has shown that it also occurs in living yeast and forms about one-fourth of the total phosphorus.Pyrophosphate added to ferment-ing zymin is rapidly hydrolysed to orthophosphate, which thenreacts in the usual way. Evidence is obtained of the presence inyeast of a pyrophosphatase which is distinct from hexosephos-phatase. The distribution of phosphorus compounds in freshEnglish brewer's yeast (as mg. of phosphorus per g. of yeast) isgiven as follows:Total phosphorus ............ 3.25 Hexosediphosphate ......... 0.38Orthophosphate ............... 1.37 Hexosemonophosphate ...... 0.72Pyrophosphate ............... 0.68 Nucleic acid .................. 0.07Organic phosphorus .......... 1.17A. Harden and M. G. Macfarlane 6O have made experiments inwhich mixtures of sand and yeast were ground for different periodsand the resultant total mass, without pressing out, was tested forthe rate of fermentation and response to phosphate.At least 80%of the diminution in the rate of fermentation which occurs is ascribedto the process of grinding, during which the yeast acquires thepower of responding to phosphate. The conclusion is drawn thatthe grinding process mainly affects the mechanism of hexosephos-phatase action. M. G. Macfarlane 61 has published a study of theaction of disodium arsenate on hexosephosphatase in which it isshown that the accelerating action takes place only in the presenceof yeast extract and cannot be obtained without an accompanyingfermentation. Although this observation does not elucidate themechanism of the accelerating action of arsenate, it does dispose59 Biochem.J., 1930, 24, 350; A., 817.Ibid., p. 1051; A., 1317.68 Ann. Reports, 1929, 26, 230.6o Ibid., p. 343; A., 818BIOCHEBIISTRY. 265of earlier theories which ascribed it to a direct effect upon thehydrolysis. In relation to tlhis question it is significant that K.Lohmann 62 has found that! the hydrolysis of Robison’s mono-phosphoric acid ester in frog-muscle pulp is accelerated by arsenateonly after the mono-ester hsts been further esterified to form theHarden and Young di-ester.The Sugar of Animal Nucleic Acid.During the past year the long-debated problem of the nature ofthe sugar component of animal nucleic acid would at least appearto have been settled. The fission of animal nucleic acid into itscomponent nucleotides presents much more difXculty than isencountered in the case of the plant acid.But despite thesedifficulties P. A. Levene and E. S. London63 have succeeded ineffecting this disruption and in isolating guanine nucleoside togetherwith smaller quantities of hypoxanthine, thymine, and cytosinenucleosides. Using combined gastric and intestinal fistuh, theysubjected solutions of animal nucleic acid to the action of theintestinal juices of the dog in vivo and subsequently for longerperiods in vitro, or alternatively the juices were collected from thefistulze and added to solutions of nucleic acid. Levene and London 64prepared from guanine nucleoside a sugar having the compositionof a deoxypentose, C,HIoO,, to which, pending its identification,they gave the name thyminose. It gave with Kiliani’s reagent thespecific colour reaction for this type of sugar, and a Willstatter-Schudel iodometric oxidation proved it to be an aldose.It formedlaevulic acid on being treated with sulphuric acid and was there-fore a straight-chain pentose. The later work of P. A. Levene andT. M ~ r i , ~ ~ and of P. A. Levene, L. A. Mikeska, and T. Mori 66 ledto the identification of thyminose as d-2-deoxyribose :qH( OH)*CH,*CH( OH) *CH( OH)*qH2The new sugar is therefore it reduction product of the pentose ofplant nucleic acid. It affords, so far as the Reporter is aware,the first instance of the occurrence of a deoxy-sugar in a naturalproduct.The Sugar of Pentosuria.--The possible relationship of the newsugar of animal nucleic acid to the sugar of pentosuria must forthe present remain speculative.In the past this sugar has been6* Biochem. Z . , 1930, 222, 324; A., 1210.63 J . Biol. Chem., 1929, 81, 711; A., 1929, 590.84 Ibid., 1929, 83, 793; A,, 1929, 1322.85 Ibid., p. 803; A., 1929, 1277. g6 Ibid., 1930, 85, 786; A., 455.I 266 CHIBNALL AND PRYDE :variously identified as dl-arabinose, I-arabinose, dl-ribose, I-ribose,d-xyloketose, or simply as a d- or an I-rotatory pentose. In viewof these curious variations, but more especially in view of the greatinterest of Levene’s new results, a re-examination of all the avail-able data seems desirable. During the past year two studies ofthe sugar of pentosuria have appeared. I. Greenwald,67 on thebasis of four cases, identifies the sugar as d-xyloketose, and P.H&ri,6* with five cases a t his disposal, finds that the sugar belongsto the xylose group but does not record very convincing evidenceregarding its optical rotation.It is perhaps worthy of note thatH. 0. Calvery G9 has isolated adenosine (adenine-d-riboside) fromnormal human urine. No trace of the phosphorylated compound(adenylic acid) was detected. The adenosine was isolated from amixed urine and the yield was small, so the nucleoside might easilyhave had its origin in the urine of a few individuals, and thereforemay not be of common occurrence. Its presence in urine maybear some relationship to the uric acid-pentose compound firstreported in urine by A. R. Davies, E. B. Newton, and S.R. Bene-dict. 70The Xugars of the Tubercle Bacillus.In the Reports for 1926 71 and 1929 72 attention was directed tothe important r6le of carbohydrates in immunological reactions.The work there summarised dealt mainly with the sugars of thepneumococcus group of organisms. In the past year much inform-ation has accumulated concerning the similarly specific sugars ofthe tubercle bacillus. From tubercle bacilli propagated in asynthetic, sugar-free medium and also from tuberculin, M. Maxim 73has isolated a polysaccharide which on hydrolysis yields d-mannoseand d-arabinose. The same two carbohydrates are reported byA. G. Renfrew 74 to be present in the polysaccharide isolated fromLong’s synthetic media in which tubercle bacilli had grown.P.Masucci and L. K. McAlpine 75 likewise confirm this and in additionfind an unidentified sugar acid. E. Chargaff and R. J. Anderson 76record the presence of mannose and arabinose together with galactoseand inositol in the polysaccharide extracted by toluene along withthe lipoids of the ehtire bacilli. R. J. Anderson and E. G. Roberts 7767 J . Biol. Chem., 1930, 88, 1 ; A., 1311.6 8 Biochem. Z., 1930, 224, 474; A., 1469.69 J . Biol. Chem., 1930, 86, 263; A., 633.71 Ann. Reports, 1926, 23, 248.73 Biochem. Z., 1930, 223, 404; A., 1219.74 J . Biol. Chem., 1930, 89, 619.7 6 Amer. Rev. Tuberculosis, 1930, 22, 678.7 6 2. physiol. Chem., 1930, 191, 172.70 Ibid., 1922, 54, 595.72 Ibid., 1929, 28, 239.7 7 J . Biol. Chern., 1930, 89, 611BIOCHEMISTRY.267find glucose, mannose, and inositol in the sugar fraction of thetubercle phosphatide described in the Report of last year. Theoccurrence of d-arabinose in these sources is of interest, sinceZ-arabinose is the usual variety found in nature. A useful reviewof the present state of these investigations will be found in thearticle by T. B. Johnson and A. G . R e n f r e ~ . ~ ~ The polysaccharideexamined by Redrew gave precipitin reactions a t a dilution of1 in 1,500,000, from which it may be inferred that the tuberclepolysaccharide is functionally similar to those of the pneumococci.A study of the molecular size of the specific polysaccharide ofType I11 pneumococcus has been made by F. R. Babers and W. F.G~ebel,’~ using the diffusion method of Northrop and Anson.80The result gives a molecular weight of 118,000.Incidentally thesame method has been applied by P. A. Levene and A. Rothen 81to the polysaccharide of ovomucoid, which appears to consistexclusively of glucosamine and mannose. The result in this caseindicates a molecular weight of 2000 and it is inferred that thepolysaccharide consists of four trisaccharide units each containing1 molecule of glucosamine and 2 molecules of mannose.The Reducing Xubstances of Blood.In view of the widespread use of various techniques for deter-mining the reducing substances of blood (usually referred to as‘‘ blood sugar ”) a paper published by J. M. Gulland and R. A.Peters 82 will be of interest. In this a study is recorded of thereducing substances of pigeon’s blood.It has frequently beenobserved that the total reducing value of avian blood determinedby the usual methods is considerably higher than that of mammalianblood. For instance, for normal hen’s blood the Hagedorn andJensen method has given values as high as 0.253%, which wouldappear to place normal avian blood within the range of humandiabetic values. Gulland and Peters find in pigeon’s blood (by thesame method) an average of 0.200%, of which only 0.135 5 0.015%can be regarded as glucose or similar reducing hexoses. Bloodfiltrates prepared by different, methods contain different proportionsof ergothioneine, uric acid, and glutathione, or other aliphaticsulphhydryl compounds. Zinc filtrates made by the Hagedorn andJensen method, which do not contain aliphatic sulphhydryl com-pounds (an observation confirmed by M.R. Everett 83), are the78 Amer. Rev. Tuberculosis, 1930, 22, 665.79 J. BioZ. Chem., 1930, 89, 387.81 J . Biol. Chem., 1929, 84, 63; A., 1929, 1478.*a Biochem., J . 1930, 24, 91; A., 360.88 J . Biol. Chern., 1930, 87, 761; A., 1201.8o Ann. Reports, 1929, 26, 241268 CHIBNALL AND PRYDE :most trustworthy for determining the reducing substances of avianblood, but these filtrates contain in addition to glucose, ergothion-eine and some other unknown substances which reduce the ferri-cyanide reagent. Some 60% of the residual (non-glucose) reducingvalue is not accounted for by ergothioneine.To judge from the number of papers published on the subject,the problem encountered by Gulland and Peters, perhaps in a lessacute form, must be kept in mind in relation to the determinationof the reducing substances of mammalian blood.That glucoseis present as such in blood has been demonstrated by L. B. Winter,s4who has been able to isolate the normal crystalline form of thehexose from blood filtrates. But it cannot be doubted that in thevarious methods used for determining blood sugar, other reducingsubstances in varying amounts are included from time to time inthe glucose figure. A useful critical study of this question inwhich the Shaff er-Hartmann alkaline copper reagent is used,together with a consideration of the effects of different deprotein-ising agents upon the determination of Flood sugar, has beenpublished by S.L. T o m p ~ e t t . ~ ~ The reducing power of glutathionein relation to this problem is dealt with by M. R. Everett,86 andthe effect of acid hydrolysis on the total carbohydrate content ofthe blood is examined by F. Silberstein, F. Rappaport, and M.W a ~ h s t e i n . ~ ~ Papers by J. Roche,88 E. J. Bigwood and A. Wuil-lot,8s and G. Font& and L. Thivolle should also be consulted.The Coagulation of Hcernoglobin and its Reversal.M. L. Anson and A. E. Mirsky91 have brought to light a veryinteresting property of haemoglobin which it probably possesses incommon with other proteins. It has been the accepted view thatwhen a protein is coagulated or denatured the change which it hasundergone is an irreversible one.Protein coagulation proceeds intwo distinct steps. The first, known as denaturation, is a changein the native protein brought about by heat, the action of acid,alcohol, or other agents, which makes the previously soluble proteininsoluble near its isoelectric point. The second step is the pre-cipitation of the insoluble denatured protein. The latter, althoughinsoluble near its isoelectric point, is soluble in acid or alkali. If,therefore, a protein is denatured in a nearly isoelectric solution, a848 58 78 889P1Biochem. J., 1930, 24, 851; A., 1306.Ibid., pp, 1148, 1164; A., 1306.Biochem. Z., 1929, 213, 355; A., 1929, 1477.Bull. SOC. Chim. biol., 1930, 12, 636; A., 1054.Ibid., 1929, 11, 1204; A., 1930, 237.90 Ibid., p. 1212; A., 237.J . Gen. Physiol., 1929,13, 121, 133; 1930,13,469,477; A,, 102,630.86 LOC. citBIOCHEMISTRY. 269visible precipitate results. But if a protein is denatured in acid oralkaline solution, no visible change results until the solution ismade isoelectric; the protein is then precipitated. The secondstep in coagulation, the flocculation of the insoluble protein, is, ashas long been known, reversible, since the flocculated protein mayreadily be redissolved. The first step in coagulation, denaturation,has hitherto not been reversed. When a solution of the coagulumin acid or alkali is brought to the isoelectric point, the protein isagain precipitated ; it is still denatured. Denaturation, therefore,is the important process in the investigation of the reversibility ofcoagulation.Anson and Mirsky have been able to show in the first placethat hzmoglobin, like all typical coagulable proteins, may becompletely denatured, the test of complete denaturation beinginsolubility at the isoelectric point.Denaturation of the nativehemoglobin has been effected in a number of different ways, byheat in the presence of acid, by acid, by urea, and by shaking, andin all cases the results are the same. From preparations of com-pletely denatured horse haemoglobin Anson and Mirsky have pre-pared native carbon monoxide-haemoglobin which is identical withthe same pigment obtained directly from native haemoglobin.‘‘ Reversed ” hemoglobin can be coagulated by heating, and inregard to its colour, absorption bands, gas affinities and otherproperties it is indistinguishable from native hzmoglobin whichhas not been through the denaturation process.By means ofcrystallographic, spectroscopic, and gas-affinity measurements thesoluble native haemoglobin prepared from coagulated horse hzmo-globin cannot be distinguished from native hzmoglobin in general,or from horse hemoglobin in particular. Denatured haemoglobinsof various species cannot be distinguished from one another byspectroscopic and gas-affinity measurements, but, on the otherhand, after reversal of the coagulation the species’ characteristicsonce more become observable. Anson and Mirsky have securedyields of 75 and 80% of the reversed protein, which would seem toremove all possibility of their results being due to the presence of aresidue of native hzmoglobin attached to the main bulk of de-natured protein.It is concluded that protein coagulation ingeneral is probably reversible. In support of this view it is shownthat, by the use of acid acetone, haemoglobin may be rapidly separ-ated into a precipitate of denatured globin and an acetone solutionof haematin. By gradual neutralisation the denatured globin maybe largely converted into a soluble native form which can combinewith haematin to form hemoglobin.It seems not improbable that the phenomenon of denaturatio2 70 CHIBNALL AND PRYDE:and its reversal may have a wide significance in the chemistry ofthe proteins as a whole. I n the living cell the reversible coagulationof proteins a t intersurfaces may play some part in determining theproperties of semipermeable membranes, or in the mechanisms ofmuscle contraction.Hcemocyanin.During the period under review a considerable revival of interestin haemocyanin has occurred and a number of interesting refer-ences to the pigment have appeared. None of the newer investig-ations supports the view that haemocyanin is constituted on linessimilar to haemoglobin, that is to say, there is no evidence of thepresence of a copper-porphyrin in its molecule.J. Roche92 hasshown that it is possible to separate the copper from haemocyaninby adjusting the solution to pH 2.5, by the addition of dilute hydro-chloric acid, followed by dialysis. Such treatment applied t ohaemoglobin transforms it into haematin and globin.No similartransformation is observed in the case of hEmocyanin. Degradedhaemocyanin, as Roche calls the copper-free substance, shows thesame isoelectric point, buffering power, and solubility as naturalhaemocyanin. It is concluded that no prosthetic group is liber-ated by this treatment, and that the presence of such in the haemo-cyanin molecule is problematical. It is thought that the viewtaken by M. Heme 93 in 1901, namely, that hzniocyanin is a copperproteinate, is much nearer the truth than some of the views advancedsince that date. Roche has also shown an interesting differencebetween the haemocyanins of Octopus and Limulus. The pigmentof the former has an isoelectric point (prr 4.8) close to that of the crust-acean pigments examined by E.Stedman and (Mrs.) E. Stedman,9*whereas the isoelectric point of Limulus haenlocyanin is muchhigher (pH 6.2-6.4), a fact which is possibly related to the muchmore archaic morphological characters of the king crab.Conclusions essentially similar to those of Roche have beenreached by J. B. Conant and W. G. Humphrey95 and by A.S ~ h m i t z . ~ ~ The former investigators find the pigment to be aprotein in combination with a complex salt of an unknown amino-acid containing sulphur, which forms highly coloured complexeswith amines, and in this respect functions in a manner similar tothe protoporphyrin of haemoglobin. Schmitz suggests that thepigment consists of a protein combined with a complex copper92 Arch.Physique biol., 1930, 7, 207. 93 2. phyaiol. Chem., 1901, 33, 370.94 Biochem. J., 1927, 21, 533; A., 1927, 689.95 Proc. Nut. Acad. Sci., 1930, 16, 543; A., 1304.96 Natu&8., 1930,18, 798; A., 1304BIOCHEMISTRY. 27 1compound which is of a peptide nature. It should be mentionedthat the methods used by Conant and Humphrey and by Schmitzin degrading haemocyanin were much more drastic than thatemployed by Roche. It is doubtful in view of Roche's resultswhether one can accept their evidence for the existence of a copper-containing prosthetic group of a peptide nature.F. Herder and E. Philippig7 give the following composition ofair-dried crystalline oxyhaemocyanin from Helix pomatia : C, 48.59 ;H, 7-04 ; N, 14-26 ; S, 0.71 ; Cu, 0.232.E. Philippi and F. Hern-ler9* have published a further paper on the action of papain ontheir purified haemocyanin, which confirms the earlier results ofC. Dh6rrB and C. Baumelerg9 regarding the rapid formation ofcrystalline oxyhaemocyanin in the presence of papain and sodiumfluoride. The ease of crystallisation is remarkable in view of theenormous molecular weight of 4,930,000 assigned by T. Svedbergto haemocyanin.Female Sexual (CEstrous-producing) Hormone.During the past two years activity in this field has greatly in-creased and in view of the fact that several workers have isolatedcrystalline products with considerable biological activities a reviewof the present position seems desirable.The presence of an estrous-producing hormone in the urine ofpregnant women was first shown in 1927 by Aschheim and Zondek.Since that date many workers have entered the field and during thepast year several claims have been made to have isolated thehormone in a crystalline form.H. Wieland, W. Straub, and T.Dorfmiiller 2 described the preparation of an active crystallinematerial which melted at about 175" and finally became liquidwith decomposition at 210°, but no claim was made that the sub-stance was pure. A. Butenandt3 prepared an active crystallinesubstance which, from the constancy of its activity after recrystallis-ation and resublimation, he believed to be the hormone itself.The substance melted at 240", with decomposition, and fromanalyses the formula C23H2803 or C24H3203 was ascribed to it.Itwas unsaturated and on account of its behaviour with aqueousalkali it was suggested that the substance was a hydroxy-lactone.A. Butenandt and E. von Ziegner * later raised the melting point to243-245", and Butenandt ti modsed his &st suggested formula to1 Ann. Reports, 1928,25,239.97 2. physiol. Chem., 1930, 191, 23; A., 1461.Compt. rend. SOC. Biol., 1929, 101, 1071.2. physiol. Chem., 1929,186, 97 ; A., 1930, 265.Naturwiss., 1929, 17, 879; A., 1930, 118.2. physhl. Chem., 1930, 138, 1; A., 646.Ber., 1930, 63, [ B ] , 659; A., 633.Ibid., p. 28272 CEIBNALL AND PRYDE :C2,H3,02 and ascribed to the compound a structure closely analogousto that of the bile acids and sterols. Finally Butenandt describednew methods of preparing the hormone and assigned to it theformula CI8H2,O2, with [ a ] , + 156" and a melting point of 250-251".It contains a hydroxyl group, a keto-group, and threedouble Wings. E. Dingemanse, S. E. de Jongh, S. Kober, andE. Laqueur described a substance which appeared to be similar tothat of Butenandt to judge from the melting point and analyticalfigures. E. A. Doisy, C. D. Veler, and S. Thayer * have also pre-pared what they claim to be the crystalline hormone, since theirmaterial possessed a constant activity and melting point after twentyrecrystallisations from several different solvents. These workerslahr ascribed to their substance the formula C,8H,1(OH), [sz'c],although their figures would agree equally well with formuhhaving one less or one more hydrogen atom.The meltingpoint was 243". One double bond is stated to be present and thesubstance is weakly acidic.I n 1929, previous to the publication of the results described inthe foregoing paragraph, G. P. Marrian described the isolationfrom the urine of pregnancy of a crystalline oestrous-producinghormone to which he a t that time, and as a result of preliminaryinvestigations, ascribed the formula CI9H3& OH), or C,,H32( OH),and a melting point of 233-235". Improved methods of isolationand purifkation of the hormone lo eventually yielded a substancewith a melting point of 281" having the constitution C18H2,03. Ithas + 38". The substance has three hydroxyl groups, oneof which is acidic and therefore presumably phenolic in character,and indeed definite reactions for a phenolic group have been obtained.Its activity is .unchanged after numerous recrystallisations.It will be observed that the formula most recently adopted byButenandt differs from Marrian's formula only by H20, and Buten-andt himself suggests that Marrian's substance may be the hydrateof his hormone.The much higher optical rotation of Butenandt'smaterial would tend to support this view, as would also the factsthat only one hydroxyl group is present whilst the remainingoxygen is present in a ketonic or similar grouping. In any caseButenandt's conclusions seem now to be much closer to those ofMarrian than they were some months ago. I n a private com-munication Marrian informs the Reporter that Butenandt has2.physiol. Chem., 1930,191, 127, 140; A., 1480.7 Deut. med. Woch., 1930, 58, 301; A., 1320.* J . Biol. Chem., 1930, 86, 499; 87, 357; A., 821, 1069.0 Biochem. J . , 1929, 23, 1090; A., 1929, 1495.10 Ibid., p. 1233; 1930, 24,435, 1021; A., 254, 821, 1320BIOCHEMISTRY, 273recently isolated, in addition to the active substance C1,H2,02,the substance C1,H2,0, (Marrian’s crystalline substance), and thathe finds the latter to be physiologically active. It would thereforeappear that both these substances are present in the urine of preg-nancy, and that at some stage two hydroxyl groups are insertedin, or lost from, the hormone as it comes from the ovary withoutmaterially affecting the physiological activity.It seems to the Reporter highly probable that all these workersare handling the same or very similar crystalline materials invarying degrees of purity.Owing to variations in the methods ofbiological assay employed by the various workers it is not possibleto make the assay figures a satisfactory basis of comparison betweendifferent preparations. But it may be mentioned that Marrian’spurest material has an activity of 8 x lo6 mouse units per gram.On conversion into the acetate the activity fell to 3.74 x lo6 mouseunits per gram. On the basis of the amount of the original sub-stance present in the acetate, this activity corresponds to 5-37 x lo6mouse units per gram. Thus the conversion of the substance intoits acetate appeared to have decreased its physiological activity toa significant extent.The material regenerated from the acetatepossessed an activity of 7.4 x lo6 mouse units per gram, fromwhich it seems cer$ain that hydrolysis of the acetate yielded theoriginal substance again.The Liver Constituent Curative of Pernicious Anemia.In the Report of two years ago attention was directed to thework of Cohn l1 and his collaborators on the concentration of thesubstance present in liver, which had a specific curative action incases of pernicious anzmia. The opinion was then expressed thatthe properties of the substance suggested those of a salt of a fairlycomplex organic acid. R. West and M. Howe l2 have now describedthe isolation of a crystalline derivative of an acid present in liverand active in pernicious anEmia. They give details of the prepar-ation of an active amorphous material from a concentrated aqueoussolution of commercial liver extract.This material is stronglyacid to litmus and to methyl-red and contains approximately 46.6%of carbon, 6.9% of hydrogen, and 10.6% of nitrogen. Free amino-nitrogen is absent, but after acid hydrolysis one-half of the nitrogenis obtained in that form. A finely crystalline quinine salt of thisacid was obtained by gently warming its solution and addingquinine gradually until the reaction was neutral or slightly alkalineto litmus. Recrystallisation was easy and to this quinine salt isassigned the provisional constitution C20H,,0~2,Cl,Hl,06N2.11 Ann. Reporta, 1928, 25, 263.l2 J , Bid. Chem., 1930, 88, 430274 CHIBNALL AND PRYDE :Physiological tests on the substance after removal of the quinineshowed it to be highly active in producing a reticulocyte responsein cases of pernicious anemia. When the quinine salt was decom-posed with soda, and the quinine removed, a solution was obtainedwhich after hydrolysis contained amino-nitrogen and gave thequalitative reactions of p-hydroxyglutamic acid. This acid wasalso isolated as its silver salt. Later, H. D. Dakin, R. West, andM. Howe l3 identified Z-y-hydroxyproline as a constituent of theoriginal active substance. The latter is therefore regarded as thedipeptide of this acid with p-hydroxyglutamic acid. From thedescription of its properties given in these communications itwould appear to have the constitution :YO*OH YH2-TH*OH(JH*NH-CO*CH CH,1-Thyroxine as a Constituent of the Thyroid Protein.C. R.Harington and W. T. Salter 14 have recorded the successfulisolation of thyroxine from the thyroid gland by means of theaction of enzymes alone. An alkaline sodium chloride extract ofthe gland was digested by successive additions of trypsin at pH 8.5,and after precipitation at p H 5 , it was extracted with acid acetoneand reprecipitated with ether. This precipitate was subjected tofurther tryptic digestion, and then precipitated at pH 5 and redis-solved in water with the aid of the least possible amount of ammonia.Pigmented impurities were removed by boiling with barium hydr-oxide. After precipitation at pR 5 and solution in alkaline 80%alcohol, further impurities were precipitated with acid acetone.The digestion product at this stage was not further acted upon byanimal erepsin or yeast peptidase.The product was dissolved inpyridine, precipitated by dilution with water, dissolved in potassiumcarbonate solution, decomposed with acetic acid, and reprecipitatedas the monosodium salt of thyroxine on cooling after boiling withsodium carbonate. The sodium salt on decomposition with aceticacid yielded Z-thyroxine. Comparative physiological tests on thedigestion product and on pure Z-thyroxine have shown that thereis no reason to presume the existence in the thyroid gland of anactive principle other than thyroxine, or of its existence there in an" activated " form.Thus the isolation of thyroxine through the13 Proc. SOC. Exp. Biol. Med., 1930, 28, 2.14 Biochem. J., 1930, 24, 456; A., 820BIOCHEMISTRY. 275unaided action of a proteolytic enzyme supplies the final proof thatthe compound is present in the gland in peptide combination as aconstituent of thyreoglobulin. Moreover the optical rotation ofZ-thyroxine isolated by the enzymic procedure proves the com-pleteness of the resolution of dl-thyroxine already described byHarington.Acetylcholine from a n Animal Source.A matter of great physiological interest is the isolation of acetyl-choline from the spleen of the ox and the horse by H. H. Dale andH. W. Dudley.16 This is the first record of the natural occurrenceof this substance in the animal body, although it has been widelyemployed in physiological experimentation.It was prepared arti-ficially by Baeyer in 1867 and there are two later records of itsisolation from plant sources. Its physiological interest lies in thecorrelation of its action with that of the parasympathetic nervoussystem just as adrenaline simulates the action of the sympatheticdivision of the autonomic nervous system. Dale and Dudley, afterdiscussing these physiological problems, say : " But there has beena natural and proper reluctance to assume, in default of chemicalevidence, that the chemical agent concerned in these effects, or inthe humoral transmission of vagus l7 action, was a substanceknown, hitherto, only as a synthetic curiosity, or as an occmionalconstituent of certain plant extracts.Many things could beexplained if the liberation of acetylcholine could be postulated ;but the minuteness of the qua,ntities required to produce the effectsin question, and the extreme instability of the substance, whileenhancing its theoretical fitness for the suggested functions, pre-cluded any hope of its chemical identification at the sites of itspossible liberation. . . . Its definite isolation from one organ hasso far altered the position that, when an extract from, or a fluidin contact with the cells of, an animal organ can be shown to con-tain a principle having the actions, and the peculiar instability, ofacetylcholine, it will be reasonable in future to assume the identific-ation." H.W. Dudley l8 has published a separate description ofthe methods used in the difficult problem of separating acetyl-choline from choline by means of their chloroplatinates. A furtherstudy by H. H. Dale and J. H. Gaddum l9 deals with the behaviourof denervated voluntary muscles in relation to the actions of thel6 Ann. Reports, 1928, 25, 261.16 J. Phyaiol., 1929, 68, 97; A., 1930, 104.1 7 The vagus nerve belongs to the parasympathetic branch of the autonomic18 Biochem. J., 1929, 23, 1064; A., 1929, 1479.Is J. Physiol., 1930, 70, 109.system.-J. P276 CHIBNALL AND PRYDE :parasympathetic system and of acetylcholine. In this importantpaper much evidence is brought forward in support of the view thatthe vaso-dilator effects of parasympathetic nerves, and of sensoryfibres stimulated antidromically, and the contractures of denervatedmuscles accompanying these actions, are due to the peripheralliberation of acetylcholine.The Vitamins.Carotin and Vitamin-A.-During the past year the main interestof investigators has centred on the problem of t'he relationship ofthe hydrocarbon carotin to vitamin-A.Earlier work in this fieldwas reviewed in the Report of lamst year.20There is now general agreement that carotin of the highestpurity so far attained, and of whatever origin, possesses intensevitamin-A activity. T. Moore21 has, for instance, shown thatcarotin prepared from the unsaponifiable matter of red palm oilwas active in doses of 0.01 mg. per day when fed to rats.It hasbecome equally clear that carotin is not itself vitamin-A. W. L.Dulidre, R. A. Morton, and J. C. Drummond 22 have described acareful colorimetric and spectroscopic differentiation of carotinfrom the vitamin-A of cod-liver oil. They found that the bluecolours produced in the antimony trichloride reaction were ofslightly different shade, that of carotin being characterised by anabsorption band at 590pp, and that of vitamin-A by a band a t608-612 pp : in regard to the ultra-violet absorption spectravitamin-A showed a band a t 320-330 pp which was absent in thecase of carotin. Moreover, pure carotin, which is deeply coloured,possesses an activity not greatly exceeding that of the best cod-liveroil concentrates, which are a t most of a pale orange colour.Theinevitable conclusion seems to be that carotin is transformed intovitamin-A in vivo. Direct evidence of this transformation hasbeen obtained by T. Moore23 and by N. S. Capper.24 Moore hasshown that the liver oils of rats suffering from vitamin-A deficiencyinvariably gave negative results when tested with the antimonytrichloride reagent. After such rats had been cured by the admini-stration of large doses of carotin, it was found that traces of yellowpigment appeared in the liver oil. At least 99% of the chromogenpresent was vitamin-A, as characterised by (a) the absence of suchintense yellow pigmentation as must have accompanied the storageof carotin as such; (b) an intensely positive antimony trichloride2o Ann.Reports, 1929, 26, 245.21 Biochem. J . , 1929, 23, 1267; A., 1930, 255.22 J . SOC. Chem. Id., 1929, 48, 518.23 Biochem. J . , 1930, 24, 692; A., 962.24 Ibid., pp. 453, 980; A., 822, 1321BIOCHEMISTRY. 277reaction showing a marked band at 610-630 pp; (c) the appear-ance of an absorption band in the untreated oil at 328 pp; (d) in-tense biological activity. Capper has shown that the band at320-330 pp is absent from trhe liver oils of rats suffering fromdepletion of vitamin-A, but is present in the liver oils of similarrats subsequently cured by massive doses of carotin. Since thisband is absent from the absorption spectrum of purified carotin,it is deduced that the substance which is responsible for the pres-ence of this band in the spectrum of the liver oils of carotin-treatedrats has been synthesised in, vivo from the carotin.In view of the foregoing results and of the importance of butteras a source of vitamin-a, R.A. Morton and I. M. Heilbron 25 haveinvestigated the presence of carotin and of the vitamin in this fat.Both were found to be present and to be capable of spectroscopicdetermination with some degree of accuracy. These workers sup-port Moore's view regarding the conversion of carotin into vitamin-Ain vivo. Further evidence bearing on this relationship has beenpublished by H. von Euler, lr. Demole, P. Karrer, and 0. Walker,26who have determined the carotin and xanthophyll contents of theether-soluble unsaponifiable matter of the extractives of the dryleaves of various plants, of the flowers of CnZthcc paZustris,.and ofmaize grains.In all cases the vitamin activity runs parallel withthe carotin content. An interesting result is that of M. Javillierand L. Emerique?' who found that a preparation of carotin fromspinach which had been kept in an atmosphere of hydrogen andexposed to feeble diffused light for 40 years exhibited the physio-logical properties of vitamin-A. The same workers 28 record thepurification of a specimen of carotin of melting point 172-173" toyield one of melting point 184-185". This was achieved by fivesuccessive purifications by dissolution in carbon disulphide, slowaddition of the solution to boiling methyl alcohol, removal of thecarbon disulphide, and filtration of the residual liquid, all oper-ations being carried out in an atmosphere of nitrogen. The purifiedproduct retained its vitamin activity.Vita;min-D.-The anti-rachitic vitamin continues to elude capture,but if anything the gap between the fugitive vitamin and its pursuersis narrowing.F. A. Askew, R. B. Bourdillon, H. M. Bruce, R. G. C.Jenkins, and T. A. Webster 29 have published a detailed study ofthe distillation of vitamin-l), or rather of the resins obtained by25 Biochem. J., 1930, 24, 870; A., 1321.26 Helv. Chim. Acta, 1930, 13, 1078; A., 1624.27 Compt. rend., 1930,190, 665; A., 647.28 &d., 1930, 191, 226; A., 1221.** Proc. Roy. Soc., 1930, [B], 107, 76; A, 1481278 CHIBNALL AND PRYDE:the removal of unchanged sterol from irradiated ergosterol. Aspecially designed still and a very high vacuum were employed.On three occasions the redistillation of one of the more volatilefractions yielded a crystalline product.Recrystallisation waspossible and the material was obtained as rather thick needlesshowing conspicuous double refraction in polarised light. Themelting point was 113-115". In each case the crystals have shownvery high anti-rachitic activity. The authors point out that, ifthere is only one substance possessing intense anti-rachitic activity,that is, only one vitamin-D, it is not very probable that thesecrystals are this substance. Resinous mixtures of approximatelythe same anti-rachitic activity as the crystals have frequently beenobtained, although their low melting points and varying absorptionspectra suggested that they were mixtures of a number of sub-stances. The interest of this problem justifies a detailed referenceto the four possibilities advanced by the authors.(1) The crystals may be an inactive substance contaminated bytraces of an intensely active oil deposited on their surfaces.This is not thought to be probable, since the cleanestspecimen obtained showed the highest activity and appearedfree from oil.If an oil film was the source of the activity,it must have had an activity of a higher order than anyyet observed.(2) The crystals may be a mixture in any proportion of two ormore substances of sufficiently similar molecular dimen-sions to form homogeneous crystals, only one of thembeing vitamin-D.This is regarded as possible in view ofthe frequent occurrence of such mixed crystals among thesterols.(3) The crystals may be a loose compound of an active and aninactive substance.(4) There may be a number of radiation products of ergosterolall possessing high but unequal anti-rachitic activities,that is, a number of compounds any one of which couldbe called vitamin-D, but would cause only a fraction ofthe total activity of the ordinary irradiation products ofergosterol. This is regarded as not improbable.It is observed that during the heating in a vacuum there isformed a new substance with intense absorption at 290 pp. Thissubstance has no anti-rachitic activity, but its appearance is associ-ated with the disappearance of the vitamin.It cannot be regardedas a simple product of vitamin destruction, since its productionbears no direct relation to the vitamin content shown, beforeThese are BfOCHEMIS!I'RY. 279distillation, by the irradiation products from which it is formed.The two conditions which favour its formation and the concurrentdisappearance of the vitamin are previous protection of the irradi-ation product from exposure to air, and the exclusion of shortwave-lengths during the original irradiation. It is tentativelysuggested, in explanation of these facts, that one of the initialirradiation products of ergosterol is an unstable substance withlow absorption and no anti-rachitic activity, that this substance isvery readily destroyed by absorption of oxygen, or by radiation ofshort wave-lengths, but that, if not previously destroyed, it isconverted by heat into the substance absorbing light at 2 9 0 ~ ~with an associated destruction of part of the vitamin present.The same authors in another paper 30 report the results of furtherirradiation of the radiation products of ergosterol. When thesecond irradiation was carried out with short rays (210-280~~)acting on the substances formed by a first irradiation with '' long "rays (longer than 280 pp), there resulted a large increase in absorp-tion a t 280yy and a decrease in anti-rachitic potency.On re-irradiation with long rays, the absorption a t 280py was slightlydecreased, whilst the activity was unchanged. The substance withhigh absorption at 2 8 0 ~ ~ can be formed by the action of shortwaves on some product of a previous action of long waves onergosterol. The results show that the product with high absorp-tion a t 280 py is not vitamin-D as previously supposed,3l and it isnow suggested that the initial effect of long-wave radiation onergosterol is the simultaneous production of at least two substances,only one of which is vitamin-D.In the Report of last year reference was made to the conditionof hypervitaminosis 32 induced by the administration of excessivedoses of irradiated ergosterol or other source of vitamin-D.Duringthe past year numerous investigations, which need not be detailedhere, have been devoted to EL consideration of this problem andmuch valuable information has been obtained.But in view of theforegoing discussion concerning the products of irradiation ofergosterol, a communication by F. Holtz and E. Schreiber 33 issignificant. It is claimed that the hypervitaminosis effects, namely,calcium deposition, hypercalcaemia, hyperphosphataemia, and circul-atory deficiency, are produced by a toxic substance, which they callthe " calcinosis " factor, and not by vitamin-D itself. The toxicfactor and the vitamin produced under various conditions ofirradiation are stated to be closely proportional. It is furtherao Proc. Roy. SOC., 1930, [B], 107, 91; A., 1481.31 Ann. Reports, 19W, 28, 249.33 2. physiol. Chern., 1930, 191, 1; A., 1481.82 Ibid., p. 251280 CHIBNALL AND PRYDE :claimed that the calcinosis factor may be isolated and its effectsstudied by heating the irradiated ergosterol to 160°, or by reductionwith sodium in alcohol, whereby the anti-rachitic vitamin alone isdestroyed.The B Vitamin Group.-Since the nature of the B vitamin com-plex was last dealt with in these Reports 34 the problem has under-gone a further development in complexity. In 1928 R.R. Williamsand R. W. Waterman 35 submitted evidence that polyneuriticpigeons, made so either by an exclusive diet of polished rice, or bya synthetic diet complete in all respects save for the vitamin-Bcomplex, require for full weight restoration or maintenance a factorwhich is distinct in properties and distribution from the anti-neuritic vitamin-B,, or the anti-pellagra vitamin-B,. In 1929V. Reader 36 presented evidence for the division of the B vitamincomplex into three components, all necessary for the nutrition ofthe rat. It was shown that two of these were destroyed by auto-claving yeast extract a t p H 9 for one hour at 120". It was later 37found possible to concentrate two of these components, and it wasthen shown that the third must still be added to the diet of rats toenable these animals to grow to maximum adult size. The Williams-Waterman vitamin and the Reader vitamin are not the same,although they were both at first called vitamin-B, by their respectivediscoverers. The matter has now been amicably adjusted and theterm B, is applied to the first-mentioned vitamin, and B, to thesecond. The present position may perhaps best be summarised inthe following table :Required bygrowing adultVitamin. rat. pigeon. Properties. + + Alkali-labile, the original anti-neuritic vitaminof Eijkmann, called torulin by Peters. + - Alkali-stable, the anti-pellagra vitamin of Gold-berger, also called the anti-dermatitisvitamin.- + Thermo-labile, the Williams-Waterman pigeonfactor.B4 3. + Alkali-labile, the rat factor of Reader.B,B'2*3W. H. Eddy, S. Gurin, and J. Keresztesy38 have extended theoriginal work of Williams and Waterman and find that yeast, wholegrains, and malt are good sources of vitamin-B,, but while maltextract often retains a good concentration of B,, its method ofmanufacture practically eliminates B,. Malt extracts made at34 Ann. Reports, 1928, 25, 269.36 Biochem. J., 1929, 23, 689; A., 1929, 1203.57 Ibid., 1930, 24, 77; A., 380.3B J. Biol. Chem., 1930, 87, 729; A., 1222.35 Ibid., p. 266'3BIOCHEMISTRY. 281temperatures as low as 60" atre practically devoid of B,, althoughthey are still very effective as sources of B,. Beef and beef liverare fair sources of B, and distinctly superior to milk, orange- andtomato-juice, spinach, and potato-juice or cane molasses in thisrespect. Vitamin-B, is much more heat-labile than B,, and, if itis submitted to alkali treatment before drying, temperatures as lowas 20" will markedly reduce the content of this vitamin in yeast.Miss Reader obtains vitamin-B,, the second alkali-labile rat factor,from the mercuric sulphate precipitate in the Kinnersley andPeters process 39 for vitamin-B,. In this precipitate some 75% ofthe original B4 is recovered. It is suggested that the vitamin formsan insoluble mercury salt, or a double salt with mercuric sulphate.In regard to the B vitamin components of longer standing thanthose just discussed, B. C. Guha and J. C. Drummond,4° and B. T.Narayanan and J. C. Drummond41 describe new methods of con-centrating B, and B2 respecbively. R. R. Williams, R. E. Water-man, and s. Gurin 42 have applied the process of Jansen and Donathfor B, to brewers' yeast, but have failed to isolate highly activematerial. On applying the method as did the original authors torice polishings, general confirmation of the Dutch results wasobtained, although the yield of active material was much lowerand crystals could not be obtained. The authors' highly purifiedbut amorphous preparations behave in all respects like the crystallinesubstance of Jansen and Donath. These results are thereforesimilar to those obtained by Kinnersley and Peters in 1927.43 Itmay be mentioned that B. C. P. Jansen4* has published furtherimprovements in his methods of fractionating the extracts of ricepolishings.Bios.-In a paper published by B. T. Narayanan45 the non-identity of " bios " with vitamin-Bi is established, and J. G. Daviesand J. Golding46 have made a similar finding with regard tovitamin-&. A. M. Copping 47 has shown that the need for " bios "depends on the type of yeast and on the nature of the culturemedium. The need is probably related to the fermentative activityof the yeast. Some of the highly cultivated brewers' yeasts willnot grow in a purified glucose-salt medium without the additionof small amounts of extracts containing an organic " bios." Nara-yanan describes a method of fractionation which provides con-39 Biochem. J., 1927, 21, 778.40 Ibid., 1929, 23, 880; A., 1929, 1496.42 J . Biol. Chem., 1930, 87, 55!); A., 1222.43 Harben Lectures, J . State &Xed., 1930, 38, 38.44 Rec. trav. chim., 1929, 48, 984.46 Ibid., p. 1803; A., 1479.4 1 Ibid., 1930, 24, 19; A., 380.45 Biochem. J., 1930, 24, 6; A., 375.47 Ibid., 1929, 23, 1050; A., 1929, 1491282 CHIBNAJLL AND PRYDE : BIOCHEMISTRY.centrates producing marked stimulation of yeast growth in dosesof the order 0.01 mg. per C.C. of an artificial sugar-salt medium. Thefinal concentrate appears to consist largely of relatively simplenitrogenous substances and contains no phosphorus. No evidencein support of the complex nature of " bios " was obtained in thecourse of these investigations, nor is there any indication thatinositol is an essential unit of " bios."A. C. CHIBNALL.JOHN PRYDE