BIOCHEM.ISTRU.THIS year has scen further definite progress in the isolation ofsubstances belonging to the groups of hormones and vitamins.The constitution of vitamin B, has been established beyond doubt ;for the first time a substance with anti-rachitic properties has beenisolated from natural sources ; a cyclic alcohol, apparently pure,with the activity associated with vitamin E has been isolated;and of a number of sterol-like substances obtained from the adrenals,one is highly active in the manner characteristic of the corticalhormone. The anti-anEmic factor in liver has not yet, apparently,been obtained pure, though considerable progress has been madetowards its isolation.Attention is directed to the attempts now being made to elucidatethe arrangement of amino-acids within the protein molecules,attempts which seem likely to prove of increasing importance andvalue as methods continue to improve for characterising andestimating specific amino-acids and for synthesising more complexpeptides.The nature of the researches involved in chemotherapy makesmaterial progress slow in this field.Nevertheless, results areemerging from prolonged systematic investigations and are makingdefinite contributions to our knowledge of the mode of action, andof the relative efficiencies of both natural and synthetic therapeuticagents.In the Plant Biochemistry section considerable space has beendevoted this year to questions relating to photosynthesis in plants.Recent additions to our knowledge of the pure chemiatry of chloro-phylll may contribute much to our understanding of the morephysiological aspects of the photosynthetic process.The manyramifications of this problem and the divergence of opinion amongauthorities on this subject, coupled with the fact that nine yearshave elapsed since the matter was dealt with in these Reports,seem to afford sufficient justification for a general review of someof the more important researches and theories on this fundamentalprocess of plant chemistry.1 Ann. Reports, 1936, 83, 362384 BIOCHEMISTRY.1. ANIMAL BIOCHEMISTRY.The Vitamins.Vitamin B,.-The constitution of vitamin B, originally suggestedhas been modified2 to that shown-in (11) by R. R. Williams (I)and the new formula has been confirmed by ~ynthesis.~The excretion of vitamin B, in human urine has been measuredby the bradycardia r n e t h ~ d , ~ the vitamin being first adsorbed onactive clay, which is then fed to B,-deficient rats for observation ofits effect on the heart rate.Normally 12-35 international units(approximately 30-90 7 ) are excreted per day, i.e., about 543%of the daily intake. In human beri-beri the excretion of vitamin Blmay almost cease, and a daily excretion of less than 12 units is held toindicate some deficiency of the vitamin in the diet. W. H. Schopferhas estimated vitamin B, by measuring the amount of growth inthe mould Phycomyces Blakesleanus on a vitamin-free syntheticmedium after addition of vitamin concentrates ; the method has,so far, however, only been applied successfully to fairly purepreparations.On the grounds that many diseases showing anorexia, cedema(with consequent heart symptoms) and peripheral nerve degener-ation (characteristic signs of experimental B1-deficiency) may bedue to a deficiency of the vitamin, and that in such cases the absorp-tion of the vitamin from the intestine may be unsatisfactory, theparenteral administration of pure vitamin B, has been advocatedby a number of workers.has obtained goodresults by this method in cases of chronic progressive neuritis,alcoholic neuritis, subacute combined degeneration of the spinalcord, etc., although in some of them oral administration had littleor no effect.W. R. Russelll a Ann. Reports, 1935, 32, 402.2 R.R. Williams, J . Amer. Chew. Soc., 1936, 58, 1063.4 L. J. Harris and P. C. Leong, Lancet, 1936, 230, 886.6 2. Vitaminforsch., 1936, 5, 67.6 E d k . Med. J., P936, 43, 315.This vol., p. 381STEWART AND STEWART. 385Vitamin B, Complex.-Although i t has been suggested that theterm B, should be restricted to lactoflavin,' there is still no uni-formity in nomenclature, and since there evidently exist morethan the two factors which led to the differentiation of the complexinto B, (flavin) and B, (curative of rat dermatitis) the tendency isto refer to the factors by their effects rather than by specific names.T. W. Birch, P. Gyorgy, and L. J. Harris distinguish a t least twoand possibly four factors in addition to flavin. They find that thefactor curative of dermatitis in rats (erroneously described as rat-pellagra) is present in considerable amounts in maize, and inmolasses, so that rats fed on typical human-pellagra-producingdiets remained free from dermatitis, and such diets cured ratssuffering from dermatitis.They conclude that this factor (= B,)is different from the factor curative of human pellagra. This, the'' P-P " or pellagra-preventing factor, is found especially in liverextract, autolysed yeast, etc., and is apparently not required bythe rat. It is not lactoflavin (which is required by the rat, ofcourse, for growth). Dogs fed on a human-pellagra-producing dietdid not develop pellagra, but " black tongue " (a condition un-affected, evidently, by B6), which was cured by a diet rich in P-Pfactor but not by lactoflavin. Xhe dog, however, needs B, as wellas lactoflavin and the " anti-black tongue " factor, as was shownby experiments with " synthetic '' diets. It seems that the blacktongue and the P-P factor may be identical, though the evidence isnot conclusive.Finally the factor curative of chicken pellagra isdifferentiated from B,, though its relation to the P-P or the blacktongue factor was not decided.S. Lepkowsky and T. H. Jukes9 found that the factor curingdermatitis in chickens could be concentrated from aqueous ricebran extracts, since inert matter, but not the vitamin, was absorbedby fullers' earth. Their experiments suggested that the factor (B6)curative of rat dermatitis was absorbed by the earth, and laterexperiments lo confirmed this.T. H.Jukes and S. Lepkowsky l1 investigated the distributionof the anti-chicken-dermatitis factor in foodstuffs. They found,for example, that wheat germ, kale, and maize contained approxim-ately equal amounts, and since wheat germ and kale contain muchmore of the P-P factor than does maize, they conclude that thetwo are different. They thus confirm the differentiation of therat-dermatitis and the chick-dermatitis factor and show that the7 Ann. Reporte, 1935, 32, 404.9 J. BioZ. Chert,., 1936, 114, 109.10 S. Lepkowsky, T. H. Jukes, and M. E. Krause, ibid., 1936, 115, 557.l1 Ibid., 1936, 114, 117.REP.-VOL. XXXIII. NBiochem. J., 1935, 29, 3830386 BIOCHEMISTRY.latter is different from the P-9 factor.The vitamin B, complex,therefore, consists of, at least, four factors, including lactoflavin.Concerning vitamin B, itself (i.e., the flavin growth factor)R. Kuhn, H. Rudy, and F. Weygand l2 have now reported thesynthesis, by a method which fixes the position of the phosphoricacid residue, of 6 : 7-dimethyl-9-d-riboflavin-6‘-phosphoric acid,which is identical with the natural lactoflavin phosphoric acid insolubility of its salts, absorption spectrum, oxidation-reductionpotential, and in growth-promoting activity (rats) whether givenorally or intra-peritoneally. R. Kuhn and H. Rudy l3 have shownfurther that the synthetic can replace the natural substance inthe “yellow enzyme.” R. Kuhn, H. Rudy, and F. Weygand14have also synthesised the E-arabinose analogue, and have shownthat it too can combine with the colloidal carrier derived from theyellow enzyme to form a chromoprotein whose catalytic activity ishigh, though less than that of the natural enzyme.Vitamin B,.-The existence of vitamin B,, originally describedby V. Reader,15 has more recently been doubted, and thc opinionexpressed that vitamin 33, deficiency (so-called) is merely a chronicdeficiency of vitamin B,, and can be cured by a sufficiently largedose of that substance.8B l6 states, however, thatin his laboratory M.Malmberg was unable to restore growth in ratsby addition to the diet of vitamin B, and lactoflavin. J. A. Keenan,0. L. Kline, C. A. Elvehjem, and E. B. Hart l8 found in 1933 thatconcentrates of the alleged vitamin B, were able to prevent thedevelopment of certain paralytic symptoms in the chick, the dietalready containing adequate amounts of vitamin B,.This workwas later confirmed and extended.19 NOW,,* it has been shownthat by the use of specially purified diets, crystalline vitamin B,,and highly potent liver concentrates as source of the vitamin B,complex, it is possible to reproduce in rats the syndrome describedby Reader and to restore growth by vitamin B, concentrates (e.g.,from peanuts) but not by crystallinc vitamin B,. These workersH. von Euler12 Ber., 1936, 69, 1643.13 Ibid., p. 1974.14 Ibid., p. 2034.16 Biochem. J., 1929, 23, 689; 1930, 24, 77, 1837.16 J. R. O’Brien, Chem. and Ind., 1934, 53, 452; L. J. Harris, Ann. Rev.Biochern., 1935, 4, 331; H.W. Kinnersley, J. R. O’Brien, and R. A. Peters,Biochem. J., 1935, 29, 701.1 7 Ann. Rev. Biochem., 1936, 5, 364.18 J . Biol. Chem., 1933, 103, 671.19 0. 1;. Kline, 0. D. Bird, C. A. Elvehjem, and E. B. Hart, J . Nutrifion,20 0. L. Kline, C. A. Elvehjem, and E. €3. IIart, Biochem. J . , 1936, 30,1936.780STEWART AND STEWART. 387consider, therefore, that vitamin B, is a real entity with demon-strable functions in at least two species of animals.Vitamin (?.-With the definite identification of vitamin C asascorbic acid, interest has shifted to such questions as its synthesisin vivo, its excretion under various conditions, the form in whichi t occurs in the tissues, the changes it undergoes there, and itsfunctions. The published results bearing on these questions are,in many ca,scs, contradictory, largely in all probability because bheusual methods for estimating ascorbic acid (titration with 2 : 6-dichlorophenolindophenol is the commonest) are by no meansspecific.Many of the published conclusions can, therefore, beaccepted only with reserve.The report of B. C. Guha and A. R. Ghosh,21 that rat tissueswere able to synthesise ascorbic acid in vitro from mannose, hasbeen contradicted by R. Ammon and G. Grave 22 and by M. Laportaand E. Rinaldi.23 On the other hand, in vivo synthesis of ascorbicacid by rats is suggested by the experiments of K. M. Daoud andM. A. S. El Ayadi2* and evidence has been adduced in supportof the view that the human foetusZ5 and the human sucMing,26but not the guinea pig foetus or suckling,27 can synthesise the vitaminto some extent.It has also been suggested by H. K. Muller 28that the eye lens is capable of synthesising ascorbic acid, butS. W. Johnston 29 finds that, although in scorbutic guinea pigs theindophenol-reducing power of the lens is merely reduced, theascorbic acid determined spectrographically has completely dis-appeared from lens and humourg, the rate of disappearance (andof re-appearance when the vitamin is administered) running parallelwith that of the other tissues. On the other hand, it has beenclaimed 30 that the indophenol-reducing substance of the lens andeye humours is ascorbic acid, since it is completely oxidised by theascorbic acid oxidising enzyme-a conclusion which is obviouslynot justified until more is known of the specificity of the enzyme.Other indophenol-reducing substances (besides those which, like21 Ann.Reports, 1935, 32, 404.sa 2. Vitaminforsch., 1936, 5, 185.23 Boll. SOC. ital. BioZ. aperim., 1935, 10, 319.24 Bioclzenz. J . , 1936, 30, 1280.25 R. Rohmer, N. Bezssonoff, and E. Storr, Compt. rend. SOC. B i d , 1936,26 Idem, Bull. Acad. Me'd., 1935, 113, 669; Compt. Tend. Soc. BioZ., 1936,2 7 G. Mouriquancl, A. Ceur, and P. Viennois, Gompt. rend. Xoc. BioZ., 1936,2 8 Klin. Tl'och., 1935, 14, 1498.2s Biochem. J., 1936, 30, 1430.30 L. Eosner and J. Bellows, Proc. SOC. E x p . BhZ. filed., 1936, 34, 493.121, 987.121, 988.121, 1005388 BIOCHEMISTRY.cysteine, reduce it slowly) do exist in nature, for one containingnitrogen, and possibly phosphorus, has been obtained from supra-renals by E.Ott, K. Kramer, and W. F a ~ s t . ~ lA similar confusion exists on the question of the state in whichascorbic acid exists in the tissues. Thus B. C. Guha and J. C. Pal,32having found that some plant extracts (e.g., cabbage) yielded moreascorbic acid on heating, concluded that ascorbic acid was presentto some extent in a combined form, whereas G. L. Mack33 andM. van Eekelen 34 attribute the phenomenon to heat-inactivationof the ascorbic acid oxidase. Van Eekelen considers that ananalogous phenomenon occurs in blood, ascorbic acid being oxidisedby the erythrocytes, but A. E. Kellie and S. S. Zilva3j deny thatintact erythrocytes are capable of oxidising ascorbic acid.Byspectrographic measurement they conclude that plasma containsno dehydroascorbic acid.A large number of papers deal with the urinary excretion ofascorbic acid, and many are concerned with the unsatisfactorynature of the available methods. M. A. Abbasy, L. J. Harris,S. N. Ray, and J. R. Marrack,36 using the method described byL. J. Harris and S. N. Ray,37 state that the urinary excretion ofascorbic acid is, in general, proportional to the intake, and fornormal adults (in England) receiving small allowances of fruit, etc.,is about 20 mg. per day. A diet is deficient in vitamin C whenthe urinary output falls below 10-15 mg. per day (per 10 stonebody weight) or when a dose of 700 mg.of ascorbic acid producesno increased excretion on the second day. The increased indo-phenol-reducing power of the urine after ascorbic acid administra-tion, and the decrease during the feeding of a scurvy-producingdiet, which has been observed by others as well as Harris and hisco-workers, certainly suggest that the reducing substance of urineis ascorbic acid or a t least a closely related substance. B. Ahmad 38found that a high meat diet caused a very considerable increase inthe excretion of indophenol-reducing substance, and concluded thatthis was probably ascorbic acid from a study of its heat stability.A failure to detect ascorbic acid by biological assay he ascribedto the presence of toxic substances in urine. Wieters39 also hasfailed to demonstrate ascorbic acid in urine by biological methods.Although these failures can be discounted to some extent byaccepting Ahmad’s explanation, it is more difficult to ignore the31 Z.phyeiol. Chem., 1935, 243, 199.33 Ibid., 1936, 138, 505.36 Biochem. J., 1936, 30, 361.3 7 Ibid., p. 71.3D Mercks Jahresber., 1935.32 Nature, 1936, 137, 946.34 Acda Brev. Ne’erl., 1935, 5, 78.as Lancet, 1935, 229, 1399.3a Biochern. J., 1936, 30, 11STEWART AND STEWART. 389chemical results reported by K. Hinsberg and R. Arnm~n.~O Theywere able to separate from urine ascorbic acid added to the extentof 1 mg. per 100 c.c., using the fact that its derivative with 2 : 4-dinitrophenylhydrazine is insoluble in cold alcohol or in ethyl hydro-gen oxalate but soluble in ethyl oxalate.From normal urine(containing more than this amount of indophenol-reducing ssb-stance) they failed to extract any of the vitamin C derivative,and from a study of the limits of their methods conclude thatnormal urine cannot contain more than 0.3 mg. of ascorbic acidper 100 C.C.The fact that ascorbic acid exists in the tissues largely, if notentirely, in the reduced form has been ascribed by several authorsto the presence of glutathione,*l which has also been shown toprotect ascorbic acid from autoxidation in vitro, provided that it ispresent in relatively large amountsj2 It is claimed that in highconcentration, glutathione can even reduce dehydroascorbic acid,provided the pH is not too F. G. Hopkins and E.J. Morgan 44have studied the relationship between ascorbic acid and glutathione,alone and in the presence of the enzyme (from cauliflower, cabbage,etc.) which A. Szent-Gyorgi45 found to oxidise ascorbic acid andnamed “ hexoxidnse.” They find that a mixture of pure ascorbicacid with pure glutathione may be quite inert, neither being oxidised(e.g., at pH 6) ; if, however, a trace of copper is present and the pHis such as to allow oxidation of the glutathione (e.g., 7.4), theformer is protected while the latter is oxidised exactly as if noascorbic acid was present. They are, therefore, inclined to ascribethe protective action of glutathione under these conditions to itsformation of a stable compound with the metal catalyst. In thepresence of the enzyme the conditions are quite different.The reactionsDehydroascorbic acid + 2GSH --+ ascorbic acid + G*S*S*GAscorbic acid -> dehydroascorbic acidare both catalysed by the enzyme, and, the second of these beingthe more rapid, ascorbic acid remains practically fully reduceduntil all the glutathione is oxidised.Hopkins and Morgan pointout that so far no enzyme capable of oxidising either ascorbic acidor glutathione has been discovered in animal tissues. Nevertheless,40 Biochem. Z., 1936, 288. 102.4 1 L. de Caro and M. Giani, 2. physiol. Chem., 1934, 228, 13; C. A. Mawson,48 Bersin, Koster, and Zmatz, 2. physiol. Chern., 1935, 235, 12.4 3 H. Borsook and C. E. I?. Jeffreys, Science, 1936, 83, 397.44 Biochem. J . , 1936, 30, 1446.45 Ibid., 1928, 22, 1387.Biochem.J., 1935, 29, 569390 BIOCHEMISTRY,their experiments in which these substances were aerated withhepatic tissue yielded some suggestion that here too glut'athioneaffords some protection to the vitamin. Other substances besidesglutathione may well be concerned in preserving ascorbic acid fromoxidation; for instance, M. Yamomoto 46 has shown adrenalin tohave this effect in vitro.S. Rusznyak and A. Szent-Gy6rgi47 report that Hungarian redpepper and lemon juice contain a substance which is closely alliedto vitamin C, curing pathological fragility and permeability of thecapillary walls to plasma proteins. They name it vitamin P, andstate that it appears to be Aavone or flavonol glucoside.Vitamin D.-The fact that antirachitic activity is the propertyof more than one compound was mentioned in these Reports lastyear.48 During the year under review the number of active sub-stances has been increased, and for the first time one has beenisolated from natural sources.The evidence leading to the acceptedconstitution of these compounds is reviewed elsewhere : it isbelieved that all the active substances so far obtained have incommon the three-ring structure with the three conjugated doublebonds of calciferol (111) .50A. Windaus 48* 51 found, over a year ago, that 7-dehydrochole-sterol, differing from ergosterol in having no double bond at C,2-C,,and no methyl group at C2*, but with the same ring structure,yielded an antirachitic substance on irradiation.He has now, withP. Schenk and F. von VVerde~-,~~ succeeded in isolating the irradi-ation product (vitamin D3) by chromatographic absorption onalumina. It has an activity of 24,000 international units per mg.(i.e., rather more than half that of calciferol). H. Brockmann 53has isolated a compound identical with vitamin D,, from funnyliver oil, and various experiments on the relative effects of different46 8. physiol. Chem., 1935, 243, 266.4 7 Nature, 1936, 138, 27.48 Ann. Reporta, 1935, 32, 405.49 This vol., p. 349.60 I. M. Heilbron, R. N. Jones, K. M. Samant, and F. S. Spring, J . , 1936,5 1 A. Windaus, H. Lettrit, and F. Schenk, Annalen, 1935, 520, 98.sa 2. physiol. Chem., 1936, 241, 100,w Ibid., p. 104.905STEWART AND STEWART.39 1liver oils in curing avian rickets suggest that it is present in otherfish liver oils (e.g., those of cod and halibut) as weIL5* G. A. D.Haslewood and J. C. Drummond 55 also have obtained a highlyactive concentrate from tunny liver oil (10,000-20,000 inter-national units per mg.), but believe it to be different from thevitamin D3 of Brockmann and Windaus.That calciferol is much less effective in curing avian rickets thanan amount of cod liver oil containing an equal number of inter-national (rat) units of vitamin D has been mentioned in theseReports before.48 It has also been found that purified cholesterol,apparently free from ergosterol, still possesses provitamin Dproperties. Cholesterol purified through the dibromide is onlyslightly active, but its activity (or rather activatability) is greatlyincreased if it is heated in presence of a little 0xygen.~~*~7 Theirradiation products of crude cholesterol and of purified heatedcholesterol resemble cod liver oil in their efficacy in avian ri~kets.~7A.G. Boer, E. H. Reerink, A. Van Wijk, and J. van Niekerk 58have isolated the provitamin from crude cholesterol, confirmed theactivity of its irradiation product as resembling that of cod liveroil with respect to avian rickets, and identified it as 7-dehydro-cholesterol. The suggestion arises, therefore, that purified chole-sterol may, under the conditions used by M. L. Hathaway andD. E. Lobb,57 undergo dehydrogenation to a small extent. Sinceirradiated plant products (cottonseed oil, wheat middlings, lucerneleafmeal, yeast, fungus mycelium) resemble calciferol in being muchmore efficacious in rat than in avian rickets, it has been suggestedthat plant and animal fats contain differeht vitamin D precursor^.^^The similarity in this respect between the unsaponifiable matterfrom lucerne oil and calciferol is confirmed by A.Black and H. L.Xa~aman,~~ who extend the similarity to irradiated “ phytosterol.”It is possible, of course, that crude phytosterols, like crude chole-sterol, may contain small amounts of dchydro-derivatives, for0. Linsert 6O has prepared 7-dehydrostigmasterol, which on irradi-ation shows definite antirachitic activity, as does 7-dehydrosito-54 M. J. L. Dols, 2. Vitnrninforsch., 1936, 5, 161; A. Black and H.L.65 Chem. and Ind., 1936, 598.66 C. E. Bills, E. M. Honeywell, and W. A. MacNair, J . Biol. Chenz., 1928,33, 251; J. Waddell, ibid., 1934, 105, 711; M. L. Hathaway and 3’. C. Koch,ibid., 1935, 108, 773.Sassaman, AnEer. J . Pharm., 1936, 108, 237.6 7 Ibid., 1936, 118, 105.68 Proc. R. Alcnd. Wetensch. Amsterdam,, 1936, 39, 662.69 R. M. Bethke, P. R. Record, and 0. H. M. Wilder, J , Biol, Chem., 1935,80 8. phy8ioE. Chem., 1936, 241, 125,112, 231392 BIOCHEMISTRY.sterol, which has been prepared by W. Wunderlich.61 The irradi-ation products of thefie substances have not yet been isolated, sotheir activity has not been measured quantitatively. It is inter-esting to note that, again, the power of acquiring antiracliitic activityis associated with the ring structure found in cholesterol, but thatagain the side chain is of little importance, for whereas stigmasterolhas the unsaturated side chain of ergosterol with, however, an ethylgroup at C24, sitosterol has the ethyl group at C,, but a saturatedside chain.The relative unimpor-tance of the side chain is furthershown by the fact that 22-di-hydroergosterol becomes antirachiticon irradiation.*** 62 The presenceof two conjugated double bonds inthe ring structure is important, andthat they must be in the ring itself is suggested by the inactivityafter irradiation of 7-methylenecholesterol (IV) prepared by B.Bann, I. M. Heilbron, and F. S. Spring.63Vitamin E.-From the unsaponifisble fraction of wheat germ oil,H.M. Evans, 0. 11. Emerson, and G. A. Emerson 64 have isolated asubstance believed to be the allophanate of P-amyrin, the allo-phanate of an alcohol, C29H50Q2, and the allophanate of " a-toco-pherol," C,,H,,O,. The alcohol from the first of these is inactive,the second shows some vitamin E activity, but a single 3 mg. doseof the regenerated a-tocopherol regularly enables vitamin E deficientrats to bear young. A dose of 1 mg., however, was insufficient toallow the regular production of litters. a-Tocopherol (tolos, child-birth, and phero, to bear) is an apparently homogeneous, oily alcohol,optically inactive, with a strong absorption band maximal a t2980 A. The relatively inactive alcohol has similar absorption,which appears to explain the observation of H.S. Qlcott 65 thatcertain concentrates with good absorption a t 2940 A. showed littleor no vita'min activity. Treatment of a-tocopherol with silvernitrate in methyl alcohol caused the disappearance of the absorp-tion band a t 2980 A., with appearance of two new bands at 2710and 2620 A., the biological activity being reduced but not lost.(Drummond et aZ.66 have stressed the presence of absorption a t2670 A. as well as 2940 A. in their active concentrates.) OllcottMePV.1 * ,-C&Ho /\/- (q3L:61 2. phySi0l. Chern., 1936, 241, 116.68 A. Windaus and R. Langer, AnmEen, 1933, 508, 105; F. G. McDonald,I'roc. Amer. SOC. Biol. Chem. ( J . Biol. Chem.), 1936, 114, lxv.63 J., 1936, 1274.64 J . Biol. Chem., 1936, 113, 319.6 5 Ibid., 1935, 110, 695.Biochem.J., 1935, 29, 2510STEWART AXD STEWART. 393(Eoc. cit.) had observed the similar persistence of activity with dis-appearance of the absorption band a t 2940 A. when his concen-trates were treated with methyl-alcoholic silver nitrate, but con-cluded that the substance responsible for the absorption Waf;,therefore, not the vitamin. A siniilar conclusion has been reachedby J. C. Drummond, E. Singer, and R. J. MacWalter,GG who foundcertain preparations to be less active than was expected from theabsorption intensity, although they had earlier G7 believed absorp-tion and activity to be parallel. They point out, however, that theband at 2940 A. may really be characteristic of the vitamin, butChat certain molecular changes may affect potency without affectingthe absorption spectrum. Evans and his collaborators, however,consider that the effect of silver nitrate merely shows provitamin Eactivity to be possessed by more than one substance, since (on thebasis of its conversion into a p-nitrophenylurethane derivative andregeneration unchanged, as well as the failure to effect any fraction-ation by solvents or adsorption on a calcium carbonate column)they consider a-tocopherol to be a chemical individual.The most potent preparations of Drummond gave analyticaldata, for both the free alcohol and its acetate, in good agreementwith those for a-tocopherol.R. H. Kimm6* also has obtained ahighly active substance which, from analysis of its P-naphthoate,appears to have the formula C,gH,,O.This is, of course, theformula of tocopherol minus H,O, but the published chemical dataare too scanty to permit reasoned suggestions as to the relationshipof the two substances. All that is known is that tocopherol is analcohol, yielding a monoallophanate and a mono-p-nitrophenyl-urethane, that it does not react with ketone reagents, that itprobably contains a condensed cyclic nuclcus, and that it containsreactive ethylenic linkages.The suggestion that vitamin E is required for growth as well asfertility is negatived by experiments 69 in which the growth of ratswas measured from weaning on a diet free from E but otherwiseadequate. The growth was as good as that of rats on a stockcolony diet and was accelerated only very slightly in males (butnot in females) when vitamin E concentrates were used to supple-ment the basic diet.Small supplements of wheat germ oil, sufficientt o ensure fertility, were without effect on growth. The inclusionof lard, however (the fat in the basic diet consisted of the ethylesters of fatty acids from hydrogenated cotton seed oil), produced67 Biochem. J . , 1935, 29, 456.6s Sci. Papers Inst. Phys. Chem. Res. Tokyo, 1935, 28, 74.60 H. S. Olcott and H. A. Mattill, Proc. Amer. Soc. Biol. Chem. ( J . Biol.Chem.), 1936, 114, lxxvii394 BIOCHEMISTRY.a significant increase in the growth rate, a fact of some interest inview of the suggestion that certain unsaturated fatty acids arecssential dictary constituents.VitcGrnin K.-H.Dam and P. Schsnheydcr 70 described, in 1934,a deficiency disease in chicks, characterised by a tendency tohemorrhage, anamia, pathological changes in the gizzard, and aprolonged blood clotting time. This was later 71 ascribed to lackof a specific fat-soluble, thermostable vitamin (K) and the sugges-tion was confirmed independently by H. J. Almquist and E. L. R.S t ~ k s t a d . ~ ~ The vitamin occurs in hog’s liver fat (one of the firstsources) and to a smaller extent in dog, chick, and cod liver ; greenvegetables are a particularly rich source.73 The vitamin is destroyedby alkali.73* 74 H. Dam and P. Schmheyder 73 have achieved apartial purification from alfalfa by extraction with various solvents,followed by adsorption on calcium carbonate or cane sugar, theirmost active preparation containing 600 to 1000 units per mg.(Aunit is defined 75 as the smallest daily dose per g. body weightwhich, given for three days, will restore the clotting time to normal.Thus a chick weighing 400 g. would require 1200 units.) H. J.Almq~ist,~* also by solvent fractionation from alfalfa, obtained aconcentrate of which 2 mg. were adequate when added to 1 kg.of vitamin-free diet ; later 76 by fractional distillation in a vacuumhe obtained a yellow viscous oil of which 0-5 mg. per kg. of dietwas adequate. Exact comparison of the degree of concentrationachieved by the two workers is not possible in the absence of dataas to weight of chicks and food consumption in Almquist’s experi-ments, though it seems likely that his preparation is the moreactive.As to the mode of action of vitamin K, P. Schmheyder 77has suggested, and H. Dam, F. Schmheyder, and E. Tage-Hansen 78support the view, that the low clotting power in the blood of chickslacking the vitamin is due to a reduced pro-thrombin content.Treatment of the serum from normal chicks by the methods ofHowell or Mellanby gives precipitates with pro-thrombin activity,though serum from K-avitaminous chicks, treated similarly, givesinactive precipitates. Vitamin K concentrates themselves, how-ever, do not accelerate clotting in vitro, but pro-thrombin from‘O Biochern. J., 1934, 28, 1935.7 1 Nature, 1935, 135, 653; Biochem. J., 1936, 29, 1273.72 J . Biol. Chern., 1935, 111, 105.73 Biochem.J . , 1936, 30, 897.74 J . Biol. Chem., 1936, 114, 241.7 5 Biochem. J., 1936, 30, 890.7 6 J . Biol. Chem., 1936, 115, 589.7 7 Nature, 1935, 135, 652.7 8 Biochem. J . , 1936, 30, 1075STEWART AND STEWART. 395normal chick serum, extracted with acetone and ether to removelipoids, itself exhibits vitamin K activity. It is possible that thevitamin may be present in pro-thrombin as a prosthetic group indefinite chemical combination with the rest of the molecule.Hormones.Adrenal Cortex.-Recent work has shown the nucleus of the sexhormones, of the sterols, the bile acids, the cardiac aglucones, andthe antirachitic vitamin to be present in ye6 another substance ofbiological importance.The necessity for life of a substance produced by the adrenalcortex was shown in the case of adrenalectomised dogs by J.M.Rogoff and G. N. Stewart,79 who later 8o found that their extractswere of some benefit in cases of Addison’s disease. Similar resultswere obtained by W. W. Swingle and J. J. PfiffnerY8l and byF. A. Hartman.82 The hormone preparations used by these workerswere very impure, but in 1934 the isolation of a crystalline substancehaving the activity of the cortical hormone was announced fromthe Mayo Clinic.83 The crystals were later found to be a rnixt~re,8~but recently H. L. Mason, C. S. Myers, and E. C. Kendall 85 haveseparated from a number of somewhat similar compounds one whichhas definite, though small, activity when tested on the rat. It isdescribed as a strongly dextrorotatory, unsaturated ketonic alcohol,C21H30Q5, m.p. 201-20S0.The fact that this preparation is less active than was expectedsuggested that it is still impure, and certainly its description doesnot tally with that of an active substance obtained by Reichstein.Early in 1936 T. Reichstein g6 reported the ready concentration ofthe cortical hormone by means of ketonic reagents after preliminarytreatment with pentane and 20% methyl alcohol. From theseactive concentrates (and in part from inactive by-products) heobtained nine crystalline substances, all apparently closely related.One of these substances, p-adrenosterone, has one-fifth of the activityof androsterone by the capon test, three more are oxidisable tosubstances with slight androsterone-like activity, a fifth is oxidised79 Amer.J . Physiol., 1928, 84, 660.80 J . Amer. Med. ASSOC., 1929, 92, 1569.81 Science, 1930, 71, 321.s2 Endocrinology, 1930, 14, 229.s3 E. C. Kendall, H. L. Mason, B. F. McKenzie, C. S. Myers, and G. A.s4 E. C. Kendall, J . Amer. Med. Assoc., 1935, 105, 1486.8 5 J . Biol. Chern., 1936, 114, 613.58 He&. Chim, Acta, 1936, 19, 29, 223, 402, 979, 1107.Koelsche, Proc. StafS Meetings, Mayo Clinic, 1934, 2, 245396 BIOCHEMISTRY.to adrenosterone and is considered, though without cortical activity,to be identical with the active substance of Kendall et al. If thisidentification is correct, it follows that the preparation obtainedby Kendall and his collaborators is a mixture containing a smallamount of the hormone.Of the nine substances obtained byReichstein, eight were devoid of cortical activity when tested onrats by the Everse-de Fremeny 87 method in doses of 0.5-2 mg.The ninth has recently 8s been found to be active, and to yield onfurther fractionation a pure crystalline compound, m. p. 180-182*,which ‘( shows to a large extent the biological activity of the rawmaterial,” and is named corticosterone. By the method of Everseand de Fremeny, 0.5-1 mg. represents the approximate rat unit;tested on dogs, 0.25-0.5 mg. of corticosterone was found to beequivalent to 1 C.C. of standard ‘( cortin ” solution. It is claimedthat the chemical formula, with the exception of a few details, hasbeen elucidated. It has not yet been reported, but the impuresubstance of which corticosterone is the main coiistituent wasdescribed as an ap-unsaturated ketone.Reichstein and his co-workers point out that the isolation of corticosterone does notexclude the possibility that other active or activating substancesmay be present in the gland.InszLZin.-From the results of electrometric titration of crystallineinsulin, C. R. Harington and A. Neuberger 89 have deduced that theinsulin mo!ecule contains 43 & 2 acid-binding groups and 60-70base-binding groups. A study of iodinated insulin suggests thatin iodination only the tyrosine groups are affected (substituted inthe 3 : 5-positions) and on this assumption it appears that theinsulin molecule contains 24 tyrosine molecules. Titration of theiodinated protein suggests that the phenolic groups are free ininsulin.Since iodiiiated insulin is almost inactive physiologicallyand activity is (nearly) proportionately restored by partial remov a1of the iodine, it seems that the phenolic groups of insulin are im-portant in relation to its physiological activity. Combining theseresults with an estimate of the amide nitrogen of insulin (34 groupsper mol.), C. R. Harington and T. H. Mead9* have shown thatinsulin contains about 38% of glutamic acid. In view of inactiv-ation which follows release of the labile axnide nitrogen or thelabile sulphur from insulin and of the suggestion that insulin maycontain a ‘( prosthetic group ” just as thyreoglobulin owes its87 J. W. Everse and P. ds Fremeny, Acta Brev.Nkerl., 1932, 2, 152.8 8 p. de Fremeny, E. Laqueur, T. Reichstein, R. W. Spanhoff, and I. E.Uyldert, Nature, 1937, 139, 26.89 Biochem. J., 1936, SO, 809.90 Ibid., p. 1598STEWART AND STEWART. 397activity to thyroxin, Harington and Mead have synthesised cysteyl-glutamine (V) and glutaminyl-cysteine (VI).QH,=SH QH,*CO*NH,QH-NH, QHz(V.) CO-NH*$X*CO-OH (?H*NH, CH,*SH 0'1.)CH,*CH,*CO*NH, CO-NH-QHCOOOHBoth of these peptides were, however, without hypoglycaemic effectin the disulphide as well as the sulphydryl form. The lability oftheir amide nitrogen was not far removed from that of insulin;their sulphur, however, was far less labile.An interesting development in the therapeutic use of insulin isdue to H. C. Hagedorn, B. N. Jensen, N. M.Krarup, and I. Wud-s t r ~ p , ~ l who find that insulin combines with protamines to formcomplexes which have isoelectric points at about pK 7.3, a t whichthey are only slightly soluble in water though rather more so inserum. Their low solubility results, after their subcutaneous injec-tion, in the slow absorption of insulin into the body fluids. Inconsequence, the hypoglyczmic effect of an injection of protamineinsulinate lasts about twice as long as that of the same amountof free insulin. These results have been confirmed r e p e a t e d l ~ , ~ ~and it has been shown that (in rabbits) protamine insulinate couldbe detected in the lymphatics five hours after injection, whereasordinary insulin had disappeared in 45 minutes.93 The publisheddata, however, indicate that there is, as yet, no general agreementas to the best way of using the new modification of insulin therapy.Indeed, it seems likely that not only will different types of dietdemand rather different ways of using standard insulin along withprotamine insulinate, but that by using, e.g., different protamines itmay be possible to produce complexes suited to particularpurposes.The Xex Normones.-The chemical relationships of the numerousactive substances which have recently been obtained are discussedelsewhere in thisD.W. McCorquodale, S. A. Thayer, and E. A. Doisyg5 haveisolated dihydrotheelin (= oestradiol) from sow ovaries and havefound it to be identical in melting point and biological assay with91 J .Arner. Med. ASSOC., 1936, 106, 177.92 E.g., H. W. Boolt et aZ., {bid., p. 180; R. D. Lawrence and N. Archer,Brit. N e d . J., 1936, i, 747; I. M. Rabinowitch et ul., Cctnudian Med. Assoc. J.,1936, 35, 124.93 H. K. Beecher and A. Krogh, Nuture, 1936, 13'7, 458.94 P. 356.9 5 Proc. SOC. E x p . Biol. Med., 1935, 32, 1182398 BIOCHEMISTRY.the substance obtained by catalytic hydrogenation of oestrone.An improved method of isolation from the aspirated liquor folliculi-as the di-a-naphthoate-leads to the conclusion 96 that at least 52%of the oestrogenic activity of the starting material is due to dihydro-theelin, which, therefore, is the chief active principle of the sow’sovary (though others may be present). The amount present isabout 0-015 rng.per kg. of ovary. Two isomeric dihydrotheelinshave been isolated from the urine of pregnant mares.97 The twoisomers should both be obtained by chemical reduction of oestrone,and McCorquodale’s material, there€ore, is a mixture or the twoisomers have equal activities.R. H. Andrew and F. Fenger98*99 have reported the isolationfrom ovaries of a substance, probably C,,H,,O,N, of which 0.00001mg. produces oestrus in rats after 96 hours. This would appear tobe the most active oestrogenic substance yet obtained.The existence in pregnancy urine of bound oestrogenic materialliberated by boiling with hydrochloric acid has been confirmed byG. van S. Smith and 0. W. Smith.1 S. L. Cohen and G. P. Marrian2have obtained from pregnancy urine a water-soluble, ether-insoluble,amorphous substance containing about 50 yo of its weight of oestriol.Its composition and reactions suggest that it is a compound ofoestriol and glycuronic acid, a suggestion which has been confirmedby later work.3Testosterone remains the most active male hormone from naturalBources, though its 17-methyl ether is stated to be more active inthe capon test and also, incidentally, to possess progesteroneactivity 4 (the two conipounds are R-O-Me and R-CO-Me, respect-ively). The activity of testosterone is said to be increased byincrease in the amount of the oily medium in which it is adminis-tered 5 or by esterification with acetic or propionic acid.6 Possiblyby these means it is protected from destruction in the animal body.Like some of the artificial male hormones (particularly those con-taining ethylenic linkages), both androsterone and testosterone aredescribed as possessing certain of the properties of the ovarian96 J .Biol. Chern., 1936, 115, 435.9 7 Proc. SOC. E x p . Biol. Med., 1935, 32, 1187.D8 Science, 1936, 84, 18.On Endocrinology, 1936, 20, 563.Arncr. J . Physiol., 1935, 112, 340.S. L. Cohen, G. F. Marrian, and A. D. Odell, Biochem. J., 1936, 30,L. Ruzicka and H. R . Roseenberg, Helu. C h k . ActCG, 1936, 19, 357.A. S . Parkes, ibid., 1936, 321, 674.2 Biochem. J . , 1936, 30, 57.2250.5 R. Deanesly and A. S. Parkes, Lancet, 1936, 230, 837STEWART AN11 STEWART. 399llor~r-~ones.~ V. Korenchevsky, M. Dennison, and S. L. Simpson *have found in prolonged experiments that large doses of andro-stcrone (or better androstanediol) caused a partial recovery of theatrophied uterus and vagina in sprayed rats.The administrationof androsterone together with oestrone was more effective (especiallyin the recovery of the uterus) than either male or female hormonealone. A similar, though smaller, co-operation between andro-sterone and ocstrone was shown in the ratc of involution of thethymus, but the two substances appeared to antagonise each otherin their effects on the adrenals (in which androsterone normallycauses reduction of weight to normal) and on the body weight.A similar co-operative effect of the two hormones was also shownin the recovery of the sexual organs (and thymus) of males.Asthc authors point out, the mutual interaction of the male andfemale hormoncs is of considerahle importance, since it is wellestablished that both are found in normal urine from males andfeiiiales. These cxperirnents have been extended to testosteroneand oc~tradiol.~ Testosterone shows the same co-operative a i dantagonistic effects with the ovarian hormones as does androsterone,the action again being much more marked in females than in males.Testosterone (and also androstanediol), i t is remarked, differs fromandrosterone in bringing about a quantitative2y normal developmentof the male sexual organs, and these substances also produce someof the changes associated with progesterone. If, therefore, theyare injected simultaneously with ocstrone, the effects in femalessimulate some of those seen dining pregnancy.Although incastrated male rats the effect of androsterone is increased byoestrone, the increase is not nearly so great as that produced bythe " X " substance of Laqueur et aE., a substance, itself inert,obtained (impure) in extracts of plant or animal tissues.1°Proteins and Amino-acids.Protein Structures.-Steady improvement in the methods avail-able for the determination of individual amino-acids in proteinhydrolysates is beginning to reveal stoicheiometrical relationships.Thus M. Bergmann l1 has extendcd his work on gelatin reported7 E. Wolff and A. Ginglinger, Compt. rend. SOC. Biol., 1936, 121, 1470;E. Wolf€, ibid., p. 1474, V. Korenchevsky, Nature, 1936, 137, 494; A.Butenandt, Nteturwius., 1936, 24, 16.8 Biochern.J . , 1935, 29, 2634.9 V. Korenchevsky, M. Dennison, and I. Brovsin, ibid., 103G, 30, 558.1" Acta Breu. Nker., 1935, 5, 84; J. Freud, ibid., p. 97; L. Ruziclra,M. W. Goldberg, and H. R. Rosenberg, HeZv. Chim. Acta, 1935, 18, 1487.11 M. Bergman and C. Niemann, J. Biol. Chem., 1936, 115, 77400 BIOCHEMISTRY.last year .12 He finds that glycine, proline, hydroxyprolinc, arginine,alanine, leucine-isoleucine, and lysine occur in the molecular pro-portions 24 : 12 : 8 : 4 : 8 : 4 : 3. On the assumption of a regulararrangement of the amino-acids in a polypeptide chain this gives aperiodicity (in the same order) of 3, 6, 9, 18, 9, 18, 24 and it ispointed out that these numbers are all multiples of 3.This period-icity of the amino-acids would be satisfied by either of the arrarrge-ments :G-P-X-G -X-X-G-P-X-G- or G-X-P-G-X-X-G-X-P-G-W. Grassmann and K. Riederle l3 have isolated Iysylprolylglycinefrom gelatin, and since the first of Bergmann's suggested arrange-ments demands the presence only of glycylproline peptides, thissupports the second. A similarly extensive stoicheiometricalrelationship has been found among the amino-acids of blood fibrin(cattle) , where glutamic acid, lysine, arginine, aspartic aid, proline,tryptophan, histidine, methionine, and cysteine (total 54.57, ofthe protein) are found to be in the molecular proportions72 : 48 : 32 : 32 : 32 : 18 : 12 : 12 : 9, with corresponding periodicities8, 12, 18, 18, 18, 32, 48, 48, 64.Similarly, R.Block and H. Vickery l4 showed some years agothat the keratins form a group of proteins with histidine : lysine :arginine molecular ratios of 1 : 4 : 12. More recently, R. Block 15has found that hzmoglobins exhibit another characteristic arrange-ment with iron : arginine : histidine : lysine in the ratios 1 : 3 : 8 : 9.Another example of attempts to gain an insight into the structureof protein is supplied by H. Bauer and E. Strauss,16 who, from astudy of iodination of globin and various derivatives, have reachedthe conclusion that globin is a complex of six units, each of molecularweight 11,680, the units being linked through the glyoxaline groupsof histidine.Canavanine and Ca.nmline.-R". Kitagawa and A. Takani l7 have con-firmed the structure of canaline as NH,-O*CH2*CH2*CI-I(NH2)*C0,H.By treatment of or-benzoylcanaline with methylisocarbamide theyobtained M- benzoylcanavanine, which yielded canavanine on hydro-lysis.NH,*C( :NH) -NH*O*CH,*CH,*CH(NH,)*CO,H,the two amino-acids having the same relationship to each other asornithine and arginine in conformity with the demonstration thatCanavanine is, thereforc, confirmed as12 Ann.Reports, 1935, 32, 418.3 4 J . Biol. Chem., 1931, 93, 113.16 Biochem. Z., 1936, 284, 107, 231.17 J . Agric. Chent. SOC. Japaiz, 1935, 11, 1077; J . Biochern. J C Z ~ C L ~ Z , 1936,l3 Binchem. Z., 1936, 284, 177.l5 Lbid., 1934, 105, 663.23, 181STEWART AND STEWART. 401they can share in the synthesis of urea.18 Incubation of canavaninewith a pig liver extract a t 37" yields canaline and y-ethylidene-canaline, which can be hydrogenated to an cc-amino-y- hydroxy-acid,C,H,O,N, and can be obtained from canaliiie and acetaldehyde.19The growth-promoting action of canavanine, shown by &I.Ogawa,20 is denied by M.Kitagawa and M. Wada.21 M. Ogawa 22now states that canavanine is not essential for growth in the laterpart of the growing period and that it is beneficial to the health ofpregnant animals though not essential for pregnancy.23a-Amino- P-hydroxybutyric Acid.-In the course of feeding experi-ments with pure amino-acids, W. C. Rose 24 found that young ratsfailed to maintain themselves when supplied with a mixture ofnineteen amino-acids instead of protein.Growth, however, occurredif the mixture was supplemented by a concentrate of the monamino-fraction of a protein hydrolysate. M. Womack and W. C. Rose 25found that the supplementary concentrate supplied two factors,one of which was identified as isoleucine (which was present in theartificial mixture, but, evidently, in insufficient amount). Thesecond essential factor has been isolated26 from fibrin and shownto be cc-amino-p-hydroxybutyric acid, since on reduction it givescc-aminobutyric acid, and since (unlike a-amino-y-hydroxybutyricacid) it does not yield a lactone when warmed in acid solution.Moreover, a-amino-y-hydroxybutyric acid (synthetic) was unableto replace the natural acid in growth experiments. H. E. Carter 27prepared a-amino-p-hydroxybutyric acid by the method of E.Abder-halden and I<. Heyns,28 but the product was without effect in growthexperiments. The synthetic material, obtained from crotonic acid,contained two of the four possible isomers. Carter, therefore, con-verted it into a mixture of the two epimers by preparing the formylderivative, heating this with sodium hydroxide and acetic anhydride,and hydrolysiiig the product with hydrobromic acid. This treat-ment yielded a mixture which supported growth, with about afifth of the activity shown by the natural acid. Attempts to con-1 8 M. Kitagawa and T. Tomita, Proc. Imp. Acad. Il'okyo, 1929, 5, 380.19 M. Kitagawa, I<. Sawada, and Y. Hosoki, J. Agric. Chem. Xoc. Japan,20 Ibid., p. 558.3 1 J .Agric. C'hem. SOC. Japan, 1935, 11, 1083.22 Ibid., 1936, 12, 256.z 3 ]bid., p. 828.24 J . Biol. Chenz., 1931-3, 94, 155; C. T. Caldwell and W. C. Rose, ibid.,25 Ibid., 1935, 112, 275.26 R. H. McCoy, C. E. Meyer, and W. C. Rose, ibid., p. 383.27 Ibid., p. 769.a8 Ber., 1934, 67, 530.1935, 11, 539.1934, 10'9, 57402 BIOCHEMISTRY.centrate the active isomer by fractional crystallisation have not yetbeen successful. A more detailed study29 of the amino-acid fromnatural sources involving its reduction to d-a-aminobutyric acid(which belongs to the L series) and its oxidation to Z-lactic acid(which belongs to the D series) indicates that it corresponds inspatial structure to d(-)-threose (VII). It therefore has theconfiguration represented in (VIII).It is proposed to name itd( -)-threonnine.TO*OHH p F * I I YHO H0.Q.H( V W H*T*OH HOPOH (VIII).CH,*OH CH3&'. Kimop, B'. Ditt, W. Heckstcdcn, J. Maier, W. Merz, and 1%.Hurlc 30 have synthesised a-amino- p-hydroxy-y-phenylbutyric acidand a-amino-p-hydroxy-6-phonyl-n-valeric acid. Their study ofthese indicates that a-amino- p-hydroxy-acids are not degraded inthe same way as simple amino-acids, but that they undergo p-oxid-ation and yield nitrogen-free acids. Presumably a-ainino- P-keto-acids are formed, and these, they say, have a much greater redoxpotential than ascorbic acid.Sulphur-containing rl mino-acids .-Dj enkolic acid 31 has beensynthesised 32 by the action of methylene dichloride on the sodiumderivative of cysteine in liquid ammonia, and its constitution con-firmed as CH,[S*CH,*CH(NH,yCO,a],.From the same laboratory %comes a synthesis of glutathione with improved yields, the methodemployed differing from that of C . R. Harington and T. H. Mead31in that the SH group of the cysteine is protected by benzylation sothat S-benzylcysteinylglycine is condensed with the a-monomethylester of N-carbobenzyloxyglutamic acid, and the final reduction isachieved by means of sodium in liquid ammonia.34 A new synthesisof methionine has been published by E. M. Hill and W. Robson,35who treat ethyl y-chloro-a-benzamidobutyrate (prepared froma-benzamido-y-butyrolactone) with sodium methyl mercaptide,followed by alkaline hydrolysis, and hydrolyse the resulting benzoyl-methionine with acid.It has been known for some time36 thatmethionine is capable of replacing cystine in a cystine-deficient29 C. E. Meyer, and W. C. Rose, J . Biol. Chem., 1935, 115, 721.3O 2. physwl. Chem., 1936, 239, 30.31 Ann. Reports, 1935, 33, 418.32 V. du Vigneaud and W. I. Patterson, J . Biol. Ch~m., 1930, 114, 633.33 V. du Vigneaud and Miller, ibid., 1936, 118, 469.34 Cf. H. S. Loring and V. du Vigneaud, ibid., 1935, 111, 38s.3b Bwchem. J., 1936, 30, 248.36 Ann. Reports, 1934, 31, 340STEWART AND STEWART. 403diet; now, however, it is suggested that the converse is not true,and that methionine is itself an “ essential ” amino-acid.37Chemotherapy.Trypanosomiasis and Sypitilis.-G. T . Morgan and E. Walton 38have studied compounds of the general formula (X)-for values ofn ranging from 1 to 8 and in which R and R’ are either hydrogen,alkyl or aryl groups-in relation to arsacetin (IX) and tryparsamide(XI), which in all probability is the most widely used of the quin-quevalent arsenical drugs.There does not appear to be any0 3 H N a As0,HNa(XI. )NH*CO*CH, NII*CO*[CH,];CO*NRR’ NH*CH,*CO-NH,well-defined relationship between the chemical constitution and thetherapeutic activity of these compounds. They exhibit varyingdegrees of curative action on experimental trypanosomiasis in mice,many of them having a therapeutic activity in trypanosome-infected mice which is at least equal to, if not greater than, thatof tryparsamide. Branching of the carbon chain tends to diminishthe activity and as the value of n in the straight chain approaches8 there is considerable rise in toxicity.W. Yorke, F. M~rgatroyd,3~and their collaborators reporb very favourably on extensive trialsof sodium succinanilomethylamidc-p-arsonate (Neocryl) (X ; n = 2 ;R - H, R’ = CH,), which is the most readily available of thesederivatives, and they regard this compound as being more activethan is tryparsarnide on trypanosomiasis in laboratory animals.They record instances of its having effected definite clinical im-provement in Nigerian sleeping sickness, in some cases with restor-ation of the cerebrospinal Auid to its normal state. They havefound too that it differs from tryparsamide in its ability toexert a definite action in primary, secondary and tertiary syphilis,as distinct from neuro-syphilis.The latter group OE workers 40 have long interested themselves inthe important problem of arsenic-resistant strains of trypanosomes,their more recent investigations of the mechanism of the action ofarsenicals on trypanosomes 40-recently summarised by W.Yorko0 (X. 137 W. C. Rose et nl., J . Biol. Chem., 1936, 114, lxxxv.38 J., 1931, 615, 1743; 1932, 2764; 1933, 91, 1064; 1935, 390; 1936, 902.39 Brit. Med. J., 1936, 1042.40 Awn. Trop. Med. and Pamsit., 1930, 24, 449; 1931, 25, 313, 351, 521;1932, 26, 215, 577; 1933, 2’9, 157; Brit. Med. J., 1933, 176404 BIOCHEMISTRY.and I?. Murgatroyd 41-being aided very materially by their funda-mental discovery 42 of a satisfactory method whereby the pathogenictrypanosomes could be maintained in vitro for twenty-four hours at37' in undiminished numbers and in a condition of unloweredvitality. Confirmation and extension of their findings are nowaccumulating from various sources.L. Launoy, M. Prieur, andA. hcelot 43 have produced an arsenic-resistant strain of T. anna-mense in the guinea pig by repeated tryparsamide treatment andhave found that, like the similarly produced arsenic-resistant2'. rhodesiense of W . Yorke and his collaborators, it is therebyrendered resistant to the quinquevalent arsenicals and to a thio-arsinite. C. H. Browning and R. Gulbransen 44 record experimentsindica$ing that the immunity which develops after treatment witha curative drug depends on the particular strain of trypanosomeand on the species of host.After studying the mode of action ofa number of organic arsenicals on rats infected with T. Zewisi andon rats infected with 2'. equiperdum, M. L. Kuhs, C. C. Pfeiffer,mid A. L. conclude that a specific relationship appearsto exist between the type of arsenical and the type of trypanosome.The same authors 46 have made the interesting observation thatinfections of T. Zewisi in rats which are easily cured by arseno-phenylglycine provided that treatment is begun within 3 4 daysof inoculation lose their arsenic susceptibility if there is furtherdelay in commencing this treatment. T. Naito and S. Oka 47 haveproduced a strain of trypanosomes resistant to orsanine (3-acet-amido-4-hydroxyphenylarsonic acid) and to the tervalent arsenicalsiieosalvarsan and neosilversalvarsan, and studied its sensitivity toother arsenical and non-arsenicaldrugs.F.R. W. K. Allen *s has treated nine cases of syphilis in Indiawith a Merck preparation Modenol, a salicylate of arsenic andmercury, and eight of these cases have shown considerable im-provement. A. B. Cannon and J. Robertson,Pg who set out todetermine the relative values of bismuth and mercury preparationswith arsphenamine in the treatment of early syphilis, have con-cluded that it is difficult to assay the relative values of bismuthand mercury, both of which are important in syphilis therapy. A41 Tram. Roy. SOC. Trop. Med. Hyg., 1935, 28, 435; Ty. Yorke. Riv.Malarial., 1935, 14, Suppl.4a Ann.Z'mp. Ned. and Parasit., 1929, 23, 501.48 Bull. SOC. Path. mot., 1935, 25, 857; 1936, 29, 769.44 J . Path. Bat., 1936, 43, 478.45 J . P b m . Exp. Ther., 1936, 57, 144.*t3 Amer. J . Nyg., 1936, 23, 10.47 2. Bakt., 1936, 137, 401.(@ J . Amer. Ned. Aaaoc., 1936, 106, 2133.48 I n d i m Med. Qaz., 1936, 329STEWART AND STEWART. 405leading article in a recent issue of the Lancet expresses the opinionthat bismuth preparations are replacing mercurials in antisyphilitictreatment and makes special reference to the very favourableresults of the tests by F. M. Thurmon 51 on some two hundredpatients in his clinic over a period of eighteen months with a fat-soluble preparation, bismuth ethyl camphorate, either alone or inconjunction with arsphenamine.In these trials it comparedfavourably both as regards local pain or discomfort and moregeneral toxic effects with the standard bismuth preparation-bismuth salicylate suspended in oil-which was in routine use onother patients in the clinic, and stress is laid on one particularlyvaluable feature of its activity, the efficiency of its mode of actionin serological tests. W. &I. Lauter and H. A. Braun 52 have pre-pared a series of bismuth trialkyl camphorates by allowing bismuthnitrate to react in aqueons glycerol with the appropriate sodiumalkyl camphorate and have determined their toxicity on intra-muscular injection into rats.Malaria.-Preliminary reports 53 on the treatment of malariawith atebrin musonate (atebrin methyl sulphonate) have beenpromising.In recent months much evidence has been publishedwhich serves to substantiate the claims of atebrin and its musonatefor wider clinical application. A. T. W. Simeons 54 has obtainedvery satisfactory results in the mass treatment of all persons in anendemic area with two injections of atebrin musonate at twenty-four-hour intervals. With atebrin, as with all synthetic prepar-ations, claims on the grounds of therapeutic efficiency are con-sidered together with cost of production. Thus J. A. Carman andR. P. Cormack 55 record the comparison of a number of cases ofmalaria in Kenya treated with atebrin musonate with an equalnumber of controls given quinine and plasmoquine. They considerthat the results were as good as those of quinine treatment, with aprobable lower relapse rate and without toxic symptoms, but theyconsider that the drug is uneconomical for use on natives.Incontradistinction to this conclusion a large-scale trial of atebrin asa prophylactic in malarial regions of the Southern States, havingshown that the drug is superior to quinine, producing completedestruction-not merely inhibition-of the parasites, has caused60 1936, 1163.5 1 New Eng. J . Med., 1936, 315.52 J . Amer. Phnm. A ~ ~ o c . , 1936, 2!j, 394; cf. M. Picon, Bull. SOC. chirn.,53 Ann. Reports, 1935, 32, 422; S. Somasundram, Tram. Roy. SOC. Trop.54 Indian Med. Gaz., 1936, 71, 132.55 Trans. Roy. SOC. Trop. Med. Hyg., 1936, 29, 381.1936, 3, 176.Med. Hyg., 1935, 29, 103; E.C. Vardy, Malayan Med. J., 1935, 10, 67406 BIOCHEMISTRY.W. W. Bispham 56 to consider such treatment not only superiorto quinine but actually cheaper in spite of the greater cost of thedrug, for the simple reason that a smaller quantity suffices toeffect a cure.For all comparative work of this nature it is well to note theobservation of F. Mietszch, H. Mauss, and G. Hecht 57 that aqueoussolutions of atebrin decompose slowly on keeping with the form-ation of an acridone and falling off of toxicity, and the conclusionwhich S. F. Seelig and W. Singh 58 have drawn from a comparisonof three methods of atebrin musonate treatment with or withoutadrenaline as to the most satisfactory method of administering thedrug. They obtained the best and quickest results by givingadrenaline, followed by an intramuscular dose of atebrin musonate,and allowing twenty-four hours to elapse before commencingregular doses of atebrin tablets.C.Ragiot and I?. Moreau 59 have used quinacrine 60 with successin cases of hematuria due to quinine. 0. J. Magidson and hiscollaborators 61 have continued to study the therapeutic activitywith acridine derivatives closely allied to atebrin in relation tovariations in chemical structure. From observations of malariain children R. Sherman 62 considers that acriquine (6-chloro-9-diethylaminobutylamino-2-methoxyacridine) has a high therapeuticvalue. B. N. Rubenstein 63p64 has found this same drug verysuccessful for the treatment of induced malaria in paralytics andregards it as having a prophylactic action on experimental malariain man.I n the study of monkey malaria anomalies in the behaviour ofquinine and the various synthetic antimalarials have been noticedfrom time to time.Working with PI. Knowlesi infections in apes,E. G. Nauck and B. Malamos 65 have furnished evidence in favourof the conception that atebrin and quinine are alike in one respect,that they exert a, direct action on the malarial parasites, but thatthey differ in their mode of action in that the morphological changeswhich the parasites undergo are quite different for the two drugsand different from the changes in the controls.66 Amer. J . Trop. Med., 1936, 16, 547; cf. H. Flack, D. C. Majumder, andK. Goldsmith, Indian J. Med. Res., 1936, 71, 373.67 Indian Ned.Caz., 1936, 71, 521.58 Records Mal. Survey Ind., 1936, 6, 171.69 Bull. SOC. Path. mot., 1936, 29, 496.60 Identical in chemical constitution with atebrin.6 1 Ber., 1936, 69, 396, 537.62 Med. Parasit. and Parasit. DiS., 1935, 4, 446.68 Arch. Schiff. Trop. Hyg., 1936, 40, 167.64 Med. Parasit. and Parasit. Dis., 1936, 5, 256.65 Klin. Woch.. 1936, 888STEWART AND STEWART. 407Antiseptics.-Noteworthy contributions have been made duringthe year under review to the problems of antisepsis. Investigationsof the means of combating infection of the urinary tract discussedin an earlier report G. H. Newns and R. Wilson 67have found that mmdelic acid in the form of its ammonium saltis an effective remedy for B.coli pyelitis in children. P. Ganguli 68has employed the sodium salt. H. 3'. Helmholz and A. E. Oster-berg 69 have studied both the urinary excretion of sodium mandelateand the bactericidal effect of this salt in various concentrations ona, number of organisms, while in continuation of his pioneer workon the subject N. L. Rosenheim 70 has given an account of thetreatment of almost one hundred cases of various types of urinaryinfection with ammonium mandelate.The Prontosil group of antiseptics, derived from paminobenzene-sulphonamide, of which Prontosil (XII) 71 and Prontosil X (XIII) 72still continue.OHare at present the most efficacious, has come into prominence andis receiving considerable attention for the treatment of strepto-coccal and staphylococcal infection^.^^ E.Fourneau, J. Tr6fouE1,F. Nitti, and D. Bovet 74 have observed that p-aminobenzene-sulphonamide has an inhibitory effect on the growth of mouldswhich is not seen in prontosil, and P. Nitti and D. B o ~ e t , ~ ~ review-ing the present position of our knowledge of prontosil and itsderivatives, note that guinea pigs cim be sensitised to prontosil but66 Ann. Reports, 1936, 32, 425.67 Lancet, 1936, 230, 1087.6 8 Indian Med. Gaz., 1936, 71, 517.69 Proc. Mayo Clinic, 1936,11,373 ; J . Amer. Med. Assoc., 1936, 107, 1794;cf. L. P. Dolan, ibid., p. 1800.70 Lancet, 1936, 230, 1083.7 1 Angew. Chem., 1935, 657.72 Ibid., p. 661.73 K. Imhauser, Klin. TYoch., 1938, 282; L. Ley, Munch. rned. Woc?~.,1936, 1092; A.Roth, Deut. med. JT70ch., 1935, 1734; C. Levaditi and A.Vaisman, Compt. rend. Soc. biol., 1035, 119, 946; 1936, 121,803; L. Colebrookand M. Kenny, Lancet, 1936, 1279, 131!1; H. Rorloin, Pror. Roy. Xoc. Med.,1936, 29, 313.74 Compt. rend. Xoc. biol., 1936, 122, 6.52.7 6 Revue d'Imrnun., 1936, 2, 450, 461408 BIOCHEMISTnY.not to thc parent substance. Prontosil and the more solubleprontosil S , administered intravenously or better orally, are graduallyestablishing themselves as successful means of combating ery-sipelas.76 H. Floch 77 has administered prontosil orally withsuccess in the treatment of elephantiasis. Numerous derivativesof p-aminobenzenesulphonamide 78 have been prepared, but thereis as yet insufficient evidence on which to base a discussion oftheir therapeutic activity.The observation of R. Hilgermann 79that alkali-metal salts of the bile acids, suitably protected by acolloid, can cure streptococcal infections opens up interestingpossibilities. c. P. s.J. S.PLANT BIOCHEMISTRY.Metabolism and Biochemical Activity of Certain Bacteria.Axotobacter.l-The production of free ammonia by these organismshas been the subject of much controversy. Earlier workers haddisagreed not only on the question of whether or not the organismdid actually produce free ammonia, but also as to whether thisammonia should be regarded as the first product of nitrogen fixationwith subsequent elaboration into bacterial protein. S. Winogradsky,2working on silica-gel to avoid secondary reactions, confirmed hisearlier adherence to the view that the nitrogen exchange of theorganisms takes the course N, --+ NH, -+ cellular protein, andthat in very alkaline media a surplus of NH, appeared in the nutrientsubstrate.S. Kostytchev and 0. Schelo~rnova,~ following upprevious investigations with A . vinelandii, added further supportto Winogradsky's theories and also demonstrated that free ammoniawas produced only in the presence of an adequate carbohydratesupply. Fixed nitrogen supplied to the organism was reduced to7 6 L. Gmelin, Munch. med. Woch., 1935, 221; W. Kramer, ibid., 1936,608; G. Scherber, W i e n . med. Woch., 1935, 284, 346, 376; E. Wehren,Schweiz. med. Woch., 1936, 06, 665 ; V. Anghelescu and collaborators, Deut.med. Woch., 1936, 1639; K.Hartl, ibid., 1936, 1641; J. Frankl, K l i n . Woch.,1936, 15, 1562.77 Bull. Boc. Path. exot., 1936, 29, 165.7 8 P. Goissedet and collaborators, Compt. rend. SOC. bid., 1936, 121, 1082 ;E. Fourneau, J. Trkfouel, F. Nitti, and D. Bovet, ibid., 1936, 122, 258:G. A. H. Buttle, W. H. Gray, and D. Stephenson, Lancet, 1936, 1286; F.Nitt,i and D. Bovet, Compt. rend., 1936, 202, 1221.79 Deut. med. Woch., 1936, 883.1 See Ann. Reports, 1933, 306.2 Ann. Inst. Pasteur, 1932, 48, 269.8 8. physiol. Chen,., 1931, 198, 105POLLARD. 409ammonia and subsequently u tilised in protein synthesis. Hefurther showed that in the absence of a suitable carbon source cellularprotein was itself deaminated with the formation of ammonia.A. Isakova,4 working with A .vinelandii and A . chroowccurn,obtained similar results and demonstrated the production ofammonia in neutral or only very slightly alkaline media in whichglucose, mannitol, or even salts of organic acids (sodium acetate,benzoate) formed the source of carbon. M. Roberg5 and alsoD. M. Novogrudski examined filtrates from Azotobacter cultureswhich were shown to contain nitrogen compounds utilisable by othermicro-organisms (notably those utilising amino-acids or ammonia).Free ammonia, however, did not appear in the filtrates until theenergy source had been exhausted by the bacteria. These resultsfall into line with an earlier suggestion of Kostytchev that ammoniafound in culture media was largely derived from bacterial protein.Presumably any ammonia derived directly from nitrogen would beutilised by actively growing organisms as fast as it is formed.Onthe other hand A. N. Bach et al. reported the production of ammoniafrom nitrogen by the expressed juice of Axotobacter cells, thus support-ing the conception of the enzymic formation of ammonia as the firststep in the nitrogen fixation process. In a series of detailed andcarefully controlled experiments D. Burk and C. K. Horner,8continuing earlier investigations, showed that the extra-cellularproduction of ammonia by both A . winelandii and A . clwoococcumprobably results almost entirely from the decomposition of cellularprotein and not to any appreciable extent from free nitrogen. Underoptimum agrobic conditions (px 7-8 and 3 0 4 0 " ) as much as 50%of the microbial nitrogen was liberated as ammonia.The presenceof nitrogen was found to be quite unnecessary for ammonia produc-tion. The course of liberation of ammonia was closely paralleledby that of oxidation of cell constituents and was inhibited by re-agents which normally check biological oxidations as well as by thepresence of even small amounts of oxidisable organic matter. It issuggested that ammonia may not even be a necessary step in thesynthesis of bacterial protein from nitrogen and that amides may beconcerned here. In this connexion G. Endres9 indicates the pro-duction of oximes in culture media of Azotobacter and suggests hydr-oxylamine as an intermediate stage in the fixation process. Burk4 Bull.Acad. Sei. U.R.S.S., 1933,9, 1493.5 Jahrb. wigs. Bot., 1935,82, 1, 65.Microbial. U.S.S.R., 1933, 2, 237.A. N. Bach, Z. V. Yermolieva, and M. P. Stepanion, Cornpt. rend. Acad.Sci. U.R.S.S., 1934,1, 22.8 Soil Sci., 1936,41, 81.@ Naturwiss., 1934, 22, 662 ; Annalen, 1935, 518, 109410 BIOOHEMISTRY.and Horner propound the following scheme of nitrogen exchangebeing most in accord with experimental data :aslllc8 stable cellular rlcnminatioliN, + organic matter -e--tz compounds of F---+ NH,.reduced N growthProm this point of view extracellular ammonia is liberated onlyunder conditions in which growth is insufficiently rapid to permit thecomplete utilisation of the ammonia produced by the oxidativedecomposition of the cellular constituents.The authors, however,point out that the data cannot be interpreted as definitely ruling outthe possibility that some ammonia may be formed in the fixationprocess.An examination of the cellular proteins of Axotobacter by R. A.Greene lo indicates that these consist largely of globulins, glutelins,and albumins. The amino-acid distribution (Van Slyke) showeddifferences among the species, but in general arginine and lysinepredominated and smaller proportions of tyrosine, tryptophan,cystine, and histidine together with approximately 40% of the non-basic fraction were found. The presence of glutathione was alsoindicated.Another aspect of the activity of Axotobacter is presented by N. R.Dhar and colleagues,ll who have shown that the fixation of nitrogenby these bacteria in tropical soils is optimum a t 35", as comparedwith 28" in temperate soils, and in both cases is negligible at 10".It would seem, therefore, that the value of Axotobacter in maintainingthe nitrogen supply in soil has been somewhat over-estimated.Theaddition of molasses to soil for the purpose of increasing fixationresulted in a rapid iiicrease in the number of organisms, but theamount of nitrogen fixed did not increase proportionally. Rapidfixation is associated with more or less stationary nunibers ofAxotobacter. The improved nitrogen-status of molasses-treatedsoils, formerly attributed to increased bacterial fixation, is repre-sented as being partly due, at least in tropical soils, to photochemicaleffects.Among investigations of the influence of different carbon sourceson the activity of Axotobacter may be cited that of S.WinogradsBy,l2in which it is shown that in addition to sugars, certain alcohols andsimpler fatty acids (2-4 C) may be utilised. T. R. Bhaskaran andV. Subrahmanian,13 working with mixed soil organisms in glucosel1 N. R. Dhar and S. P. Tandon, Proc. Nat. Acnd. Sci. India, 1936, 6,35; N. R. Dhar and E. V. Seshacharyulu, ibid., p. 99; N. R. Dhar and S. K.Mukerji, J . Indian Chem. Xoc., 1936, 13, 155.10 Soit? S&., 1935, 39, 327.l2 Compt. rend., 1936, 203, 10. Is Current Sci., 1935, 4, 234POLLARD. 41 1media, noted an initial period of activity, in which carbon dioxideand organic acids were formed from glucose, but the amount ofnitrogen fixed was much less than that to be expected.Moreovermuch of this nitrogen was in a soluble form. Subsequently theorganic acids were decomposed and apparently normal fixationproceeded. In a later paper l4 fixation of nitrogen by the mixedflora was shown to be facilitated by addition of organic acidsobtained in the nnagrobic decomposition of sugar. Utilising purecultures of A . chroowccum, Bha.skaran l5 found no relationshipbetween the presence of these sugar-decomposition products andthe fixation of nitrogen, the course of which differed from thatoccurring with mixed soil cultures. It appears possible, however,that the acids contributed largely to the production of cellularconstituents or perhaps served as energy sources.In young culturesthe accumulation of carbon in the slime and bacterial cells was muchmore rapid than that of nitrogen. Later the ratio narrowed some-what. G. Guittonneau and R. Chevalier l6 record that pure culturesof Axotobacter can utilise sodium sslicylate and continue the fixationprocess .Carbon Metabolism of, and Nitrogen Fixation by, Rhizobia.The close relationship between the carbon metabolism and nitro-gen exchange of these organisms referred to in a previous Report 17continues to receive considerable attention at the hands of variousresearch workers. Interesting data relating t o the effect of nutri-tional factors on the respiratory quotient of cultures have now beenobtained. have examined theoxygen consumption of R. meliloti on glucose media, and have shownthat the carbon dioxide produced corresponds to approximatelyone-third of the carbon in glucose, the amount being somewhatgreater when ammonia than when nitrate forms the nitrogen source.The pH optimum for growth of R. meliloti and R.japonicum appearsto be less than that for re~pirati0n.l~ In a further publication 0. R.Neal and R. H. Walker 2O record that the oxygen consumption ofR. meliloti was substantially the same on glucose, mannitol, andsucrose media, but was very much greater on arabinose-nitratemedia. Galactose was more effectively utilised than was glucose onboth ammonia and nitrate media, whereas maltose, lactose, inositol,Thus 0. R. Neal and R. H. Walker14 Proc. Indian. Acad, SCi., 1936, 4, B, 163.l5 Ibid., p.67.l6 Compt. rend., 1936, 203, 211.l7 Ann. Reprta, 1934, 347.19 D. W. Thorne and R. H. Walker, J . Ract., 1935, 30, 33.20 Ibid., p. 173.Proc. Iowa Acnd. Sci., 1934, 41, 1674 12 BIOCHEMISTRY.dulcitol, and sorbitol with both forms of nitrogen, and raffinose anderythritol in ammonia media, proved inferior energy sources.For this species ammoniacal nitrogen was in general more readilyutilised than was nitrate. The reverse was true of R. japonicurn,which also exhibited characteristic differences in its ability to utilisecarbohydrates. Arabinose proved the best energy source, followedby glucose, galactose, and xylose, which were equally effective.Maltose, lactose, sucrose, mannitol, and erythritol were of little orno value in this respect.Subsequently Thorne, Neal, and Walker 21determined changes in respiratory quotient with time for five speciesof Rhixobia, using different sources of nitrogen, and found character-istic species-differences in this case also. Comparing 24- hour cul-tures, the mean quotients for four nitrogen SOiirces for R. Qaponicumand R. leguminosarum were definitely lower for R. rneliloti, R. trifolii,and R. phuseoli in glucose media. In the absence of the sugar thesedifferences disappeared. The mean quotients of the five specieswere similar on nitrate and on ammonia media with glucose, butwere lower when yeast or asparagin was used to supply nitrogen.In the absence of glucose respiration in nitrate and ammonia mediawas largely endogenous and the quotient approached the theoreticalvalue for protein, wix., 0.13.Asparagin and yeast provided somecarbon supplies and the quotients were lower in these cases, especiallywith yeast.The much-discussed dependence of the nodulation of leguminousplants on the carbon : nitrogen balance of the plants themselves hasbeen further examined by P. W. Wilson,22 who traces characteristiceffects of differences in C : N balance on the size and distri'uution ofnodules, the amount and rate of nitrogen fixation, and the influencethereon of external conditions. On the basis of these effects a systemof classification of plants in respect of their carbohydrate : nitrogenratio is developed. An interesting review of the significanceof the carbohydrate supply of the plant in the symbiotic relationshipis given by F.E. Allison 23 who emphasises that, provided soil con-ditions are suitable, the activity of organisms within nodules isprimarily controlled by the availability of carbohydrates, and thatit is only when these become inadequate that the bacteria may makea direct attack on plant tissue. C. E. Georgi,a in examining thewell-known effect of a supply of fixed nitrogen in reducing nodulation,showed that this condition is reflected in a temporary increase in theamount of carbohydrates and a decrease in that of nitrogenousal Arch. Mikrobiol., 1936, 7 , 477.22 Wisconsin Agric. Exp. Sta. Res. Bull., 1935, No. 129, 40 pp.s3 Soil Sci., 1935, 39, 123.24 J . Agric. R e . , 1935, 51, 597POLLARD.413matter in the sap of red clover. The inhibitory action is diminishedby further increasing tho carbohydrate supply, e.g., by increasing thesupply of carbon dioxide to the leaves. Similar conclusions arereached by E. W. Hopkins 25 in the case of soya-bean organisms,the carbohydrate : nitrogen balance in this case being altered byvarying the period of exposure and light, by partial shading, and byalteration of the nitrate supply of the plants. Whatever com-bination of external factors was adopted, the general conclusion isdrawn that accumulation of soluble nitrogen in the plants restrictedand that of carbohydrates favoured nodule formation. F. S.Orcutt and P. W. Wilson,26 also working with soya bean to whichvarying supplies of nitrate were given, record similar results.Stressis laid, however, on the indirect effect of nitrates on nodulation, thecarbohydrate level in the plant sap varying with the nitrate concen-tration in a manner similar to the intensity of nodulation. It isinferred that nodule formation is directly related to the carbohydratesupply and is affected by other conditions only to the extent to whichthese conditions influence the carbohydrate concentration in theplant. There is evidence that a definite rate of nitrate supply to theplant can be associated with the cessation of nitrogen fixation by thebacteria. Below the limiting value, the nitrate concentration in-creases or decreases nodulation according to whether photosynthesisor nitrogen supply becomes the limiting factor in protein formation,i.e., whether there is a surplus of carbohydrate in the sap or a surplusof nitrogen, which is necessarily accompanied by a very low level ofcarbohydrate.H. G. Thornton and H. Nicol27 support this view byshowing that in sand-cultured lucerne the yield and nitrogen contentof lucerne were not affected by varying (within limits) the amountof nitrate supplied. Presumably the limiting factor here was thecarbohydrate produced by photosynthesis, and the nitrogen supplywas derived from nitrate or from nodule organisms to meet require-ments. Above certain concentrations of nitrate in the nutrient thenumber and size of nodules diminished. In other concentrationsnitrates actually prevented the infection of growing roots by noduleorganisms.This was to some extent counteracted by addition ofglucose to the media.28 In the latter paper Thornton recorded astimulation of growth and an increase in the number of root hairs dueto secretions from the nodule bacteria.In many instances stimulation either of nodulation or of growth ornitrogen fixation by free cultures of nodule organisms has beenobserved following the addition of various substances. Thus S.26 Soil Sci., 1935, 39, 297.27 J . Agric. Sci., 1936,26, 173.B8 H. G. Thornton, Proc. Roy. SOC., 1936, B, 119, 474.26 lbid., p. 289414 BIOCHEMISTRY.Winogradsky 29 found the addition of simple nitrogen compounds,e.g., ammonia, amines, amides, t o otherwise nitrogen-free culturesimproved the development and sugar consumption but caused nomarked increase in the amount of nitrogen fixed.Addition of moresubstances such as extracts of yeast or of plant organs further in-creased development and initiated a normal rate of fixation of nitro-gen. Similar results were obtained by A. Itano and A. M a t s ~ u r a , ~ ~the activity of extracts being in the order seedlings > germinated> ungerminated seed, and among corresponding extracts of differentspecies in the order, nodulc-bearing legumes > non-legumes > non-nodule-bearing legumes. In a later paper 31 the same authors foundthe actual principle from bean nodules to pass into the cathodechamber on electrodialysis. In this respect it differed from yeastextract, in which the accessory substance showed no tendency tomigrate.No relation was apparent between the activity of acoessorysubstances and their nitrogen contents. Their action was that of atrue growth-promoting substance rather than that of a nutrient.P. E. Allison and S. R. Hoover 32 attribute the increased growth ofRhixobia by natural, but not by synthetic, huniic acid to the presenceof co-enzyme R. This is the reverse of the view previously expressedby D. W. Thorne and R. H. Walker,33 who ascribed the stimulatoryeffects of plant and yeast extracts to nutrient matter present anddiscredited the intervention of any co-enzyme in the activity of theorganisms. A somewhat different view of this question was pre-sented by C. A. Ludwig and F. E. Allis0n,3~ who observed increasednodulation of soya bean and lucerne when grown in sand cultures inthe presence of other plants, e.q., wheat or maize.Additions of sugaror small amounts of nitrogen compounds sometimes producedsimilar effects under these conditions, but extracts of the sand inwhich the plants had been growing were inactive. The excretionof stirnulatory agents by the plant roots seems therefore excludedfrom consideration. The authors suggest that the presence of otherplant roots induces in the rhizosphere conditions conducive to thedevelopment of the bacteria, possibly including the formation of abacterial growth-promoting substance. Thorne, Neal, and Walker(Zoc. cit.) support the view that yeast extracts exert a stimulatoryaction on the growth and respiration of Rhizobia, which can bedifferentiated from that attributable to the carbonaceous and nitro-genous nutrients which they contain.The action of yeast is, how-29 Ann. Tmt. Pasteur, 1936, 56, 221.3O Ber. O h r a Irzst. larzdw. Forsch., 1936, '7, 185.31 J . Ayric. C h e m SOC. Japan, 1936, 12, 457.33 Proc. Iowa Acccd. Sci., 1934, 41, 63.34 J. Arner. SOC. Agronomy, 1935, 27, 895.'2 Soil Sci., 1936, 41, 333POLLARD. 415ever, ascribed to its ability to act as an effective hydrogen-donatorto the organisms. This is in agreement with the observation ofW. P. Allyn and I. L. Baldwin35 that yeast, unlike potassiumnitrate, when used as a nitrogen source for Rhixobia cultures, tendsto maintain in the media an oxidation-reduction potential which isvery favourable to the growth of the organisms.In a recent paper 36Thorne and Walker record that reducing agents, e.g., cysteine andthioglycollic acid, increased the growth and oxygen consumption ofRhixobia.The nitrogen exchange of nodule organisms, especially in relationto the mechanism of the fixation process, and to the observed excre-tion of nitrogenous substances, has formed the subject of manyinvestigations. According to A. I. Virtanen and M. Tornianen 37the nodular proteins yield tryptophan, arginine, tyrosine, asparticacid, and some diamino-acids. In culture media, following thegrowth of the inoculated legumes there are prescnt aspartic acid,lysine, and smaller amounts of simpler compounds, e.g., nitrite,nitrate, hydroxylamine or ammonia.Excretion of these compoundsis dependent on appropriate supplies of air to the root^,^^,^^ andceases when these are immersed in stagnant liquid media. Thevitality of the organisms is not destroyed, however, since on re-aera-tion nitrogen fixation and excretion of fixed nitrogen continue.Virtanen concludes that the aspartic acid and probably the lysineexcreted are not derived from nodular protein (the proteolyticactivity of the organisms is apparently small 40), but represent prim-ary products of nitrogen fixation.41* 42 It is suggested that asparticacid may be formed from hydroxylainine and oxalacetic acid.43S. Winogradsky (Eoc. cit.) observed the elimination of ammoniafrom cultures to occur only during fixation of nitrogen.G.Bondt4 in an examination of the nitrogen exchange in soyabean, established that a very considerable proportion of the totalnitrogen fixed by nodule organism (in some cases probably SO--SO%)diffuses into the cytoplasm of the host plant and is translocatedinto the plant system. In a discussion of the fixation process theauthor considers this to resemble a type of respiratory activityrather than a stage in the synthesis of bacterial protein.3 5 J . Bact., 1932, 23, 369.37 Sumen Kern., 1936,9, B, 13.38 A. I. Virtanen, J. Agric. Sci., 1935, 25, 278, 290.39 A. I. Virtanen and S. von Hausen, ibid., 1936, 26, 281.40 A. I. Vistanen and T. Laine, Biochem. J . , 1930, 30, 377.4 1 Idem, Suomen Kern., 1936, 9. B, 12.42 A. I. Virtanen and M. Tornianen, Zoc.cit., ref. (37).43 A. I. Virtanen and T. Laine, Suomen Kern., 1936, 9, B, 5.44 Ann. Bot., 1936, 50, 659.36 Soil Sci., 1936, 42, 231416 BIOCHEMISTRY.Photosynthesis in Plants.InflzLence of External Factors.-The complex problem of the effectsof the quality and intensity of light, of the carbon dioxide concen-tration of the surrounding atmosphere, and of temperature on therate of carbon assimilation of plants forms the subject of a greatnumber of publications of the last few years. Much of this workfollows along lines which are not altogether new in general principlebut either develop an improved technique, expand the detail ofexperimental data, or, in the light of advancing knowledge, leadto new interpretations of already accepted facts.The applicationof the theory of limiting factors either as put forward by Blackmanas a development of the “ law of minimum,” or with the modification(introduced by Harder) of the idea of relative minimum, servedfor a number of years as a working basis for much experimentalwork. More recent research tends to explore the limits of applicabil-ity of these theories. No attempt can be made in the space of thisReport to give a comprehensive review of this field of enquiry, butsome indication of the general trend of recent work seems desirable,especially in view of its ultimate bearing on the more purely chemicalconsideration of the mechanism of the photosynthetic process.W. H. Hoover, E. S. Johnston, and F. S. Brackett 45 record dataagreeing within limits with Blackman’s law, and show that in wheatplants carbon assimilation exhibits a straight-line relationship withcarbon dioxide concentration in an excess of light, and with lightintensity in the presence of an excess of carbon dioxide.Theyindicate, however, a range of conditions between zones in which eachlimiting factor becomes dominant and conclude that this intermedi-ate range is wider for higher plants than for algz. 13. N. Singh andK. N. La1,46 using wheat, linseed, and sugar cane plants, compare theresults of much experimental work with the theories of Blackmanand of Harder. The form of carbon dioxide concentration-assimil-ation curves obtained shows no sharp change of direction at a criticalconcentration such as would be anticipated if assinilation werecontrolled entirely by the level of supply of the factor in minimum(Blackman).The gradual and regular change of direction ismore in accord with the view that factors other than that inminimum influence to a definite though relatively smaller extentthe rate of carbon assimilation (Harder). The principal observationsemerging are that under conditions of low carbon dioxide concen-tration and low light intensity the rate of assimilation is controlledby carbon dioxide. With high light intensity, however, irrespective45 8mithsonian Misc. CoEI., 1933, 87, No. 16.46 Plant Physwl., 1935, 10, 245; Proc. Indian Acad. Sci., 1935, 1, B, 909,754POLLARD. 417of the level of carbon dioxide present, light controls assimilation.The influence of temperature on assimilation rates is also examined 47and relationships are found to assume the same general nature.The temperature range over which assimilation takes place in radishleaves is recorded as 12.647.4", with maximum values at 30".It isconcluded that under no conditions is assimilation determined by anyone factor alone, but that, on the other hand, the theory of " relativeminimum " is only of limited application. It is held that withoutconsideration of the internal cellular mechanism of photosynthesis,no relationship between environmental factors can be applied as ageneral principle under all conditions.The extent to which light of different wave-lengths can be utilisedby plants varies somewhat with the species examined. Differencesin the colour and thickness of leaves are partly concerned here(see further, under nutritional factors).G. R. Burns 48 shows thatwhite pine and spruce utilise all the visible spectrum except theviolet and part of the blue. Other plants, however, utilise the blue-violet range, and with this as sole illumination, assimilation is pro-portional to its intensity.4s In general, however, rates of photo-synthesis are highest in the red-orange region and decrease steadilytoward the 51 As is to be expected, the percentage utilis-ation of light by different plants shows considerable variation. Aninstance of this is shown by recent work of Gabrielsen (Zoc. cit.), thealga ChZoreZZa exhibiting a notably greater efficiency in this respectthan mustard.The influence of the quality, as distinct from in-tensity, of light on the assimilation process has obvious practicalbearings on greenhouse practice, and is frequently of prime import-ance in research work under conditions in which light of constantintensity must be obtained from artificial sources. In this connexionR. H. Dastur and K. M. Samant 62 have examined the relative ratesof carbohydrate formation in leaves exposed to different sourcesof light. Their investigations suggest that in addition to the actualphotosynthetic process the subsequent elaboration of carbohydratesmay also be affected. Thus the amount of starch produced in arti-ficial light was approximately 30% of that produced in daylight,whereas sucrose production was similar with both light sources.In diffused daylight the total carbohydrate production was doublethat obtained in artificiallight.In plants producing no starch, e.g.,4 7 B. N. Singh and K. Kumar, Proc. Indian Acad. Sci., 1935, 1, B, 736.48 Plartt Physiol., 1933, 8, 247; 1934, 9, 645.4s R. H. Dastur end R. J. Mehta, Ann. Bot., 1935, 49, 809.50 LOC. cit.5 1 E. K. Gabrielsen, Planta, 1935, 23, 474.52 Ann. Bot., 1933, 47, 295.REP.-\'OL. XXXIII. 418 BIOCHEMISTRY.AlZium cepu, sugar production in diffused daylight was approximatelythree times that in artificial light. In these varied effects of differentlight sources, quality, rather than intensity, appears to be the domin-ant factor. On somewhat similar lines J. M.Arthur and W. D.Stewart 65 compared the efficiency of various artificial light sourceson the basis of dry matter production in buckwheat plants. It isnoted that the order of efficiency thus obtained differs from thatshown by calculations on a basis of equal energy radiated within thevisible spectrum. The efficiency of dry matter production in theplants did not appear to depend upon any relationship between theemission bands of the lamps and the absorption bands of chlorophyllpigments. Gaseous-discharge lamps (sodium, mercury, neon) bycomparison with ordinary filament bulbs produced greener leaves andplants having lower stem : leaf ratios.The ill-effect of ultra-violet light on the photosynthetic processunder certain conditions is examined by W.Arnold 54 in the case ofChlOreZh pyrenoidma, and is ascribed to its action in rendering in-active an unidentified unit in the mechanism of photosynthesis.Neither the chlorophyll nor the respiratory process is affected.0. Jirovec,55 as a result of experiments with green and colourlessstrains of Euglena grmilis, concludes that chlorophyll normallyaffords partial protection against ultra-violet rays.Plane-polarised light appears to cause no abnormality in the carbo-hydrate content of leaves.56The influence of external factors on the relative ratio of carbonassimilation and of respiration becomes a matter of considerableimportance in the investigation of photosynthesis. Not only do thetwo processes take place simultaneously with the same end-products,but also the " compensation point " in a closed system, Le., thecondition in which respired carbon dioxide is quantitatively utilisedin photosynthesis, is often regarded as an index value in studies ofthe influence of light or temperature on carbon assimilation.F. vander Paauw 67 showed that temperature produced parallel effects onrespiration and assimilation in the green alga Stichococcus bacillarisand also, a t temperatures less than 22", in Oocystis. At highertemperatures in the latter and at lower temperatures in Chlamy-domoru;cs respiration responded more than assimilation to changes oftemperature, With many other plants there was very close parallel-ism between rates of assimilation and respiration over the range63 Contr.Boyce l'hompon Inst., 1935, 7, 119.64 J . Ben. Phy8wl., 1933, 17, 135.55 Protophrna, 1934, 21, 617.66 R. H. Dastur and R. D. Asans, Ann. Bot., 1932, 46, 879.67 Plan@, 1934, 22, 396POLLARD. 41910-30*. Moreover mild stimulation or retardation of both processescould be effected by appropriate treatment with potassium cyanide.68Somewhat similar results were obtained by E. S. Miller and G. 0.Burr,59 who determined the compensation point for a number ofplants, using a special apparatus in which the upper portions of theplants were subjected to light of high intensity but the roots werekept relatively cool. Plants of various species quickly reduced theconcentration of carbon dioxide in the circulating air to a levelof O-Ol% (vol.), which was maintained for periods of 24 hours.Thisvalue did not change with temperature in the range 5-35". Sincethe respiration rate is known to increase by as much as 15 timesover this range, and under the conditions of the experiment light wasalways in excess, it is concluded that the limiting factor in assimil-ation is not the primary process of light absorption but the intermedi-ate reaction which is controlled by the carbon dioxide concentration ;also that the temperature coefficient of this reaction is identical withthat of respiration. The utilisation of the increased products ofrespiration without increase in carbon dioxide concentration isascribed to the assimilation of an intermediate product of respirationbefore any carbon dioxide is liberated from it, a possibility whichwas suggested previously by Warburg. At 35-37' the above rela-tions appear to break down rapidly and the gas exchange of differentspecies shows wide variations.The dependence of the rate of photosynthesis on the water contentof the leaf is examined by a number of workers.60.62 I n general amore or less direct relationship is established up to a critical watercontent, beyond which assimilation tends to decline. Dastur(h. cit.) determines the " assimilation number " of various plants(ie., H,O content/C02 assimilated) and finds this to increase withrising water content to an optimum value and subsequently todecrease. Comparison with Willstiitter's " assimilation number "(i.e., chlorophyll content-CO, assimilated) shows a much closerrelationship of assimilation with the water than with the chlorophyllcontent.The lack of proportionality between chlorophyll content of leavesand assimilation observed in many cases by Willstatter and by sub-sequent workers does not apparently obtain in the case of ChZoreZh,in which a direct ratio is established by W.E. Fleischer,63 even whenthe chlorophyll content is artificially varied by controlling the supply58 F. van der Paauw, Rec. Trav. bob. nterl., 1932, 29, 497.5s Plant Phpiol., 1935, 10, 93.60 R. Melville, Ann. Re@. Exp. Stcb. Cheshunt, 1933, 87.61 B. N. Singh and K. N. Lal, Ann. Bot., 1935,49, 291.62 R. H. Dastur and B. L. Desai, ibid., 1933, 47, 69.63 J . Gen. Physwl., 1935,18, 573420 BIOCHEMISTRY.of iron. R.Emerson,64 with somewhat different experimental con-ditions, had made similar observations, and further recorded thatlight conditions giving optimum assimilation rates in leaves rich inchlorophyll were also optimum for those poor in chlorophyll.Possibly this case resembles that of Elodea densa, examined by E. vonEuler, B. Bergman, and H. H e l l ~ t r o r n , ~ ~ in which the chlorophyllcontent per chloroplast and the number of chloroplasts per cell weresubstantial 1 y constant.Mechanism of Photosynthesis.-The complexity of the effects ofenvironmental conditions and of internal plant factors on carbonassimilation observed directly by plant physiologists has beenparalleled by the more purely chemical investigations of thecomplexity of chlorophyll.Recent years have brought a much more complete understandingof the chemistry of chlorophyll 66 and related compounds, but themeans by which chlorophyll brings about the conversion of carbondioxide into carbohydrate is still the subject of controversy.It isvery generally accepted that formaldehyde represents an intermedi-ate stage in the conversion and that the change CO, + H,O -+CH,O + 0, is effected (probably in the absence of light) with the aidof light energy previously absorbed by the chlorophyll. Theories ofthe mechanism of photosynthesis differ considerably in detail, butare centred round considerations of whether the fundamental energyexchange involves carbon dioxide, water, oxygen, or the chlorophyllitself.Warburg originally supposed that the chlorophyll (andcarotene) in leaves was transformed into an isomeric substance duringexposure to light and in the subsequent (dark) reaction the isomer,a reducing agent, caused the transformation of carbonic acid intoformaldehyde and water. By contrast among the earlier theories,that of Thuiiberg supposed the absorbed light energy to act on thewater associated with chlorophyll rather than on carbon dioxide, thechain of reactions being :2H,O + chlorophyll + light --+ H, + H,02co, + H, + 5 2 0 2 -4- 0, + H4CO2(1 methyleneglycol)H,CO, + CH,O + H,OAs will be shown, these and other early theories of assimilation havereappeared in recent years, modified or extended to bring them intoaccord with newly observed facts.E.C. C. B a l ~ , ~ ' after prolonged investigation of relevant catalytic6* J . Gen. Physiol., 1929, 12, 609; Proc. Nat. Acad. Sci., 1929, 15, 281.e5 Ber. deut. bot. Ges., 1934, 52, 458.G6 Ann. Reports, 1935, 362.67 Proc. Roy. SOC., 1936, B, 117, 218POLLARD. 42 1actions, has now formulated the primary photosynthetic reactionas depending in the absorption of carbon dioxide by chlorophyll-a,the photosensitised complex changing to chlorophyll-b and formalde-hyde. The cycle is completed by reduction in the dark (Blackmanreaction) of chlorophyll-b to chlorophyll-a, the reducing agentsuggested being carotene. Thus :C,,H720,N4Mg-C0,,H20 light C55H7006N4Mg,H20 + CH20chlorophyll-a-CO, complex. chlorophyll-b.C,5H7006N4Mg~H20 + c40135G C55H7205N4Mg + C40HijG02ch loroph y ll- b.carotene. chlorophyll -a. xanthophyll.The kinetics of these reactions are shown to be in accord with pub-lished experimental data relating to the carbon assimilation ofChlorella,By mathematical consideration of the effects of temperature andlight intensity on carbon assimilation G. E. Briggs 68 indicates asystem involving the formation of a chlorophyll-CO, complex whichin light undergoes molecular rearrangement to a peroxidised sub-stance. This is decomposed through the agency of plant catalysts,yielding carbohydrate and oxygen, or, in part, decomposes aninhibitor (present in the cell) which inactivates the catalyst. Theclassic researches of Willstatter and StoH had previously led tosimilar views.More recently there has appeared some divergencebetween the theories of Willstatter and of Stoll, the differencescentring on the position of oxygen as a necessary agent in the firststages of the assimilatory process. A. Stoll 69 assumes that chloro-phyll-a and -b contain a ‘‘ supernumerary ” double linking outsidethe conjugated system, which permits catalytic hydrogenation with-out significant change in the absorption spectrum.70 The dihydro-derivative is easily dehydrogenated and serves as reducing agent forthe chlorophyll-CO, complex , yielding formaldehyde. Rehydro-genation of the chlorophyll results from the fission of water closelyassociated with it,H2O + H + OH (-+ HZOz),the hydrogen peroxide being subsequently decomposed by the cata-lase of the leaf.I n a later publication 71 Stoll brings his scheme moreclosely into line with recent developments in the elucidation of thestructure of chlorophyll. The catalytic hydrogenation of chloro-phyll is now known to yield a dihydro-derivative by addition at the68 Proc. Roy. SOC., 1933, B, 113, 1.69 Nnturwiss., 1932, 20, 965.70 LOC. tit. ; also R. Kuhn and A. Winterstein, Ber. deut. bot. Ges., 1932, 65,71 Naturwias., 1936, 24, 53.1737422 BIOCHEMISTRY.double bond of the vinyl group, a position in which mobility or theeasy interaction with carbon dioxide co-ordinatively attached to thecentral magnesium atom seems unlikely, Accepting Fischer’sformula for chlorophyll (I), Stoll now associates photoactivity withthe mobility of the hydrogen atom in position 10 (Fischer havingnow introduced the two ‘i extra” hydrogen atoms into thestructure).An enolisationis indicated as related to the brown phase in the chlorophyll cycle.The hydrogen attached to the carbon C,, in (11) may be replaced byhydroxyl and in this substance the hydrogen on C,, becomes mobilein daylight (but not in darkness) provided atmospheric oxygen isexcluded.Stoll, therefore, although reconstructing his earliermechanism, still retains his primary conception that oxygen is not animmediately active agent in photosynthesis, which is more accur-ately represented as involving a photolysis of water as a result of theprincipal energy exchange.On somewhat similar lines K. Shibata and E. Yakushiji T2 assumethe co-ordination of four water molecules with the central magnesiumatom of the chlorophyll.These become activated by the absorbedlight in such a manner that the reaction,is facilitated. Here again oxygen is not immediately concerned andthe associated water molecules are the vehicle of the energy exchange,72 hTaturwk., 1933, 21, 267.HZCO, + 4(H . . . OH) = 2H20 + CH,O + 40H( = 2H,O,POLLARD. 423four quanta being involved. The Blackman (dark) reaction isrepresented by the decomposition of hydrogen peroxide by catslase,this being the sole source of oxygen in the system.H. Gaffron 73 supports the conception that the assimilation processdoes not necessitate the intervention of an activated or metastableoxygen atom.By contrast to the above Willstatter’s later work 74 leads him toassume the necessity of a t least a small amount of oxygen to initiatethe activity of chlorophyll. He also differs from Stoll in supposingthe photosynthetic cycle to involve the formation of dehydrogenatedchlorophyll derivatives.The formation of actively reducing hydro-gen atoms is represented schematically by an initial reaction of chloro-phyll with oxygen, yielding monodehydrochlorophyll :chlorophyll + 0, ---+ (0,H) -t monodehydrochlorophyll.The wandering of a hydrogen atom into the Mg-CO, complex yieldsdehydrochlorophyll. Then follows,dehydrochlorophyll + H,O -> OH -+ monodehydrochloropl~ylland againmonodehydrochlorophyll _I, dehydrochlorophyll + H,the H going to the Mg-CO, complex. This cycle is completed fourtimes, yielding the necessary four hydrogen atoms for the reductionof one molecule of carbon dioxide.The process is assumed toproceed in stepwise manner, each hydrogen atom reacting singly.Regeneration of chlorophyll in light is effected thus,dehydrochlorophyll + H,O --j OH + monodehydrochlorophyllmonodehydrochlorophyll + H,O + OH + chlorophyll.The radicals 0,H and OH formed intermedially either take part in afurther hydrogenation cycle or yield H,O, 0, and H,O, accordingto the environmental conditions which regulate the intensity of thevarious stages of the reaction.J. Franck 75 supports WillstBtter’s view of the necessity of oxygenfor the process, but considers that photochemical reactions in solu-tion are more likely to occur by means of reactions of activatedmolecules than through the formation of radicals 76 as indicated byWillstSCtter.With this in view and with the purpose of adjustingthe cycle of changes to accord more satisfactorily with calculatedenergy relationships, Franck modifies Willstgtter’s system by assum-ing a somewhat different series of intermediates, and, adopting73 Biochem. Z., 1935, 280, 337; 1936, 287, 130.74 R. Willstiitter, NatWr~i88., 1933, 21, 262.7 5 Naturwiss., 1935, 23, 226.719 J. Franck and E. Rabinowitsch, Trans. Paraday Soc., 1934, 153, No. 30424 3IOCHEMISTRY.Fischer’s views, includes in the chlorophyll molecule the two looselybound hydrogen atoms. To make the comparison with Willstiitter’sscheme more evident, he designates ordinary chlorophyll as HH-chlorophyll, monodehydrochlorophyll as H-chlorophyll, dehydro-chlorophyll as “ chlorophyll,” and dehydrochlorophyll, t o which ahydroxyl group is loosely attached, as OH-chlorophyll.The pre-liminary (light) reaction involves dissociation of one of the twoloosely bound hydrogen atoms,HH-chlorophyll + hv --+ H-chlorophyll + Hthe hydrogen atom being taken up in a series of changes with waterand oxygen (as in Willstatter’s scheme) with the ultimate formationof hydrogen peroxide, and H-chlorophyll combining with carbonicacid. The actual assimilation process is represented as :OH (i) H-Chlorophyll . . . OH>C=O + hv --+OH-chlorophyll . . . og>CxO(ii) OH-chlorophyll . . . ‘g>C=O + H,O + hv -+H-chlorophyll . .. O:>C=O + H20,(iii) H-chlorophyll . . . O:>C=O + hv -+H OH-chlorophyll + H>C-O(iv) OH-chlorophyll + H,O + hv ---+ H-chlorophyll + H,O,By assuming differences in the energy of combination of hydrogenand of hydroxyl in themselves, and in relation to position in theco-ordinated magnesium complex or the chlorophyll residue,Franck brings the above cycle of reactions into line with the acceptedenergy balance of the complete assimilation process.Evidence of a vital part played by oxygen in the photosyntheticprocess is put forward by H. Kautsky and colleagues and is based onphenomena of an entirely different character. Following an exten-sive study of photosensitised surface reactions, including those con-cerned in fluorescent conditions, Kautsky concluded that in manysurface oxidations the presence of an activated or metastable oxygenatom is essential to effect the energy transfer.A green leaf whichhas been placed in the dark for a period and afterwards exposed toultra-violet light exhibits a temporarily increased fluorescence,followed by a steady decline to a low level which is substantiallyconstant in unchanged external conditions. The process, lasting a POLLARD. 425most a few minutes, may be repeated indefinitely by alternate ex-posure of the leaf to darkness and to light.In examining these changes H. Kautsky, A. Hirsch, and F.Davidshofer 77 associate the fluorescence with changing intensitiesof different stages of the photosynthetic process. The photosensit-ised chlorophyll is able to transfer it!s energy only to a molecule ofdefinite type.Of those present in the leaf system, only oxygenfulfils this requirement, and the presence of activated or metastableoxygen in the leaf plastids is assumed.78 The transference of energyto oxygen lowers the intensity of fluorescence. The energised oxygeneffects the building up of the chlorophyll-carbon dioxide-peroxidecomplex,79 resulting ultimately in the production of carbohydrateand oxygen. The increase in the oxygen supply brought about inthis way results in a still further decrease in fluorescent intensity(declining portion of curve) until a balanced condition is reachedbetween light absorption, fluorescence, and oxygen transfer, which ischaracteristic of the normal condition of assimilation.The rate ofinitial increase in fluorescence under these conditions is controlled bythe intensity of irradiation, but is unaffected by temperature.80 Thesecond stage of the assimilation process (reactions involving thechlorophyll-CO, complex and the final production of carbohydrate)is definitely restricted by decreased temperature and also by treat-ment with hydrocyanic acid or toluene, and such restriction,by lowering the supply of photosynthetically derived oxygen, causesa prolongation or an increased intensity of the fluorescence.81 Anartificially increased concentration of carbon dioxide in the atmo-sphere (to 1%) has no influence on the course of fluorescence, butvariations in the oxygen concentration cause corresponding changesin intensity, A critical point is apparently reached with 0.5% ofoxygen, below which fluorescence does not increase with the intensityof irradiation.82 I n an atmosphere free from oxygen leaves fluorescewith high and constant intensity until liberation of oxygen causes adiminution to the normal equilibrium In a later and moredetailed examination of fluorescence curves Kautsky 84 traces thecourse of activation of the chloroph-yll-0, complex and finds it tobe of a unimolecular type.I n the light of this and previous observ-77 Ber., 1932, 65, 1762.7 8 H. Kautsky, H. de Bruijn, R. Neuwirth, and W. Baumeister, Ber., 1933,79 H. Kautsky and A. Hirsch, Naturuliss., 1931, 19, 964.80 H. Kautsky and H.Spohn, Biochem. Z . , 1934, 274, 435.8 1 H. Kautsky and A. Hirsch, ibid., 1935, 277, 260.82 H. Kautsky and W. Flesch, ibid., 1936, 284, 412.83 H. Kautsky and A. Hirsch, ibid., 1935, 278, 373.84 H. Kautsky and A. Marx, Naturwiss., 1936, 24, 317.66, 1588426 BIOCHEMISTRY.ations he considers that in the darkened leaf the equilibrium, chloro-phyll + 0, =+= chlorophyll-0, complex, [ChIO,, in the plastids isnormally balanced almost completely to the right, The complex isdissociable but non-fluorescent. On irradiation [ChIO, undergoesrearrangement to form a new non-dissociable but fluorescent complex[ChO,], probably a peroxidised form. This unimolecular changecorresponds with the initial increase in the fluorescence curve.J. Franck 86 doubts the physical soundness of Kautsky's views of theproperties of a metastable oxygen molecule, and considers the fluores-cent changes are more in agreement with the existence of mobilehydrogen atoms bound to the chlorophyll molecule.The risingpart of Kautsky's fluorescence curves may well be represented by thechangeHH- chlorophyll -+ H- chlorophyll.II. Gaffron a6 also criticises Kautsky's interpretation of the experi-mental data, and advances evidence, based on the carbon assimil-ation of Chlorella, in support of his view that neither free nor looselybound oxygen is necessary for the initiation of the assimilationprocess.recently reports the isolation of two crystallinefluorescentl substances from leaves, both of which actively absorbultra-violet light, especially in the shorter wave-lengths.Bothresist saponification, but gradually lose their characteristic propertieson exposure to air. The fluorescent spectra indicate the absence ofester, carboxyl or hydroxyl groups. The fact that one or both of thesesubstances occurred in leaves of all plants examined suggests thatthey may have an interesting bearing on Kautsky's observations.From considerations of the photobleaching of fluorescent dyes inan oxygen-free atmosphere by the action of ferrous salts J. Weiss 88suggests the reactionFe" + HOH + hv = Fe"' + OH' + Hmay explain the vital change taking place in the case of chlorophyllas with the photosensitised dyes, and recalls the observation of K.Noack a9 that chloroplasts in leaves contain appreciable proportionsof ferrous iron.An interesting discussion of the energy relationships in the varionstheories of the photosynthetic mechanism is given by H.Gaffron andH. H. StrainK. w0hi.908 5 O p . cit.; also J . Franck and H. Levi, Naturwiss., 1935, 23, 229.8 6 Biochem. Z . , 1933, 264, 270; Naturwiss., 1935, 23, 528.8 7 Nature, 1936, 137, 946.88 Ibid., 1936, 136, 794.89 2. Bot., 1930, 23, 957. Naturwiss., 1936, 24, 18, 103POLLARD. 427Condition of Chhophyll in the Plant.It has long been assumed that in the leaf chlorophyll exists in acolloidal condition. The fact that chlorophyll cannot be extractedfrom dried leaves by certain organic solvents until water has beenadded, has suggested that in the chloroplast chlorophyll occurs insome form of labile combination or elaborate physical state €romwhich it is released or dissociated by means of water.Moreover it has been shown91*92*93 that in the assimilationprocess some 1500-2000 molecules of chlorophyll (Emerson andArnold’s “ photosynthetic unit ”) must be present to effect the trans-fer of the four light quanta (necessary for reduction) to one moleculeof carbon dioxide.This contributes to the view that some diEerenti-ation between the chlorophyll molecules is likely in respect of theirphysical or chemical condition.Recent observations, among which may be cited those of W.M e n ~ k e , ~ ~ L. G . M. Baas-Becking and H. C. K ~ n i n g , ~ ~ and J. G.Wakkie,96 lend further support to the conception that the physicalcondition of chlorophyll in leaves is a somewhat complex one.Acolloidal state seems unlikely and the absorption and fluorescencespectra indicate that chlorophyll cannot be present in simple solution.Mencke, suggests that two phases are present: one, a lipoid phasein which the chlorophyll is dissolved in the lipoid constituents of theplastid, and an aqueous phase in which the lipoid solution is dis-persed. B. Hubert 97 indicates that if chlorophyll is in solution itmust be in a medium of very high refractive index, and concludesthat a condition of adsorption is probable.Discussing Mencke’s views, J. Weiss 98 points out that, if a lipoidsolution of chlorophyll is dispersed in an aqueous phase in the chloro-plast, a considerable portion of the absorbed light may be stored bymolecules of chlorophyll in the interior of the lipoid phase as a formof electronic excitation energy, and may be passed from moleculeto molecule by a “ resonance ” effect.Only those molecules a t thelipoid-aqueous interface react with carbon dioxide, but in time thewhole of the energy stored by the “ internal ” molecules may reachthe “ surface” molecules and bring about the formation of thechlorophyll-C0, complex. I n weakly assimilating leaves, energyreaching the surface may not be entirely utilised and will appear as91 R. Emerson and W. A. Arnold, J . Gen. Physiol., 1932, 16, 191.92 W. A. Arnold and H. I. Kohn, ibid., 1934, 18, 109.99 H. 1. Kohn, Nature, 1936, 13’7, 706.94 Protoplatma, 1934, 21, 279.95 Ibid., 1935, 38, 1082.97 Ibid., 1934, 37, 694.Proc.K . Akad. Wetenach. Ameterdam, 1934, 37, 674.Nature, 1936, 137, 997428 BIOCHEMISTRY.fluorescence. This accords with Kautsky’s observation that fluores-cence in some cases bears an inverse relation to the rate of assimil-ation. The “ photosynthetic unit ” may also be regarded as directlyrelated t o the ratio of ‘‘ surface ” : “ internal ” molecules, in whichcase some 400-500 molecules of chlorophyll are present in the in-terior of the lipoid phase for each actively assimilating “ surface ’’molecule.The possibility of labile compounds of chlorophyll and protein isexamined by R. S. Hilpert and K. H e i d r i ~ h , ~ ~ who show that a de-finite portion of “ mobile ” protein, different from the general proteinof the leaf, can be associated with chlorophyll in all organs of theplant and in all stages.A.Stoll discuss a further development of the chlorophyll-proteincomplex theory. I n the light of Willstatter’s explanation of theactivity of lactoflavin, wiz., the formation of a “ Symplex,” lacto-flavin-phosphoric acid-colloid carrier, Stoll suggests the presence inthe plastids of a symplex (“ chloroplastin ”), chlorophyll-colloidcarrier [ ? protein]. The symplex may be assumed to dissociate inthe presence of water containing dissolved electrolytes from the leaf.The insolubility of chlorophyll from dried leaves in certain organicsolvents is thereby explained. The point of attachment of the col-loid is possibly the double bond of the vinyl side chain.On thisbasis the actual absorption of light and its transformation intopotential chemical energy is associated with the chlorophyll moleculeand is independent of temperature. The subsequent production offormaldehyde (temperature sensitive) may be regarded as anenzymic reaction since the symplex has an enzyme-like structure.The chlorophyll-colloid (protein) combination may thus be regardedas a specific assimilating enzyme.Chlorophyll Formation in Plants and Nutritional Factors affecting it.Nitrogen.-In addition to the environmental influences alreadydiscussed, genetic,2 nutritional and physiological factors areconcerned in the production of chlorophyll in plants. Recent workhas in many cases thrown further light on the manner in which theseindirect and often less obvious factors affect the photosyntheticactivity of plants.An adequate supply of nitrogen to plants is obviously necessary99 Ber., 1934, 67, 1077.1 Naturwiss., 1936, 24, 53.2 H.von Euler et al., Svensk Kem. Tidskr., 1934, 46, 301; 2. physiol.Cheqn., 1935, 233, 81 ; 234, 151.3 W. Mevius, Jahrb. wiss. Bot., 1935, 81, 327.4 B. N. Singh and K. N. Lal, Ann. Bot., 1935,49, 291; W. E. Loomis andK. €3. Burnett, Proc. Iowa Acad. Sci., 1931, 38, 150POLLARD. 429for the actual elaboration of the chlorophyll molecule. It is alsogenerally recognised that conditions favouring rapid vegetativegrowth are in general those which favour chlorophyll production. Agenerous nitrogen supply is a prominent factor in this.It mighttherefore be anticipated that a deficient nitrogen supply couldoperate as a limiting factor in chlorophyll production on much thesame lines as it does in gross dry-matter formation in the growingplant. A relationship of this kind is indicated by the work of R. K.Tamm and 0. C. Magi~tad.~ In pineapple leaves the chlorophyllcontent tended to increase uniformly with the amount of nitrogenousfertiliser applied, up to a limiting amount. Very large applicationsresulted in a decrease in chlorophyll production. F. M. Schertz,Gexamining chlorotic mottling in leaves, found this could be correctedby treatment with sodium nitrate. Moreover the customary cor-rectives, iron and manganese, for chlorosis were ineffective if thenitrogen supply was inadequate.It was later shown that the levelof nitrogen supply could be correlated directly with pigment form-ation in the chloroplast. J. D. Guthrie ' also records that nitrogendeficiency had little influence on the chlorophyll content of plants inwinter (when factors other than nitrogen limit growth), but duringrapid spring growth chlorophyll production was restricted by apartial deficiency of nitrogen. The form in which nitrogen is sup-plied to the plant affects the .nitrogen-chlorophyll as well as thenitrogen-growth relationship. G. B. Ulvin,* working with sugar-cane, found that nitrate-fed plants produced more chlorophyll thandid those supplied with ammonium salts.The observation by G. Gassner and G. Goeze of a direct relation-ship between the protein and the chlorophyll content of cereal plantsseems to add further confirmation of the significance of nitrogennutrition in the formation of chlorophyll.Potassium.-The close relation between potassium and theassimilatory process in plants is very generally recognised.It isusually considered that potassium acts in this respect by regulatingenzyme activity rather than through any direct influence on thechlorophyll itself. Such an influence is, moreover, difficult to estab-lish experimentally owing to the varied ways in which potassiuminfluences the functional activities of the plant.LundegBrdh 10 observed that at moderately high temperatures(20-30") assimilation in potassium-deficient leaves was greater than6 Plant Physiol., 1935, 10, 159.6 Bot.Gaz., 1921, '91, 81; Plant Physiol., 1929, 4, 269.7 Arner. J . Bot., 1929, 16, 716.8 Plant Physiol., 1934, 9, 59.9 Ber. deut. bot. Ges., 1934, 52, 321.1" " Die Nahrstoff aufnahme der Pflanzen," 1932430 BIOCHEMISTRY.in those adequately supplied and that this difference was related tothe higher chlorophyll content of the deficient leaves. I n olderleaves from which much of the normal potassium content had beeneliminated, assimilation was restricted. Gassner and Goeze l1associate (( moderate ” potassium deficiency with a maximumchlorophyll content in wheat leaves, and simultaneously with maxi-mum assimilation and respiration. Amore severe deficiency of potass-ium has a definite inhibitory effect on all factors. It seems possible,therefore, that the potassium supply may influence the amount ofchlorophyll formed as well as its activity, although the apparentlyreciprocal effects of potassium and nitrogen on chlorophyll pro-duction12 tend to introduce an element of doubt in this respect.D. Miiller and P. Larsen l3 regard the lowered ratio of assimilationof potassium-deficient plants as being due to ‘( protoplasmic ” factorsrather than to direct effects on chlorophyll.In a review of the effects of potassium deficiency on carbon assimil-ation in plants G. Rohde l4 points out that deficient leaves aresmaller, thicker, and more bluish-green than those which are ade-quately nourished, and that these factors influence the intensity ofabsorption of light by chlorophyll in regard to total absorption andto the proportional absorption of light of different wave-lengths inthe visible spectrum. Willstatter’s earlier observations show that inyellow-green leaves (low chlorophyll content) much more carbondioxide is assimilated per unit chlorophyll than in blue-green(chlorophyll-rich) leaves. I n the former, assimilation is probablylimited by the proportion of chlorophyll present and in the latter byenzymic activity. Hence, although the importance of potassium inthe assimilation process is manifestly great, its apparent directinfluence on chlorophyll may in some cases be attributable tosecondary effects.Iron, Manganese, and Magnesium.--It has long been recognisedthat deficiency of any of these elements may result in chloroticconditions in plant leaves. Iron and manganese are not constituentsof the chlorophyll molecule, but are doubtless concerned in its form-ation, probably by acting as oxidation-reduction catalysts. I nmany respects iron and manganese have been shown to differ in theireffects on chlorophyll formation.15 I n general, iron has by far thegreater stimulative action, although the work of G. B. Ulvin16suggests that manganese to some extent supplements the effect of iron.Rohde l7 suggests that the observed effects of manganese in increas-l3 Ann. Reports, 1936, 438.l4 2. Pfianz. Diing., 1936, A , 44, 1.l6 Plant Physdol., 1934, g, 69.l1 LOC. cit. ; also 2. Bot., 1934,27,257.l3 Planta, 1935, 23, 501.l5 Ann. Reports, 1934, 355.17 LOC. citPOLLARD. 43 1ing carbon assimilation depend very frequently on the stimulationof the later enzymic stages of the process, although there is littledoubt that this element can affect the chlorophyll content of leaves,and probably also its photo-oxidative properties.l8Deficiencyof iron leads to chlorosis, but chlorotic plants, the condition of whichmay be remedied by treatment with iron, do not always show anotably low iron content. Evidently iron may exist in the plantand yet be unable to play its normal part in chlorophyll production.J. Oserkowsky19 in an attempt to determine the “active ” ironfinds a general correlation between the chlorophyll content of leavesand the amount of iron extracted by N-hydrochloric acid. A smallbut definite amount of ‘‘ inactive ” iron probably dissolves in theacid. No relationship is apparent between the total and the‘‘ active ” iron contents or between total iron and chlorophyll con-tents. Moreover the active iron within the leaf is not all in a water-soluble condition. Prolonged iron deficiency is shown to lead toserious breakdown in the chloroplasts, thus explaining the frequentfailure of iron treatment to cure chlorosis when applied late in theseason. There is evidence that iron is concerned in the formationof pyrrole compounds, utilised in synthesising the central pyrrole-niagnesium nucleus of the chlorophyll molecule. G. Polacci,20working with ChZoreZZa, observes that the presence of magnesiumpyrrole-2-carboxylate in the nutrient medium obviates the necessityof supplying iron. Magnesium supplied as sulphate in an iron-freemedium cannot induce chlorophyll production. The apparentlycatalytic effect of iron on pyrrole ring-formation is not produced ifmanganese or titanium 21 is used in its place.The action of iron is much more definitely established.A. G. P.A. G. POLLARD.C. P. STEWART.J. STEWART.li. Noaok, Naturwiss., 1926, 14, 383.Is Plant Physiol., 1933, 8, 449.2u Ber. deut. bot. Ges., 1935, 53, 540; C. Polacci, B. Oddo, and M. Gallotti,Boll. SOC. itd. Biol. sperin~, 1935, 10, 665.21 0. L. Inman, G. Barclay, and M . Hubbard, Plant Physiol., 1936, 10, 821