BIOCHEMISTRY.SOME of the most interesting developments in biochemistry during thepast year have had to do with the following topics : vitamins, intermediatemetabolism of proteins, biochemistry of muscle, intracellular gels, bio-chemical systems in plants, carboxylation and decarboxylation in bacterialsystems, metabolism of moulds. The specialists invited to cover the corre-sponding reviews in these pages are Dr. L. J. Harris, Dr. A. Neuberger,Dr. Dorothy Needham, Dr. J. F. Danielli, nr. F. W. Norris, Dr. H. Krebsand Dr. Marjory Stephenson, and Professor H. Raistrick.1. VITAMINS.In consequence perhaps of the special needs of war-time most attentionseems to have been given of late to two problems of immediate practicalimportance, namely, the devising of methods, first for the more accuratedetermination of vitamins in foodstuffs (see also the Report on AnalyticalChemistry), and secondly for assessing the nutritional status of humans.The old idea that vitamins were only “ qualitative ” factors, and that theamount present in the food did not matter provided they were there in themerest trace, has gone by the board.A definite minimal quantity of eachvitamin is now recognised as needed to prevent deficiency disease, and alarger quantity to prevent ‘ ‘ subclinical deficiencies. ” Dietetic essentialsnot recognised until a few years ago are now well established and havealready found important clinical uses ; such are nicotinamide, riboflavinand vitamin K : knowledge is accumulating too about the significance ofthe still “newer ” vitamins, for example, adermin (vitamin BJ, biotin(vitamin H), choline, p-aminobenzoic acid, inositol.Particularly interestingis the recent work relating to choline, the absence of which producesremarkable changes in the animal organism.Water-soluble Vitamins : Vitamin B,.Methoas of Assay.-Probably the most comprehensive study of techniquefor estimating a vitamin in foodstuffs which has so far appeared is that ofL. J. Harris and Y. L. W8ng.l They describe a series of modifications inthe thiochrome method, based on the procedure previously used for urine.2Their results were checked by biological assays, three different methodsbeing used, on more than 50 foodstuffs of all types. With the improve-ments recommended, the chemical and biological values generally differedby less than 15%.The reliability of this modified chemical method hasalso been confirmed by E. C. Slater and other workers. Another procedure1 Biochem. J., 1941, 35, 1050; Chem. and I n d . , 1942, 61, 27.2 Y . L. Wang and L. J . Harris, Biochem. J., 1939, 33, 1356; 1941, 35, 1068.3 Aust. J . Exp. Biol. Med., 1941, 19, 29F€ARRIs: VITAMINS. 229which is being made increasingly specific, and is likewise relatively simpleto perform, depends on a measurement of the carbon dioxide evolved duringthe fermentation of yeast, which is stimulated by the addition of vitamin B,.To distinguish between specific fermentation due to the vitamin and thenon-specific “ blank,” tests are done before and after addition of ferricyanide,or better of sulphite, to the unknown.These substances destroy the vitaminand the products formed have no activity in the ferrnentati~n.~have confirmed theobservation 6 that an elevation in pyruvic acid may be used to reoord thepresence of a deficiency of vitamin B, in the experimental animal. Fordetecting partial deficiencies in the human subject, as in animals, tests oftolerance to carbohydrate loading 7 were recommended and these haveproved of practical use clinically.8R61e of Vitctmin B, in Carbohydrate 4letabolism.-Observations of E. S. G.Barron and co-workers 9 just published may help to remove confusionabout the chemical reactions brought about in mammalian tissues byvitamin B,. It has long been accepted that in yeast, vitamin B,, in theform of its pyrophosphate ester, is the co-enzyme for the decarboxylationof pyruvic acid, an essential intermediate in the metabolism of carbo-hydrates. In animal tissues the reactions have been found much lesssimple to study.The point is made in the papers just mentioned that inorder to examine the effect of vitamin B, on animal tissues in vitro, it isnecessary to incubate the vitamin first with the tissue, in order presumablyto convert it into the active pyrophosphate derivative. With this proviso,it can then be shown that vitamin B, catalyses a variety of reactions, suchas oxidation, dismutation and condensation, all of which, however, seemto involve decarboxylation a t some stage. In the latter respect, there-fore, animal tissues now seem to come fundamentally into line with yeast.Much other important work10 on the biochemical function of vitamin B,as a co-enzyme has appeared, and it will be more convenient to reviewthe whole problem when a longer perspective is possible after the lapse ofanother year.Assessment of Level of Nutrition.-Several workersA.S. Schultz, L. Atkin, and C. N. Frey, J . Biol. Chem., 1940, 136, 713; A. S.Schultz, L. Atkin, C. N. Frey, and R. R. Williams, J . Amer. Chem. SOC., 1941, 63, 633;H. Laser, Biochem. J . , 1941, 35, 488.M. E. Shills, H. G. Day, and E. V. McCollum, J . Biol. Chem., 1941, 139, 145;H. A. Harper and H. J. Deuel, ibid., 1941, 137, 233.G. G. Banerji and L. J. Harris, Biochem. J., 1939, 33, 1346.See Ann.Reports, 1940, 31, 387.E. 0’s. Elsom, F. D. H. Lukems, and E. H. Montgomery, J. Clin. Inveat., 1940,19, 153. ’ E. S. G. Barron and C. M. Lyman, J . Biol. Chem., 1941,141, 951 ; E. S. G. Barron,C. M. Lyman, M. A. Lipton, and J. M. Goldinger, ibid., p. 957; E. S. G. Barron, J. M.Goldinger, M. A. Lipton, and C. M. Lyman, ibid., p. 976.lo E.g., F. J. Stare, M. A. Lipton, and J. ill. Goldinger, ibid., p. 981; H. A. Sober,M. A. Lipton, and C. A. EIvehjem, ibid., 1940, 134, 605; M. A. Lipton and C. A.Elvehjem, aid., 1940, 136, 637; S. Ochoa, ibid., 1941, 188, 761; J. H. Quastel andD. M. Webley, Bhltem. J., 1941, $6, 192230 BIOCHEMISTRY.Vitamin B, Complex.RiboJlavin.-The polarograph is proving of increasing use in thedetermination of vitarnins,ll and offers a convenient method, for example,with riboflavin.12 With attention to detail, the chemical method (measure-ment of fluorescence) also gives results in agreement with biological assays.13Cheilosis (angular stomatitis) has in the past year or two come to beconsidered as the characteristic sign of riboflavin deficiency in man, but tojudge from the clinical observations of T.E. Machella l4 it may be a relativelynon-specgc lesion, the primary cause being sometimes a deficiency ofvitamin B,, sometimes nicotinic acid or sometimes even ascorbic acid.Nicotinamide.-Pellagra has been very prevalent in Spain since thecivil war,15 and, possibly as a result of the present war or perhaps only asa result of better searching for it, an increased incidence is recorded inNorthern Ireland.16 To assess the level of nutrition in the anti-pellagravitamin,17 the modified procedure recommended by E.KQdicek and Y. L.Wang l8 involves dosing with nicotinamide while the patient is kept on a dietfree from trigonelline, and measurement of the urinary response in excretionof trigonelline plus nicotinic a ~ i d . 1 ~ An observation which may throwlight on the mode of action of the vitamin is that it inhibits the breakdownof co-zymase by the nucleotidases present in animal tissues.20Microbiological Methods.-Relatively specific methods now exist fordifferentiating and determining the various components of the vitamin-B,complex by means of their stimulating action on various micro-organismsunder appropriately standardised conditions.Recent literature describingmch methods for pantothenic acid 21 and for nicotinic acid 22 may be citedas excellent examples from an important and quickly growing literature.Vitamin C.Methods of Assay.-Further evidence 23 is available of the applicabilityof the polarograph for estimating vitamin C.24 A source of error whichhad too often been overlooked in the chemical determination of vitamin Cl1 E. Kodicek and K. Wenig, Nature, 1938, 142, 35.l 3 J. J. Lingane and 0. L. Davis, J. Biol. Chem., 1941, 137, 567.l3 F. 0. Van Duyne, ibid., 1941, 139, 207.l4 Amer. J. Med. Sci., 1942, 1, 114.l5 Quoted by Lancet, 1941, ii, 458.l6 J. Deeny, Brit. Med. J., 1942, i, 157.l 7 Cf. L. J. Harris and W. D. Raymond, Biochein.J., 1939, 33, 2037.l 8 Nature, 1941, 148, 23.See further W. A. Perlzweig, H. P. Sarett, and J. W. Huff, J . Bid. C'kem., 1941,2o P. J. G. Mann and J. H. Quastel, Nature, 1941, 147, 326; Biochem. J . , 1941,21 M. J. Pelczar and J. It. Porter, J. Biol. Chem., 1941, 139, 111.28 H. Isbell, J. G. Wooley, R. E. Butler, and W. H. Sehrell, ibid., p. 499; E. E.23 T. Osterud, Tek. Ulcebhd, 1939, 86, 216.24 Cf. E. Kodicek and K. Wenig, Nature, 1938, 142, 35.140, proc. C; H. P. Sarett, J . Nutrition, 1942, 23, 23.35, 502.Snell and L. D. Wright, ibid., p. 675TTARRIS : VITAMTNS. 23 1is that any sulphur dioxide present as a preservative in fruit pulps, juices,etc., will seriously interfere unless special precautions 25 are taken.War-time Considerations.-With the diminished supply of importedfruits, our bodies’ reserves of vitamin C are likely to be far lower than theywere before the war, and it is therefore useful to have the authoritativememorandum dealing with the preparation and cooking of green vegetables,which gives rules for treatments calculated to cause the least loss.26 Incooking sour fruits a limited amount of alkali (sodium bicarbonate) cansafely be added to save sugar.27 Valuable compilations by M.Olliver 28and W. B. Adam29 deal comprehensively with the effects of cooking andcanning respectively on the nutritive value of vegetables.“ Partial De$ciency ” of Vitamin C.-Various views are taken about thereality of sub-clinical deficiency. On the one hand S. S. Zilva 3O has argued :“ As far as the civilian population of this country, leading a normal life, isconcerned, the natural supply of vitamin C during the greater part of theyear is so superabundant that, even allowing the widest margin for destruc-tion in the cooking and the preparation of the food, the intake is more thanadequate to supply the vitamin C requirements.” On the other hand,tests in Germany proved that the addition of extra vitamin C (50 mg.perday) to the diet of over a million children increased their resistance toinfection and improved their yearly gains in weight and height-althoughthere had been little or no actual clinical ~urvey.~l Similar tests have beendone in a large training school in Britain on 1500 adolescents : in the controlgroup receiving no added vitamin C there occurred 17 cases of pneumoniaand 16 of rheumatic fever, whereas there was no case of either in the groupgiven added vitamin C ; tonsillitis occurred in both groups but the averageduration of illness in the control group was much longer, the difference beingof undoubted statistical significance.31a Addition of vitamin C to the diethas also been found to diminish the incidence of gingivo-stomatitis, or aidin its cure,31b and to promote healing after dental extraction.31c Again thehealing of wounds after surgical operation is usually found to be better insubjects receiving ample vitamin C32 than in those whose intake is lower.There is in fact ample evidence that in man, as in animals, sub-clinicaldeficiency of vitamins produces a variety of somewhat ill-defined but nonethe less real faults in nutrition 33-f0r vitamin C these may include sub-25 L.W. Mapson, Ghem. and Ind., 1941, 60, 802.36 Accessory Food Factors Committee, Medical Research Council, Brit. .&led. J.,97 L. W. Mapson and J. Barker, Ghem and Ind., 1941, 60, 661.2 8 Ibid., p. 586.30 Biochem. J . , 1941, 35, 1240.31 Bull. War Med., 1941, 2, 6.310 A. J. Glazebrook and S. Thomson, J. Hyg., 1942, 42, 1.311 F. S. Roff and A. J. Glazebrook, J . Roy. Nav. Med. Sew., 1939, 25, 340; Brit.1941, ii, 26.2s Ibid., p. 627.Dent. J., 1940, 68, 135; H. G. Campbell and R. P. Cook, Brit. Med. J., 1941, i, 360.H. G. Campbell and R. P. Cook, Brit. Dent. J., 1942, 72, 6.C. C. Lund and J.H. Crandon, Ann. Surgery, 1941, 114, 776.33 See L. J. Harris, Proc. Nutrition SOC., 1941, No. 1 ; also Food, 1942, 11, 23232 BIOOHEMISTRY.optimal growth, diminished resistance to infection, impaired formation ofscar tissues, and irregularities in teeth and gums.Therapeutic Uses of ‘Vitamin C.-Apart from its value in securing theadequate healing of there is evidence also that an abundantsupply of vitamin C prolongs the life of experimental animals submittedto severe bleeding This it may do bysecuring a more adequate supply of oxygen to the tissues.Mode of Action of Vitamin C.-In the last-mentioned connection it maybe noted that S. S. Zilva and his colleagues36 have codrmed the findingof D. C. Harrison 37 that addition of ascorbic acid in vitro to the liver tissuesof scorbutic animals augments their uptake of oxygen.The exact meaningof this finding is not yet clear, but G. A. Snow and S. S. Zilva discuss itspossible connection with carbohydrate metabolism. Various types ofenzyme systems are however also activated by vitamin C, e.g., liver esterase,succinic dehydrogenase, and to a less extent cytochrome oxidase, andphosphatases from the kidney and intestine; 38 the degree of specificity ofsuch activations still remains in doubt. In cauliflower juice dehydroascorbicacid is reduced by SH-glutathione, and the enzyme which catalyses thereaction has now been separated : it is suggested that it may have a r61eas an ‘‘ end syetem ” in plants.39or to low oxygenFat-Soluble Vitamins : Vitamin A.Factors influencing Utilisation of Vitamin A .-It is becoming apparentthat the efficiency with which vitamin A or carotene prevents deficiencydisease in animals (and presumably in humans) varies with other factors inthe diet : e.g., the utilisation of carotene by the rat depends on the chemicalnature of the oil administered with it ; 40 prolonged deficiency of vitamin Ecauses a secondary deficiency of vitamin A ; 41 and the quantity of foodconsumed of course also influences the growth rate with any given intake ofvitamin A.42Assessment of Level of Nutrition.---S. Yudkin43 has made a careful re-investigation of dark adaptation as a means of detecting sub-clinical deficiencyof vitamin A.Of 24 subjects tested for “night blindness,” all exceptthree improved with vitamin A.The method, which is in any case relativelyspecific, can thus be made completely so by taking as the criterion animprovement or otherwise after dosing with vitamin A.44 S. Yudkin4334 C. P. Stewart, J. R. Learmonth, and G. A. Pollock, Lancet, 1941, i, 818.35 B. G. B. Lucas, quoted by J. M. Peterson, Nature, 1941, 148, 84.36 A. E. Kellie and S. S. Zilva, Riochem. J., 1941, 35, 783; G. A. Snow and S. S.37 Ibid., 1933, 27, 1501.Zilva, ibid., p. 878.C. J. Harrer and C. G. King, J . Biol. Chem., 1941, 138, 1 1 1.E. M. Crook, Biochem. J., 1941, 86, 226.40 W. C. Sherman, J . Nutrition, 1941, 22, 153.4 1 A. W. Davies and T. Moore, Ndure, 1941, 147, 794.42 K. D. Muelder and E. Kelly, J . Nutrition, 1941, 21, 13.43 Lamed, 1941, i i 787.44 See, e.g., Ann. Rqwt.8, 1939, 36, 338EKARRIS : VITAMINS. 233emphasises that it may be necessary to give massive doses, and that withsmaller doses especially the cure may be only transient. For any givensubject there is a critical low level for the vitamin A in the blood, and belowit dark adaptation is adversely affected ; unfortunately, however, thereappeared to be no such standard level applicable for all subjects. Benz-edrine or alcohol produced transient improvement, but the supposed adju-vant action of vitamin C45 could not be confirmed. An alternative pro-cedure, biomicroscopy with the ~ l i t - l a m p , ~ ~ has been used by H. D. Kruseand his fellow investigators in the course of their impressive surveys onthe evaluation of nutritional status; 47 but Kruse is rightly a t pains topoint out that the ocular lesion (keratosis) is not the sole, first or mostimportant abnormality : '' xerophthalmia is not synonymous with avita-minosis A."Efseects of De$ciency.-E.Mellanby 48 has published the detailed accountof his observations on the skeletal changes produced in young dogs bydeficiency of vitamin A. The bony overgrowths in the skull and vertebralcolumn may produce deformities in the brain and spinal cord. Mellanbyconcludes that vitamin A controls the degree of activity of the osteoblastsand osteoclasts.Vitamin A in Blood and Urine.-Reference to vitamin A in the bloodand its connection with dark adaptation in humans has been made above.Similarly it has been shown that in dogs the level of vitamin A in the bloodis proportional to the intake in the diet, and during depletion the level dropgradually : a low level in the blood cannot however be taken to indicatethat the reserves have become depleted.49 In humans vitamin A is notfound in the urine except in certain diseases, notably pneumonia and chronicnephritis. It is probably significant that in these two diseases, amongothers, there is a great disappearance of the reserves of vitamin A from theliver.A strange phenomenon, still lacking explanation, is that in dogs,by contrast with humans, vitamin A is a normal constituent of the urine.60Vitamin D.InJEuence on Metabolism.-Extended observations by S. H. Liu51 inChina on patients with osteomalacia have proved once again that vitamin Dis the primary factor controlling cure of the disease, and have confirmedalso that its mode of action is to decrease the fzecal loss of calcium.52Factors inJEuencing Rachitogenesis.-It has been emphasised 53 that fat isone of many factors-others include the acid-base reaction, the Ca-P ratio,presence of heavy metals, sugars, etc.-which have an effect on the pro-45 Cf.C . P. Stewart, Edinburgh Med. J., 1941, 48, 217.46 Milbank Memorial Fund Quarterly, 1941, 19, 207.47 Ibid., 1939--1941.49 P. C . Leong, Biochem. J., 1941, 35, 806.61 Chinese Med. J., 1940, 57, 101.62 Cf. L. J. Harris, Lancet, 1932, i, 1031.69 *4. Knudson and R. J. Fbody, J . Nutrition, 1940, a0, 317.'* J . PhpiOl., 1941, 99, 467.N.R. Lawrie, T. Moore, and K. R. Rajagopal, ibid., p. 825234 BIOCHEMISTRY.duction or healing of rickets. Phytic acid is another such, and important,factor, and to it the rickets-producing action of cereals is attributable.The cause of the deleterious action of the phytic acid seems to be partlythat it precipitates the calcium in the intestine and partly that it rendersthe calcium non-ionised and thus impedes its absorption.54 But, fortun-ately, when wheat flour is baked with yeast, an enzyme, phytase, presentin the flour largely destroys the phytic acid.55Other Pat-soluble Vitamins.Of importance to all researchchemists and analysts working on vitamins is the adoption of racemica-tocopherol acetate as the international standard for vitamin E.56 Thetwo chemical methods for estimating the vitamin, potentiometrically withauric chloride, or colorimetrically with the ferric reagent, have been sys-tematically reinvestigated by Karrer and his colleagues and found to giveresults in satisfactoryIt is pleasing for once to be able to recorda diminution instead of an increase in the apparent number of the vitamins.A variety of symptoms in various species, sometimes attributed in the pastto lack of still unidentified factors, have been found to respond to syntheticvitamin E.There is accordingly no longer any need to call in hypotheticalnew vitamins to account for the following disorders : " nutritional ence-phalomalacia " of chicks, " nutritional myopathy " of ducklings and " giz-zard disease " of turkeys, " muscular dystrophy " of guinea pigs, rabbitsand young rats,58 generalised edema in " alimentary exudativediathesis " in chicks.6oAlthough admittedly lack of vitamin E causes muscularatrophy in experimental animals, it seems dangerous to jump to the con-clusion, as has sometimes been done, that vitamin E is therefore useful ina wide range of neuromuscular disorders in man.Controversy on this issuestill continues.61 For habitual abortion in women, however, a statisticaltreatment of the results of treatment is distinctly encouraging.62An observation which for the firsttime promises to throw light on the mode of action of vitamin F (nutri-54 E. F. Yang, Nature, 1940, 145, 745; D. C. Harrison and E. Mellanby, ibid.,p.745.5 6 E. M. Widdowson, ibid., 1941, 148, 219.6 6 E. M. Hume, ibid., pp. 472, 473.67 p. Karrer, W. Jaeger, and H. Keller, Helv. Chint. Acta, 1940, 23, 464; see alsoA. Emmerie, Rev. Trav. china., 1940, 59, 246; F. Grandel and H. Newman, 2. Untem.Lebenam., 1940, 79, 57-6 8 A. M. Pappenheimer, J . Mt Sinai Hosp., N.Y., 1940, 7, 65; H. M. Evans andG. A. Emerson, Proc. Xoc. Ezp. Bid., Med., 1940, 44, 636; C. G . Mackenzie, J. B.Mackenzie, and E. V. McCollum, J . Nutrition, 1941, 21, 225.6s H. R. Bird and T. G. Culton, Proc. SOC. Exp. Biol. Med., 1940, 44, 543.60 H. Dam and J. Glavind, Naturwiss., 1940, 28, 207.61 see, e.g., Lancet, 1941, ii, 619; Brit. Med. J., 1941, ii, 618.62 A. L. Bacharach, Brit. Med. J . , 1940, i, 890; 1941, ii, 709.Vitamin E.-#tandards and methods.Symptoms of avitaminosis E .CZinicaZ uses.Vitamin F.-Physiological actionHARRIS : VTTAMNS.235tionally essential fatty acids) is that in its deficiency there is an impairedabsorption of ordinaryReports continue to appear stressing thevalue of vitamin K in preventing hzemorrhage after operation for obstruc-tive jaundice, or in new-born infants ; the comprehensive monograph onthis vitamin by H. R. Butt and C. T. Snell 64 will be welcomed.Vitamin K.-Clinical uses.The Newer Vitamins.Enumeration.-The ‘‘ vitamin-B, complex ” includes, by definition, ribo-flavin and nicotinamide (pellagra-preventing factor), both of which havealready been referred to above, and also adermin (pyridoxin, vitamin B,)and pantothenic acid (filtrate factor, bios IIA), which have been discussedin a recent issue of these Reports.65 Other vitamins more recently charac-terised, which could logically be classified as of the “B, group,” includevitamin H (biotin, bios IIB, co-enzyme R), choline, inositol (bios I), andp-aminobenzoic acid (possibly identical with the ‘‘ anti-grey-hair factor ’,),Vitamin H (Biotin, Bios IIB).-Vitamin H is the factor needed by ratsor chicks for protection against the nutritional injury which arises whentheir diet contains much raw egg-white.It is identical with biotin (orbios IIB, a factor needed by yeast and other micro-organisms) and alsowith the so-called ‘‘ co-enzyme R ” which stimulates the growth of micro-organisms in the nodules of the roots of certain leguminous plants.66 Theaction of the raw egg-white in inducing the deficiency in rats has beentraced to a component in the crude protein which unites with the vitamin Hand thus renders it unavailable to the organism.67 This component hasbeen named ‘‘ avidin,” and methods have been worked out for fractionatingit, and estimating i t .G 8 Vitamin H itself has also been isolated and itsempirical formula established (C,,H,,0,N2S) ; 69 its behaviour to variousreagents has been ~tudied,~O and it has been shown to be a carboxylic acidcontaining an “’-substituted cyclic urea group and a-cozH thioether linkage (annexed formula).71 As to the physio-C,H,, -zz>CO logical nature of the ‘‘ egg-white injury ” and the actionof vitamin H in preventing it, it may perhaps be ofsignificance that G.Gavin and E. W. McHenry 72 havefound that biotin given to rats induces fatty livers, whereas the simul-taneous administration of egg-white (or of inositol) prevents this effect.Cho1ine.-Perhaps the most spectacular work on vitamins during thepast two or three years is that relating to choline. This substance is underI,,,63 R. H. Barnes, E. S. Miller, and G. 0. Burr, J. BioZ. CJLem., 1941, 140, 773.64 ‘. Vitamin K,” 1941.6 5 Ann. Reports, 1940, 37, 389, 390.6 7 R. E. Eakin, E. E. Snell, and R. J. Williams, J . BioZ. Chem., 1940, 136, 801.6 8 Idem, ibid., 1941, 140, 535.19 V. du Vigneaud, K. Hofmann, D. B. Melville, and P. Gyorgy, ibid., p. 643.70 C. B. Brown and V. du Vigneaud, ibid., 1941, 141, 85.7 1 K.Hofmann, D. B. Melville, and V. du Vigneand, ibid., p. 207.TZ Ibid., p. 620.66 Ibid., p. 392236 BIOOHEMISTBY.certain conditions a dietary essential for rats,73 absence of it caushg anexcessive deposition of fat in the liver and hzemorrhages in the kidney; 74it is needed also by poultry for the prevention of p e r ~ s i s . ~ ~ It seems thatcholine is concerned in the organism in carrying out transmethylation.For this purpose it can be replaced either by methionine or by betaine :the more of the latter substances there are in the diet the less choline isneeded. On the other hand the need for choline increases when there isincreasing cystine (or fat) in the diet. Viewed from another angle, cholineor betaine, as biological methylating agents, enable the animal to utilisehomocystine in place of methionine.76 There may be similar inter-relationsbetween oholine on the one hand and vitamin B,, nicotinic acid, or other" B " vitamins on the other.72* 77 As yet there is no evidence whethercholine (or its biological equivalents) is needed by man.Amti-grey-hair fuctor ( ? p-aminobenzoic acid). Loss of pigmentation inthe f w of rats kept on deficient diets had been noted by numerous inves-t i g a t o r ~ . ~ ~ Work by A. F. Morgan et aZ.,79 G. Lunde and H. Kringstad,mand J. J. Olsen et uLma made it clear that a hitherto unidentified vitaminwas concerned in preventing this disorder. It has been shown that silverfoxes, dogs and guinea pigs also are susceptible to the deficiency.81S.Ansbacher 82 claims to have identified p-aminobenzoic acid, previouslyrecognised as a growth-promoting factor for plants, as the anti-grey-hairvitamin. Clinical applications for it have already been put f0rward,~3 butthere is still a conflict of evidence whether p-aminobenzoic acid, or maybe73 C. H. Best, M. E. Huntsman, E. W. McHenry, and J. H. Ridout, J . Physiol.,1935, 84, 38.74 W. H. Griffith and N. J. Wade, J . Biol. Chem., 1939, 131, 567; B. Sure,J . Nutrition, 1940, 19, 71; R. W. Engel and W. D. Salmon, ibid., 1941, 22, 109.7 6 0. D. Abbott and C. U. DeMasters, ibid., 1940, 19, 47; T. H. Jukes, J . Biol.Chem., 1940, 134, 789; J . Nutrition, 1940, 20, 445; A. G. Hogan, L. R. Richardson,H. Patrick, and H. L. Kempster, ibid., 1941, 21, 327; J .Biol. Chem., 1941, 138, 459.76 J. P. Chandler and V. du Vigneaud, ibid., 1940, 135, 223; H. J. Channon, M. C.Madfold, and A. P. Platt, Bi0~he7la. J., 1940, 34, 866; A. D. Welch, J . Biol. Chem.,1941, 137, 173; H. P. Jacobi, C. A. Baumann, and W. J. Meek, ibid., 1941, 138, 571;W. H. Griffith, J . Nutrition, 1941, 21, 291 ; W. H. Griffith and D. J. Mulford, J . Amer.Chern. Xoc., 1941, 63, 929.77 p. Gyorgy and R. E. Eckardt, Biochem. J., 1940, 34, 1143; P. Gyorgy and H.Goldblatt, J . Exp. Med., 1940, 72, 1; W. H. GriEith and D. J. Mulford, J . Nutrition,1941, 21, 633; J. C. Forbes, ibid., 1941, 22, 359.78 For early literature, see A. Bakke, V. Aschehoug, and C . Zbinden, Compt. rend.A&. Sci. U.R.S.S., 1930,191, 1157; F.J. Gorter, Nature, 1934,134, 382; 2. Vitumin-forsch., 1935, 4, 277; G. A. HartweI1, Biochem. J., 1923, 17, 547; P. Gyorgy, aid.,1935, 29,741.'70 A. $. Morgan, B. B. Cook, andH. G. Davison, J . Nutrition, 1938, 15, 27.80 2. physiol. Chm., 1939, 25'7, 201; J . Nutrition, 1940, 19, 321.800 J. J. Oleson, C. A. Elvehjem, and E. €3. Hart, Proc. XOC. Exp. BioZ. Med.,1939, 42, 283.81 G. Lunde and H. Kringstad, Naturwiss., 1939, 27, 755; A. F. Morgan andH. D. &nuns, J . Nutrition, 1940, 20, 627.82 Science, 1941, 93, 164.83 B. F. Sieve, ibid., 1941, 94, 257NEUBERGER : THE METABOLISM OF NITROGENOUS COMPOUNDS. 237alternative €actora--e.g., biotin, pantothenic aeid-do or do not in factcure rats of their grey hairs.84Anti-alopecia Factor for .Mice ( ? Inositol, Bios I ) .-Inositol, cyclohexane-hexol, has been known since 1928 s5 to be a growth stimulant for yeast,classified as “ bios I.” I n 1940 D.W. Woolley,86 by feeding mice on arestricted diet, produced in them a disease characterised by hairlessness ofthe trunk and cessation of growth. Later he stated that the protectivesubstance was inosit01.~~ Certain related substances, e.g. , inositol hexa-acetate, cephalin, and phytic acid (inositol hexaphosphate), were foundactive.88 We seem therefore to have the entertaining anomaly that, whereassmall amounts of phytic acid act as a. vitamin for mice, or as a growthstimulant for yeast, yet larger amounts act as a “ toxamin ” or ‘‘ anti-vitamin.” g9Folk Acid, Grass Juice Factor.-Folic acid, a substance necessary forthe nutrition of yeast, occurs abundantly (as its name implies) in theleaves of plants.It has recently been isolated,g0 and its possible relationto the grass juice factor>l needed by guinea pigs, still remains to be decided.Classijkation of ‘‘ Bios ” Factors.-Since the various components of“ bios ” (the growth-promoting stimulant for yeast whose effects were firstdescribed in 1901 92) have lately been identified with certain vitaminsneeded by mammals, the following table of synonyms may help to removeconfusion :Bios I1Biosdimethylbutyryl- p -alanine)IIB = Biotin (vitamin H, co-enzyme R)L. J. H.2. THE METABOLISM OF NITROGENOUS COMPOUNDS.Recent advances in our knowledge of the intermediary metabolism ofnitrogenous compounds have been due mainly to the application of threedifferent methods : (1) the study of nutritional requirements of animals(mainly non-ruminants) and the replacement of naturally occurrhg sub-stances by related compounds, (2) the investigation of biochemical reactionscatalysed by tissue slices, cell extracts and purified enzymes, (3) the use of“ labelled ” compounds, i.e., substances containing heavy or radioactive84 K.Unna, G. V. Richards, and W. L. Sampson, J . Nutrition, 1941, 22, 553;L. M. Henderson, J. M. McIntre, H. A. Waisman, and C. A. Elvehjem, ibid., 1942, 23,47; R. R. Williams, Science, 1940, 92, 561; P. Gyorgy and C . E. Poling, Proc. SOC.Exp. Biol. Med., 1940, 45, 773.1E. V. Eastcott, J . Physical Chew,., 1928, 32, 1094.86 J.Bid. Chem., 1940, 136, 113.D. W. Woolley, Science, 1940, 92, 384; J . Biol. Chrn., 1941, 139, 29.Idern, ibid., 1941, 140, 461.H. K. Mitchell, E. E. Snell, and R. J. Williams, J . Amer. Chern. XOC., 1941, 63,See Ann. Reports, 1940, 37, 393. 9a E. Wildiers, La Oelhle, 1901,18,313.Cf. above, p. 234.2284238 BIOCHEMISTRY,isotopes.space a.vailable, but a few general problems may be discussed.It is impossible to review the whole field adequately within theEssential Amino-acids.It has been known for a considerable time that animals, with thepossible exception of ruminants, require certain amino-acids in their diets.These " essential " amino-acids may be necessary for two purposes : theyhave to serve as the building material of the body proteins (" structural "requirements) and will therefore be particularly important for the growinganimal, or may be needed for some specific chemical function (" functional ''requirements).In some cases such a specific function is known, e.g., arginineis necessary for the formation of creatine and methionine is needed as supplierof methyl groups. I n other cases, however, a specific biochemical functioncan be inferred, although its precise nature may be still obscure. Valinedeficiency, e.g., leads to severe nervous symptoms in the rat,l from whichit appears likely that this amino-acid is important for some specific biologicalreaction. The distinction between essential and non-essential amino-acidsis, however, not very sharp, The absolutely non-essential amino-acids aremainly those which are related to keto-acids occurring in the metabolismof carbohydrates, such as glutamic and aspartic acids and alanine; othernon-essential amino-acids are serine, proline and hydroxyproline.A secondgroup of amino-acids consists of compounds which may be called " semi-essential " ; thus arginine, although it is synthesised by the rat,2 is not formedfast enough to permit optimal growth ; for the chick arginine is indispensable,since it cannot form it at all or only very slowly.4 Glycine also is not essentialfor the rat and is apparently necessary for optimal growth in the chick.5Choline is another nitrogenous compound which is synthesised by animalsin amounts insufficient for metabolic requirements a t least under certaindietary conditions.' Another type of " semi-essential " amino-acid is onlydispensable if a related essential amino-acid is provided in the diet in amountssufficient to cover the requirements of both substances.Thus cystine, whichcan be made from methionine, is without growth effect if large amounts ofmethionine are fed ; 8 cystine stimulates growth, however, if methionine issupplied in suboptimal amounts.9 A similar position may possibly existfor tyrosine and its relationship to phenylalanine.The third group comprises the truly " essential " amino-acids, leucine,isoleucine, valine, threonine, histidine, lysine, phenylalanine, tryptophanW. C. Rose and S. H. Eppstein, J . Biol. Ghem., 1939, 127, 677.C.W. Scull and W. C. Rose, ibid., 1930, 89, 109.W. C. Roae, PhysioZ. Rev., 1938, 18, 109.A. Arnold, 0. L. Kline, C. A. Elvehjem, and E. B. Hart, J . Biol. C'hem., 1936, 116,H. T. Almquist and E. Mecchi, ibid., 1940, 135, 365.H. P. Jacobi, C . A. Ba~unann, and W. J. Meek, ibid., 1941, 138, 577.See the review by W. J. Griffith, J . hrutrition, 1941, 22, 239.M. Womack and If'. C. Rose, ibid., 1941, 141, 375.699.* M. Womack, K. S. Kemmerer, and W. C. Rose, J . Biol. Chem., 1937, 121, 403NEURERGER : THE METAROLTSI~T OF NTTROGENOUS COMPOUNDS. 239and methionine, which are indispensable for growth3 and cannot be madefrom substances normally present in animal diets. It has been claimed thatall these nine amino-acids are also necessary for maintenance of nitrogenequilibrium in the adult; 10 E.W. Burroughs, H. S. Burroughs, and H. H.Mitchel1,lf however, find that all the essential amino-acids are not equallyimportant for maintenance ; thus threonine and isoleucine occupy somewhata key position, whilst lysine and histidine are considered non-essentialfor the adult rat. These authors also consider phenylalanine not essentialfor the adult if tyrosine is provided. The duration of these experimentsseems, however, too short for such very definite conclusions to be drawn.Investigations using Isotopes.This particular field has been reviewed recently by R. Schoenheimer andD. Rittenberg l2 and only those results are discussed here which are of a verygeneral character. If isotopic nitrogen is fed to rats in the form of ammoniumsalts,13 I ( - )-leucine,l* d( +)-leucine,l5 glycine 16 or dZ-tyrosine,17 a consider-able part of the marked nitrogen is recovered from the tissue proteins.The isotopic nitrogen was found to be present in all amino-acids with theexception of lysine.The authors concluded that a rapid and extensiveinterchange of nitrogen takes place between dietary amino-acids and tissueproteins, involving the opening of peptide linkages, deamination, reaminationor possibly transamination of amino-acid residues. It would follow thatthe old distinction between endogenous and exogenous nitrogen metabolismwhich is due to 0. Folin 18 must be abandoned. This old conception, whichhas recently been restated and defended,lg can certainly not be maintainedin its original form, since i t has been shown that a considerable fraction of thenitrogen of creatinine which was supposed to be a measure of the endogenousmetabolism is provided by normal constituents of the food.There is,however, evidence that some proteins, such as the serum proteins, aremetabolically more active than others, and there may even be some proteinswhich are completely inert.This interchange between dietary nitrogen and the amino-acid residuesbound in the tissue proteins is interpreted as involving the oxidative de-amination of amino-acids to keto-acids and resynthesis. That such a forin-ation of amino-acids from the corresponding keto-acids actually takes placeis shown by the fact that most keto- and a-hydroxy-acids replace theircorresponding essential amino-acids.20 Lysine, however, cannot be replacedlo P.A. Wolf and R. C. Corley, Amer. J . PhysioE., 1939, 127, 589.l 3 G . L. Foster, R. Schoenheimer, and D. Rittenberg, J . Riol. Chem., 1939, 127, 319.lJ R. Schoenheimer, S. Ratner, and D. Rittenberg, ibid., 1939, 130, 703. ’’ s. Ratner, R. Schoenheimer, and D. Rittenberg, ibid., 1940, 134, 653.l6 D. Rittenberg, R. Schoenheimer, and A. S. Keston, ibid., 1939, 128, 319.I ’ R. Schoenheimer, S. Ratner, and D. Rittenberg, ibid., 1939, 127, 333.J . Nutrition, 1940, 19, 363, 385. l2 Physiol. Rev., 1940, 20, 218.Amer. J . physiol., 1905, 13, 117.Em w. Burroughs, H. S. Burroughs, and H. H. Mitchell, J . Nutrition, 1940, 19, 271.2o biT. C. Rose, Phpiol.Rev., 1938, 18, 109240 BIOOEEMISTRY.by or-hydroxy-s-aminohexoic acid; 21 this fact is in accordance withthe results obtained with the use of isotopes,22 which shows that for lysinedeamination ia irreversible. The availability of the hydroxy- or keto-acidanalogue of threonine has not been examined.d-Amino-acids can be deaminated in vitro by the d-amino-acid oxidase;it is therefore to be expected that all essential Z-amino-acids which can bereplaced by their corresponding keto-acids should also be replaceable bytheir d-isomerides. This is actually the case for histidine, tryptophan,phenylalanine and methionine ; Z-leucine, Z-isoleucine and Z-valine cannot,however, be replaced by the d-compounds,2* although in vitro experimentsindicate a fairly high rate of deaminati~n.~~ The reason for this discrepancyis quite obscure.The observation that phenylalanine can cover the dietary requirementsof both phenylalanine and tyrosine for the growing animal must mean thatphenylalanine can be converted into tyrosine and that the reaction isirreversible.This interpretation was confirmed by A. R. Moss and R.Schoenheimer,24 who fed dZ-deuterophenylalanine to rats and recoveredisotopic tyrosine from the tissue proteins. The conversion of hydroxy-acidsinto the corresponding amino-acids which follows from the feeding experi-ments mentioned has also been demonstrated directly by feeding deutero-dZ-P-phenyl-lactic acid and the recovery of labelled t y r ~ s i n e . ~ ~The special function which a particular amino-acid may have to servehas been very clearly demonstrated in the case of methionine. This amino-acid is necessary to build up the tissue proteins and it can replace-asmentioned above-cystine.H. Tarver and C. L. A. Schmidt 26 have investi-gated the fate of methionine containing radioactive sulphur and were able toshow the presence of isotopic sulphur in the cystine isolated from the tissues.But the most interesting function of methionine is probably its ability tosupply methyl groups for two important biosyntheses, the formation ofcholine and creatine.Choline Metabolism.It has been known for some time that dietary choline can prevent theaccumulation of fats in the livers of animals fed on certain diets.27 It wasnoticed later that methionine has a similar " lipotropic " effect,28 whereascystine produces an increase in the deposition of liver fat.29 I n the meantime,evidence of the inter-relationship between choline and methionine wasobtained by du Vigneaud and his co-workers. They demonstrated thathomocystine and homocysteine are incapable of supporting growth of animals*l 0.A. McGinty, H. B. Lewis, and C. S. Marvel, J . Biol. Chem., 1924, 62, 75.22 N. Weissman and R. Schoenheimer, ibid., 1941, 140, 779.23 H. A. Krebs, Biochem. J., 1935, 29, 1620.a6 A. R. Mom, ibid., 1940, 137, 739.27 C. H. Best and T. M. Hershey, J . Physiol., 1932, 75, 56.28 H. F. Tucker and H. C. Eckstein, J . Biol. Chem., 1937, 121, 479.*@ H. J. Channon, M. C. Manifold, and A. P. Platt, Biochem.J . , 1938, 32, 969;24 J . Biol. Chem., 1940, 135, 416.26 Ibid., 1939, 130, 67.A. W. Beeston and H. J. Channon, ibicl., 1936, SO, 280NEEDHAM : THE MECHANISM OF MUSCLE CONTRACTION. 241on a diet devoid of cystine and methionine; administration of chohe,however, enables the animal to utilise homocystine instead of methi~nine.~~The conclusions drawn from these experiments that methyl groups can betransferred reversibly between methionine and choline and that the presenceof donors of labile methyl groups is essential in diets, have been confirmedby later work using labelled methyl groups. If methionine containing adeuteromethyl group is fed to rats, choline containing a high concentrationof deuterium in its methyl groups can be isolated from the tissues.31Choline deficiency may also lead to a severe haemorrhagic renal de-which can be prevented by choline, methionine and betaine 33and is aggravated by cystine and chole~terol.~~Creatine Metabolism.H.Borsook and J. W. Dubnoff 35 showed that guanidoacetic acid wasslowly converted by tissue slices into creatine and that methionine aloneof many substances tested increased greatly the rate of this reaction. Theactual transfer of the methyl group from methionine to creatine in vivowas later clearly demonstrated by du Vigneaud et aZ.31 with the aid of labelledmethyl groups. Guanidoacetic acid is formed in the body mainly fromarginine and g l y ~ i n e , ~ ~ but other nitrogenous substances act only as potentialprecursors of creatine in so far as their nitrogen is utilised for the synthesisof glycine and arginine.It could also be shown that arginine provides theamidine part of the molecule, and glycine supplies the sarcosine moiety.The shift of methyl groups which is demonstrated by all these investigationsis, however, not completely reversible. Methyl groups are interchangeablebetween choline and methionine and both choline and methionine can functionas methyl donors to creatine. The latter reaction appears, however, to beirreversible.37 A. N.3. THE MECHANISM OF MUSCLE CONTRACTION.Knowledge of the nature of the protein myosin, of which the contractilemuscle fibril is composed, has been greatly extended during the last fewyears. have studied the amino-acid contentof myosin.Bailey estimated the cystine, methionine, tyrosine and tryp-tophan, and Sharp the basic amino-acids, dicarboxylic acids and mono-amino-acids, with the result that 85% of the protein is now identified.30 V. du Vigneaud, J. P. Chandler, A. W. Moyer, and D. M. Keppel, J. Biol. Chem.,1939, 131, 57.31 V. du Vigneaud, M. Cohn, J. P. Chandler, J. R. Schenck, and S. Simmonds, ibid.,1941, 140, 625.32 W. H. Griffith and N. J. Wade, ibid., 1939, 131, 567.33 W. H. Griffith, J . Nutrition, 1941, 21, 291.34 W. H. GrifFith and 0. J. Mulford, ibid., p. 633.a6 J . Biol. Chern., 1940, 132, 559.s6 K. Bloch and R. Schoenheimer, aid., 1941, 138, 167.37 V. du Vigneaud, J. P. Chandler, and A. W. Moyer, ibid., 1941, 139, 917.K. Bailey 1 and J.G . SharpBiochem. J . 1937, 31, 1406. a Ibid., 1939, 33, 679242 BTOCHRMTSTRY.Myosin contains very little cystine sulphur, most of the sulphur being inthe form of methionine; some of the cystine is present in the reduced -8Hform even in native myosin and the effect of various treatments upon the--SH content has been much studied.3* 4*have made an important X-ray and elastic study of strips of myosin film,made by drying the sol, and prepared in such a way that the myosin chainmolecules are oriented. When oriented but unstretched, they give an a-photograph, almost indistinguishable from that of a-keratin ; when stretched,they give a p-photograph. The X-ray and elastic properties of myosinresemble those of keratin that has suffered breakdown among the crosslinkages, including S--S bridges, of the polypeptide grid, i.e., the super-contracting form of keratin.Quantitative study by means of X-rays ofthe structural changes accompanying super-contraction of myosin filmsshows that the effect must be due to further regular folding of the poly-peptide chains, and cannot be due merely to disorientation of long thinunits. The hypothesis is put forward that muscle contraction arises fromsuper-contraction of its myosin component,. Astbury ' 1 * has suggesteda new view of the nature of the intramolecular fold in a-keratin and a-myosin; in the transformation from the flat polypeptide grid to the newa-configuration, all the experimental and structural conditions are satisfied,in particular, space is available for the side-chains standing out alternatelyon one side and the other of the plane of the fold.In view of the structural importance of myosin, it is of very great interestto learn that enzyme properties are also associated with this protein,especially as this enzyme activity consists precisely in bringing about thatreaction (the hydrolysis of adenosine triphosphate, henceforward referredto as ATP) which has for some time past been recognised as the chemicalchange providing the energy for the muscle contraction.V. A. Engelhardtand M. N. Ljubimova and Ljubimova and Engelhardt lo first showedthat adenosine triphosphatase activity is associated with the myosin fractionof the muscle proteins ; whch the myosin is purified by several precipitations,the power (shown by muscle brei and less purified myosin) to split off thesecond phosphate group is lost..The action of the myosin thus consists insplitting off one phosphate group from ATP with formation of adenosinediphosphate (ADP). These fundamental observations have been confirmedand extended by workers in several lahoratories.ll* 12* 13* 14, lQQ Bailey l4W. T. Astbury and S. DickinsonA. T. Todrick and F. Walker, ibid., 1937, 31, 392.A. E. Mirsky, J . Gen. Physiol., 1936, 19, 559.Proc. Roy. SOC., 1940, B, 128, 307.4 J. P. Greenstein and J. Edsall, J . Biol. Chem., 1940, 133, 397.7 Nature, 1941, 147, 696.* Nature, 1939, 144, 668. 8 Chem. and Ind., 1941, 60, 491.lo Biochimia, 1939, 4, 716.l 1 M. N. Ljubimova and D.Pevsner, &id., 1941, 6, 178.I f A. Szent-Gyorgyi, and I. Banga, Science, 1941, 93, 158.Is J. Needham, S. C. Shen, D. M, Needham, and A. 8. C. Lawrence, Nature, 1041,1' K. Bailey, in the press.147, 766.14a D. M. Needham, in the pressNEEDHAM THE MECHANISM OF MUSCLE CONTRACTION. 243has studied the kinetics and specificity of the reaction, and its activation bymetallic ions, of which Ca" is the most efficient. Needham, Shen, Needham,and Lawrence 13 found a large reversible fall in flow birefringence whenATP was added to myosin sol. This large effect, specific for ATP amongstmany substances tried, may indicate a combination between the myosinand the ATP, such as might be expected between enzyme and substrate.When the stimulus to contraction reaches the muscle fibril, the im-mediate change providing energy seems to be the breakdown of ATP withformation of ADP and inorganic phosphate.(It is at present uncertainwhether this breakdown is simultaneous with the shortening of the fibrils,or whether it is the first of the " recovery processes " bringing about lengthen-ing and '' recharging " of the contracted fibrils.) It is well known that theATP thus broken down is built up again by three distinct processes : (a)by reaction with creatine phosphate ; ( b ) by reaction with phosphopyruvate ;( c ) by esterification of phosphate coupled with the oxido-reduction betweentriose phosphate and pyruvate. The nature of the last process has recentlybeen elucidated by 0. Warburg and W. Chri~tian.1~ It had beenshown 16, 17, 18. 19 that the part of this oxido-reduction essential for theesterification is the reaction between triose phosphate and co-enzyme I :Triose phosphate + Co + ADP + H,PO, --+Phosphoglyceric acid + CoH, + ATP .. (1)D. M. Needham and R. K. Pillai,m using extracts of muscle acetone powder,had shown that arsenate affects the course of the reactions in that theoxido-reduction continues in its presence while the disappearance of inorganicphosphate and the formation of ATP is prevented. Meyerhof et aZ.,19using more purified enzyme preparations from yeast, had shown that thereaction between triose phosphate and co-enzyme alone proceeds onlyslowly and soon stops. The reaction goes further if ADP and inorganicphosphate are present, and practically to completion if glucose is alsopresent.Meyerhof et ul.19* 21 had recognised that the whole process (equation1) is reversible; further, they had found that in presence of arsenate, evenin absence of ADP, the reaction between triose phosphate and co-enzymegoes to completionTriose phosphate + Co --+ Phosphoglyceric acid + CoH, . . (2)E. Adler and G. Gunther,22 using partly purified preparations from brainand yeast, had also drawn attention to the fact that this reaction (2) comesto a standstill before the reactants are exhausted, as if at an equilibriumBiochem. Z., 1939, 305, 40.l6 D. M. Needham and R. K. Pillai, Biochem. J., 1937, 81, 1837.0. Meyerhof, W. Schulz, and P. Schuster, Biochm. Z., 1937, 298, 309.D.M. Needham and G. D. Lu, Biochem. J . , 1938, 32, 2040.0. Meyerhof, P. Ohlmeyer, and W. Mohle, Biochem. Z., 1938, Zg7, 90.2n Nature, 1937, 140, 165.2 1 0. Meyerhof, P. Ohlmeyer, and W. Mohlo, Biochem. %., 1938, 297, 193. '' Z . physiol. Chem., 1938, 258, 153244 BIOUHEMTSTRY .point. This equilibrium, however, cannot be altered by adding phospho-glyceric acid, and they had concluded that an unknown intermediate mustbe involved. They also had found an effect of arsenate, which caused thereaction to go rapidly to completion. Warburg and Christian were ableto link together and explain all these facts. They obtained from yeast apure crystalline preparation of the enzyme responsible for the oxidationof glyceraldehyde phosphate by co-enzyme. Like Adler and Gunther,they observed an apparent equilibrium point not affected by addition ofphosphoglyceric acid, and in presence of arsenate the reaction went tocompletion.They observed also that, in absence of arsenate, phosphateis necessary for the reaction, and phosphate concentration affects theequilibrium point. These observations led to the isolation by E. Negeleinand H. Bromel 23 of the intermediate 1 : 3-diphosphoglyceric acid. Further,by addition of another specific enzyme preparation to the crystalline triosephosphate dehydrogenase, a system was obtained which could transferphosphate from the diphosphoglyceric acid formed to ADP. The seriesof reactions involved in the coupled phosphorylation of ADP may thereforebe formulated thus :Glyceraldehyde phosphate + H,PO, 2 Glyceraldehyde diphosphateGlyceraldehyde diphosphate + Co 2 Diphosphoglyceric acid+CoH,Diphosphoglyceric acid + ADP 2 Phosphoglyceric acid + ATPThe effect of arsenate is explained by Warburg and Christian in the followingway.Arsenate can replace phosphate in the reaction and an arsenylatedglyceraldehyde phosphate is formed and oxidised ; the arsenylated phospho-glyceric acid is, however, unlike the diphosphoglyceric acid, unstable underthe experimental conditions, and breaks down irreversibly. 1 : 3-Di-phosphoglyceric acid thus takes its place with creatine phosphate andphosphopyruvic acid in the chemistry of muscle contraction as one of thesources of energy and of phosphate for the reconstitution of ATP. Thewhole subject of the importance of the guanidine phosphate, enolphosphate and carboxyl phosphate groupings for transfer of energy has beenconsidered by H.Kalckar.= D. M. N.4. PHYSICOCHEMICAL PHENOMENA.Intracellular Gels and Studies on Gel-forming Systems of the Cytoplasm.In the last two or three decades many interesting studies were made ongelatin sols and gels. These studies have been profitable from a physico-chemical point of view but, except in the most abstract manner, have notcontributed to our knowledge of the part played by gels in biological systems.For this there are two causes : gelatin is not, so far as we know, one of thesubstances responsible for intracellular gelation ; nor has the informationavailable on the cytological level made it at all plain, until quite recently, what23 Biochem.Z., 1939, 303, 231.24 Chem. Rewiewa, 1941, 28, 71; Biol. Rev., 1941 17, 28DANIELLI : PHYSICOCIHEMICBL PHENOMXNA. 245importance must be attached to intracellular gels, or from what point of viewthe intracellular gels may most profitably be studied. In the last five or tenyears there has been a substantial incregse in our knowledge of the functionof gels in cells, and also a growing volume of work on the properties of myosin,the chief gel-forming substance in muscle cells.Micro-dissection 1 has made a most useful contribution by demonstratingthat the cortical cytopltwmic layer of many cells is gelled and that in theresting cell the bulk of the remaining part of the cytoplasm is comparativelyfluid. The asters, spindle and chromosomes of dividing cells are gelled elasticbodies, and in some instances are known to be birefringent.2 One of the mostvaluable results is that of E.N. Harvey and D. A. Mar~land,~ who observedcells through the microscope during the process of centrifugation. They foundthat, even in the “ fluid ” part of the cytoplasm, intracellular particles maymove in an intermittent fashion under centrifugal force ; this is an indicationthat even the fluid part of the cytoplasm may be weakly gelled or undergointermittent sol gel transformations. These various observations havemade it certain that gels exist in living cells, and that many of the observablebodies in the interior of cells are elastic gels : from birefringence studies ithas been concluded that many of these bodies consist of adlineated needle-shaped protein particles.2 Some of these gels are known to be thixotropic.1The importance of these observations on the physicochemical conditionof the interior of the cell has been emphasised by a series of studies on theeffect of moderately high pressure on cells.Earlier work in this field has beenreviewed by McK. CatteL4 The important feature of more recent work is thedemonstration that in a wide variety of cell types the application of pressurereduces the cytoplasmic viscosity and eventually liquefies the cortical gellayer. The liquefaction usually occurs at a pressure of the order of 400 atms. ;it is followed immediately by profound changes in behaviour.Cells ofirregular shape round up into ~pheres,~ long tentacles break up under surfacetension forces into a string of spherical droplets,6 protoplasmic streamingceases,’ and dividing cells which have reached the “ dumbell ” stage revertto the spherical state.* The liquefaction of the gel is reversible. For eachincrement of 68 atms. pressure a number of cell characteristics were found tobe diminished to 0.76 of their former value. This is true of the cytoplasmicviscosity of ova of two species of Arbacia, the viscosity of two species ofA m d a and of the leaf cells of Elodea ; the rate of impingement of the cleav-age furrow on the division axis in two species of Arbacia; and the velocityof protoplasmic streaming in Elodea. The view has been advanced that theliquefaction of the gels is due to hydration of protein molecules; but, asR. Chambers, 1924, in “ General Cytology,” Ed.Cowdray, Chicago ; J . Cell. Comp.L. E. R. Picken, Biol. Rev., 1940,15, 133.J . Cell. Comp. Physwl., 1932, 2, 75.Physiol., 1938, 12, 149.4 Biol. Rev., 1936, 11, 486.6 D. A. Marsland and D. E. S . Brown, J . Cell. Comp. Physiol., 1936,8, 167, 171.8 J. A. Kitching and D. C. Pease, aid., 1939,14,133.7 D. A. Marsland, &id., 1938,12,57. D. A. Marslsnd, ibid., 1939,13, 15,23246 RTOCHEMISTRY.Cattell remarks, at present the possibility has not been eliminated that " thechanges in viscosity are secondary to changes resulting from stimulation orchemical reactions." It is known that even globular proteins such as oval-bumin, serum albumin and insulin form thixotropic gels and show anisotropyof flow when denatured, or when their SS bonds are reduced to SH.g What-ever the explanation of these changes may be, it is now clear that sol * gelchanges play an essential r61e in cell behaviour, and that physicochemicalstudies on the molecules concerned in these changes are of great interest.These studies have so far been restricted, with few exceptions, to observ-ations on myosin, the protein forming the spontaneously birefringent gel ofmuscle fibres.* Several observers lo have found that when a muscle passesinto rigor a large part of the myosin is no longer soluble in potassium chloridesolution.A. E. Mirsky 11, 12, l3 has investigated the nature of this changeand its relation to muscular activity.When the thixotropic gel formed bynative (soluble) frog myosin is heated to 38-39", its solubility is lost. TheQ1, for this reaction is about 1,000. There is little change in the SH groups.A small diminution in transparency of the gel occurs. If the temperatureis raised further ( 4 1 4 5 " ) , the gel-structure is destroyed, opaque clumpsforming, and SH groups appearing. The reaction resulting in appearanceof SH groups has a much lower Qlo than the reaction producing insolubility.With previously studied proteins these two steps (loss of solubility andappearance of SH groups) appear to be more closely linked than with myosin.Physiological processes corresponding to these changes are thermal (revers-ible) shortening of frog muscle,14, l5 which has a sudden onset a t 38".Thisthermal contracture, which Mirsky considers to be due to the first step inmyosin denaturation (loss of solubility), is of the same order of magnitude asthe contraction in an electrically produced tetanus. A further shorteningoccurs when the muscle is heated from 39" to 45" ; this process is irreversibleand is regarded by Mirsky as due to the change in insoluble myosin which isaccompanied by appearance of SH groups.I n vitro the initial denaturation change (loss of solubility) may also beproduced by simple physical means, such as dehydration by cold and bysubstances such as caffeine, nicotine, chloroform and glycocholate. Vera-trine does not initiate or accelerate denaturation.This may be regarded asstrong evidence for the view that the initial stage of denaturation is involvedin muscular contraction, for the first group of substances throw a muscle intoD W. G. Myers, Cold Spring Harbor Sympot&rn, 1938, 6, 120; H. Neurath, ibid.,10 P. Sax], Beitr. Chem. Physiol., 1907, 9, 1 ; A. E. Mirsky, J . Qen. Physiol., 1935,11 J . Gen. Physiol., 1937, 20, 455.12 Ibid., p. 461.13 Cold Spring Harbor Symposiun~, 1938, 6, 153.14 E, Gottschlich, Pflugers Arch., 1893, 54, 109; 55, 339.16 P. Jensen, ibid., 1914, 160, 333.* The physical chemistry of the gels of the nucleus has had some slight attention,p. 1 1 8 ; C. Stern, ibid., p. 119 ; A. White, ibid., p. 265.19, 571 ; E. B. Smith, Proc. Roy. SOC., 1937, B, 124, 136.but not sufficient for discussion hereDANIELLI : PHYSICOCHEMICAL PHENOMENA.247a reversible contracture 16 (sustained single contraction), whereas veratrineproduces a similar result, but by acting on the nervous system to give atetanus 17 (many consecutive twitches).A similar change in solubility after activity has been demonstrated in themyosin of crab limb muscle,l* and in a myosin-like protein found in sea-urchin eggs.19A second approach to the subject has been made by Needham et aZ.,20who have endeavoured to link the phosphorylation cycles, whereby it is nowbelieved that the energy obtained from carbohydrate oxidation is transferredto the contractile apparatus of the muscle, with the long-established fall inbirefringence of a muscle occurring during contraction.It had previouslybeen shown by A. Muralt and J. T. Edsal121 that myosin solution shows doublerefraction of flow. More recently W. A. Engelhardt and M. N. Ljubimova 22have found that myosin is so closely associated with the enzyme adenylpyro-phosphatase that it is possible that myosin is itself the enzyme. As thebreakdown of adenylpyrophosphate is believed to be the nearest in time tocontraction of the known chemical processes occurring in active muscle,studies were made of the effect of adenylpyrophosphate on the flow birefring-ence of myosin. These experiments were carried out with a sol containingabout 3% of myosin in O.75~-potassium chloride and other solutions of equiv-alent ionic strength at pa: 7. Adenylpyrophosphate causes a large andprolonged fall in birefringence, which occurs in less than one minute at about18" and takes considerably longer a t 0".This change is reversible. Thebirefringence returns to its original value in about two hours at 37", andmore slowly at lower temperatures.This observation may well prove to be a key to the connection betweenmuscle intermediary metabolism and the transformation of energy intomechanical work, especially if it proves possible to combine studies of viscosityand birefringence with X-ray studies. W. T. Astbury and S. Dickinson 23have now shown that a process occurs in muscle protein during contractionwhich is closely analogous to supercontraction of keratin in which the cross-linkages of adjacent polypeptide chains of keratin have been broken byreduction of SS bonds : observation of the occurrence of such processes inrelation to the action of adenylpyrophosphate would clinch the argumentthat this in vitro action of adenylpyrophosphate is closely analogous to theprocesses occurring in living muscle. In addition, X-ray studies are probablyessential for making a detailed analysis of the action of adenylpyrophosphate,16 K.J. A. Secher, Arch. exp. Path. Pharm., 1914, 77, 83; H. N. Langley, J PI~ysiol.,1907, a, 347; G. Schwenker, Pjluqers Arch., 1914, 157, 443.1 7 G. Lamm, 2. Biol., 1911, 56, 223; 1912, 68, 37.18 J. F. Danielli, J . Physiol., 1938, 92, 3P. A . E. Mirsky, Science, 1936,84,333.20 J. Needham, S . C. Shen, D. M. Needham, and A.S. C. Lawrence, Nature, 1941,147,'1 J . B i d . Che))i., 1930, 89, 315, 351.22 Nature, 1939, 14, 668;23 Proc. Roy. Soc., 1940, B, la, 307.766.Biochiwia, 1939, 4, 716: A. Szent-cyorgi an<i 1. Bang,Science, 1941, 93, 158248 BIOCHEMISTRY.for this probably cannot be achieved from studies of viscosity and doublerefraction only.Other studies of the action of ions on myosin flow birefringence have beenmade by Edsall and his colleague^.^^ The properties of threads of gelledmyosin have also received attention.2sAll of the studies so far reported have, implicitly or explicitly, left theproblem of intermicellar forces untouched. Since myosin in the muscle fibreis gelled, these forces must be of significant magnitude, and it may be thattheir study will enable a connection to be established between the responseof a muscle fibre to a stimulus, and the observed changes in myosin bire-fringence and X-ray scattering during contraction : this connection is a tpresent quite obscure. Important theoretical studies of intermicellar forceshave been made by Langmuir and Levene.26 J.F. D.5. SOME PLANT PRODUCTS AXD ENZYMES.With the advent of the United Stateg as a full belligerent, the diminutionin the number of publications observable in recent months will doubtlessbecome more marked. There appears to be no major development in plantbiochemistry during the past year, but steady advances in knowledge havebeen maintained. The plant growth substances are amongst the mostinteresting and important of plant products. I n this field, following thepreparation of crystalline biotin, the constitution of this somewhat elusivemember of the bios group is likely to be determined in the near future.Knowledge of the chemistry of the hydrolytic enzymes has lagged behindthat of those enzymes concerned in oxidation and allied processes, Theisolation in the crystalline state of increasing numbers of the former and thestudy of the properties of the pure enzymes are leading steadily to morecomplete understanding.These topics, together with some notes on plantproteins, are discussed in the following pages.Growth Substances.-A concise review of growth substances in theirpractical and commercial aspects is contributed by M. A. H. Tincker.1The methods of detection and estimation of growth substances are under-going continuous modification and elaboration; but a tendency is alsot o be noted towards simplification.Thus, the Avena method of auxindetermination requires elaborate apparatus and technique not readilyavailable to many workers. An upright-growth method which can beapplied to cut pea shoots is described by E. G. Brain,2 who has found i t ofvalue in comparative experiments under ordinary greenhouse conditions,24 J. P. Greenstein and J. T. Edsall, J . Biol. Chem., 1940,133, 397; J. T. EdsaIl andJ. W. Mehl, ibid., p. 409.26 H. H. Weber and K. Meyer, Biochem. Z . , 1933, 266, 137 ; H. H. Weber, PJEiigersArch., 1935, 235, 205; W. A. Engelhardt, 3%. N. Ljubimova, and R. A. Meitina, C m p t .rend.U.R.S.S., 1941, 30, 644.26 I. Langmuir, J. Chem. Physics, 1938,6, 873; S. Levene, Proe. Roy. SOC., 1939, A ,17'0, 145.Nature, 1941, 147, 439. ' Ibid., 1941, 148, 666NORSCIS : SOME PLANT PRODUCTS AND ENZYMES. 249and although not as exact as the more refined methods there are indicationsthat its accuracy may be increased as the result of further experiments.G . E. Turfitt3 has described a rapid method of testing substances forphytohormone activity, using yeast growth as the criterion, such growthbeing assessed by multiplication rates based on cell counts. p-Indolyl-,a- and p-naphthyl-, and phenyl-acetic acids in concentrations of i$ to 1 partper million cause varying degrees of stimulation; further increases in con-centration may cause a diminution or even inhibition of growth.As nostimulation is observable with washed yeast, it is thought that the action ofsubstances such as those mentioned is combined with that of the biossubstances, which would be removed by washing.Experiments by E. J. Kraus and J. W. Mitchell,47 5 on bean plants, whichshowed characteristic responses, indicate that a-naphthylacetamide may beadded to the extending list of plant growth substances. Treatment oncut stems was carried out with the compound in lanolin, or in aqueousemulsion with lanolin ; seedlings were also sprayed.The wound hormone, traumatic acid, which promotes wound peridermformation in potato, has been shown to be Al-decene-1 : 10-dicarboxylicacid and it is of interest to enquire whether acids of the same general typeare equally effective.J. English has prepared by synthesis a number ofanalogues of the acid of the general type C02H*[CH2]n*CH:CH*C02H andCQ,H*[CH2],~CH2*CH,*C0,H. A5-Undecene- I : 1 P-dicarboxylic acid andA1 : 7-octadiene-l : 8-dicarboxylic acid were also prepared and examined.All the acids thus synthesised were found to be active plant wound hormones.Bios.-As indicated in last year’s Annual Reports,7 it has been sug-gested that biotin is identical with vitamin H, a substance which protectsrats from “ egg-white injury.” Full confirmation of the identity appearsto be forthcoming as the result of experiments which demonstrated that theactivity of the vitamin in stimulating yeast growth is identical with that ofbios, and conversely that biotin may be used to remedy deficiency of thevitamin in rats.These confirmatory results were obtained by V. du Vigneaud,D. B. Melville, P. Gyorgy, and C. S. Rose.s It is also probable that coenzymeR, a growth factor for many strains of legume nodule bacteria, is identicalwith biotin and vitamin H (see also this vol., p. 235).The isolation of biotin as the free acid rather than the ester marksa considerable step forward, since not only is it desirable in some biologicalinvestigations t o employ the free acid, but a line of chemical attack onconstitution has been opened up. The isolation of the free acid in crystallineform is described by V. du Vigneaud, K. Hofmann, D. B. Melville, andJ. R. Ra~hele.~ The empirical formula ascribed to it is ClOHl6O3N2S,and titration curves indicate that the compound is a monocarboxylic acid.There is no specific absorption in the ultra-violet.Biochem.J., 1941, 35, 237.J. W. Mitchell and W. S. Stewart, ibid., p. 410.J . Arner. Chem. SOC., 1941, 63, 941.Science, 1940, 92, 62.Bot. Baz., 1939, 101, 204.Ann. Reports, 1940, 37, 393. * J . Biol. Chern., 1941, 1Q0, 763250 BTOCHRMTSTBY.Using the crystalline preparation above, G. B. Brown and V. diiVigneaud lo have studied the stability of biotin towards a variety of reagentsand treatments, employing yeast growth as a criterion of activity. It ha?sbeen found that alkali or acid treatment results in inactivation, and that,whilst biotin is inactivated by many reagents which react with a-amino-acids, it is not affected by ninhydrin, a fact held to indicate strongly thatbiotin is not an a-amino-acid.Acylating, alkylating and carbonyl reagentsdo not inactivate biotin. Aeration under various conditions with air oroxygen has no effect, but activity is rapidly lost under the action of strongeroxidising agents such as hydrogen peroxide or bromine water ; it is concludedthat biotin contains an easily oxidisable group or groups. These experimentson activation have led to a direct chemical attack on the crystalline biotinwith a view to disclosing the nature of the functional groups present in themolecule. The conclusion arrived at in the previous communication, thatno a-amino-groups are present in the molecule, is confirmed by K.Hofmann,D. B. Melville, and V. du Vigneaud; 11 no nitrogen is produced whenbiotin is treated with nitrous acid by the van Slyke method, and the ninhydrinreaction is negative. Treatment of biotin with baryta at 140" for 20 hoursled to the isolation as sulphate of a " diaminocarboxylic acid," C9Hls02N2S,which gave a dibenzoyl derivative, contained two free amino-groups and musthave been derived from the parent biotin with the loss of one carbon and oneoxygen atom. The most likely explanation of such a change suggests that acleavage of a cyclic urea derivative is involved as below :+02H + C8H13S -NH2 ( [ zzo2H) 'SH13'[ ~~~~The nature of the sulphur atom was indicated as follows : Biotin containsno alkali-labile sulphur and does not liberate hydrogen sulphide when treatedwith zinc dust and hydrochloric acid. Further, no positive nitroprussidetest could be obtained in presence or absence of sodium cyanide.Thestability of the sulphur therefore indicates a probable thioether structure andexperimental evidence supports this. Thus, biotin (I) on treatment withhydrogen peroxide and glacial acetic acid gives a crystalline sulphone (11)in 90% yield. There is no loss of carbon or hydrogen involved in the change,but two oxygen atoms are added. The production of a strong yellow colourwhen biotin and the diamino-carboxylic acid are treated with tetranitro-methane, and the failure to produce a colour when the oxidation product issimilarly treated, are also consonant with the suggestion that a thioetherstructure is involved, At this stage, then, the authors conclude that biotinis a carboxylic acid containing an NN'-substituted cyclic urea grouping,and sulphur in thioether linkage.-C02H--NH -NH>C 0 (11.) 1 -so,lo J .Biol. Chem., 1941, 141, 85. l1 Ibid-, p. 207NORRIS : SOME PLANT PRODUCTS AND ENYZMES. 251M. J. Pelczar, jun., and J. R. Porter 12 have shown that pantothenic acidis one of the growth factors for Proteus morganii, and in a later paper l3observe that this organism is specially suitable for the biological assay ofpantothenic acid in natural materials. The response of the organism topantothenic acid is extremely sensitive and specific, since 0.0002 pg. of thecalcium salt is sufficient to initiate visible growth.In a number of teststhe amounts indicated by the result of the assay corresponded closely withthe amounts known to be present. The extreme sensitivity of the organismrenders it specially suitable for dealing with small amounts of the materialto be examined.Methods of estimation of inositol involving isolation of the product fromthe material under investigation have not proved satisfactory, and more-over are unsuitable when dealing with small quantities. D. W. Woolley l4has developed a method of estimation based on the growth response of yeastto the presence of inositol in a medium' which in its absence supportedpractically no growth. Graded amounts of inositol were found to inducegraded responses, such growth being measured by a turbidimetric method.Quantitative results were obtained with a number of natural products.I>.W. Woolley l5 has also studied the biological specificity of meso-inositolin respect of mice and of yeast, and shown that in the latter case suchspecscity is virtually absolute. Thus for yeast, the following substancesare inactive : d-inositol, Z-inositol, pinitol, quebrachitol and quercitol ;inositol hexa-acetate, phytin and soy bean cephalin; quinic acid andinosose. Mono- and tetra-phosphates of inositol were only 5 and 20,;respectively as potent for yeast as inositol, and mytilitol (probably methylinositol) had about one-tenth of the activity of inositol.Additions to the list of growth factors for lower organisms are still forth-coming. Thus, following the observation of E. E.Snell and W. H. Peterson l6that there existed a new growth factor, probably a purine, for Lactobacilluscasei, E. L. R. Stakstad 1' reports the isolation of a dinucleotide or mixtureof nucleotides from solubilised liver. This was obtained by adsorption onnorit, followed by elution with a dilute solution of ammonia in 70% methanol.The product was purified until no increase in activity could be observed.It had the properties of a nucleotide, since it contained nitrogen, phosphorusand a pentose (not deoxyribose). Hydrolysis showed that the factorcontained a purine and a pyrimidine nucleotide, the purine base beingguanine, the pyrimidine base still awaiting identification. The " dinucleo-tide " may be partially replaced by guanine and thymine, and the formercould be effectively replaced by adenine, hypoxanthine and xanthine. Uracilor cytosine could not replace thymine.This work is in line with that ofE. E. Snell and H. K. Mitchell,l8 who have found that both purine andpyrimidine bases are essential factors in the growth of Lnctobacillus nra binosus,Lactobacillus pentosus and Leuw nostoc rnesenteroides.l2 Proc. Xoc. Exp. Biol. Med., 1940, 43, 151.l4 Ibid., 1941, 140, 453. l6 Ibid., p. 461.l3 J. Bid. C'hein., 1941, 139, 1 1 1 .16 J. Bact., 1940, 56, 273.J. BioE. Chem., 1941, 139, 478. l* Proc. Nat. Acad. Sci., 1941, 27, 1252 BIOOHEMISTRY.In concluding this section, reference may be made to an excellent review ofgrowth-promoting nutrilites for yeasts by R.J. Williams.19Following the preparation of crystalline papainby A. K. Balls and H. LineweaverY2O a further communication by A. K.Balls and E. F. Jensen 21 describes the preparation of a new crystallineenzyme, which the authors propose to term chymopapain, by analogywith the chymotrypsin of M. Kunitz and J. H. N ~ r t h r o p . ~ ~ The newenzyme is extremely stable a t 10" and pH 2.0, and this fact was utilised inits preparation. At pn 2.0 an extenaive precipitation of protein from asuspension of coagulated papaya latex is effected by hydrochloric acid, andthe protein remaining in solution is proteolytically active, but an inertfraction is still present. This is precipitated by half saturation with sodiumchloride at pH 4.0, and the active crystallisable material may be precipitatedby addition of hydrochloric acid to the solution fully saturated with sodiumchloride. The crystals obtained were much more soluble than the originalpapain under the same conditions, showed a strong positive nitroprussidereaction, and the amount present in latex was much greater than that ofpapain.In further studies of the action of papain on proteins, H.Lineweaverand S. R. Hoover 23 find additional support for the suggestion that enzymeaction is greater on denatured proteins than on the native protein. Thus,in the case of papain, it was found that the initial rate of digestion of haemo-globin in the presence of a t least six molar concentrations of urea is verymuch greater than in water solution only.The increase in digestibility issimilar to, but not exactly parallel to, the decrease in solubility of haemo-globin when denatured by urea. The increase in rate of hydrolysis ofproteins when denatured appears to be comparable to the increase in re-activity of -SH, S - S - and tyrosine phenol groups. The method wherebydenaturation is effected seems to have little effect on the rate of digestion.The increase in rate of digestion of a denatured protein is different for eachenzyme, and that for a number of diff erent proteins treated by a single enzymeis also different.The widely adopted oxidation-reduction theory of papain activationis not in harmony with experimental facts observed by J. S. Fruton andM. Bergmann,24 and, in a later paper, by these authors and G.W. Irving,jun.,Z5 who do not accept this explanation. In the first instance, papainwas activated by hydrocyanic acid, inactivated by precipitation withhopropy1 alcohol, and the whole process repeated, at the end of whichalmost complete recovery of the original activity of the enzyme was observed.These facts are difficult of interpretation in terms of the disulphide-sulphydryltheory; moreover, the action of hydrocyanic acid on disulphide linkageswill produce only one sulphydryl group : R-S-SR' + HCN + R*SH +Enzymes.-Papain.lS Biol. Rev., 1941, 18, 49.z1 J . Bid. Chern., 1941, 137, 459.48 J. Biol. Chern., 1941, 137, 325.f6 IN., 1941, 130, 669.20 Ann. Reports, 1940, 37, 429.aa J . Gen. Physiol., 1935, 18, 433.z4 Ibid., 1940,133, 153NORRIS : SOME PLANT PRODUCTS AND EXZYMES.263R’oSCN. The authors suggest that a better explanation is afforded bysupposing that hydrocyanic acid combines with papain to form a dissociablepapain-hydrogen cyanide compound corresponding to the hydrogen cyanideactivated enzyme. On precipitation with isopropyl alcohol, the compounddissociates and the precipitate consists of the hydrogen cyanide-free enzyme,inactive towards synthetic substrates. In the later paper it is pointedout that natural activators are usually present in preparations of proteinaaes,and minute quantities of these activators profoundly affect the response t oadded activators. In the case of papain it was found that, if the naturalactivators were removed by careful dialysis, subsequent addition of hydro-cyanic acid involved no activation of the enzyme.The activation normallyobserved with hydrocyanic acid thus depends on the presence of naturalactivators, which may occur only in minute traces. Benzoyl-Z-arginineamidebeing used as artificial substrate in the study of reaction kinetics, it has beenfound that the component of papain (and of beef spleen cathepsin) whichhydrolyses the substrate may exist in two inactive forms. The a-form isnot activated by hydrocyanic acid, but may be converted into the p-form,which is, and the activation consists in the formation of dissociable com-pounds of the activator and the p-form. It is noteworthy also that theactivation does not involve the mutual transformation of disuIphide andsulphydryl groups, nor is there any evidence of reduction or oxidationprocesses.Urease.-The presence of sulphydryl groups in urease has been established,and the activation of the enzyme by certain reducing agents, and its inactiv-ation by oxidising agents, suggest that the activity of the enzyme may be afunction of oxidation-reduction potential.The activity of crystallineurease was determined by I. W. Sizer and A. A. Tyte11,26 who employedsubstrates which were adjusted a t varying Eh by a number of oxidisingand reducing agents used separately or in mixture, and also by the use ofsodium sulphide and potassium permanganate at various concentrations.Curves similar to the familiar activity-p, curves were obtained in all cases,and an optimum Eh of + 150 mv.was indicated. The activity of crudeurease, in contrast to that of crystalline urease, was unaffected by variationsin Eh of the substrate. In a note to the paper it is pointed out that the Ehof the jack bean after soaking in water is + 190 mv., a value in such closeagreement with the optimum Eh of the enzyme as to suggest some phy&o-logical significance. The authors stress the somewhat empirical nature oftheir observations; but this is probably the first example of the productionof activity-& curves, and examination of other enzymes in this aspect mayyield important results.An improved method for the preparation of crystalline urease is de-scribed by A. L. D o ~ n c e , ~ ~ whose modification of the original method ofJ. B.Sumner 28 involves a very considerable shortening of the time requiredfor crystallisation, thus avoiding denaturation of the enzyme.26 J . Biol. Chern., 1941, 138, 631.28 Ibid., 1926, 70, 97.t 7 Jbid., 1941, 140, 307254 BTQCREMISTRY.RibonucZease.4rystalline ribonuclease isolated from fresh ox pancreasdigests yeast nucleic acid, the products being of low molecular weight.M. Kunitz describes the isolation and properties of the crystalline enzyme,which appears to be a protein of the albumin type of molecular weightabout 15,000. The enzyme is stable over a wide range of pPa and particularlyover the range pE 2 . 0 4 . 5 . Denaturation of the protein comprising theenzyme involves a corresponding loss in its activity.Similar values for themolecular weight have been found by A. Rothen,3O who gives 12,700 fromrate of sedimentation and diffusion data, and 13,000 from equilibriummeasurements. The purified crystalline enzyme had an isoelectric pointat pa 7.8 by electrophoresis. All these values are in good agreement withthose computed by I. Pank~chen,~l who has made crystallographic andX-ray studies of the enzyme and calculates 15,700 and 13,700 for the hydratedand the anhydrous protein respectively.F. W. Allen and J. J. Eiler 32 also have prepared the crystalline ribonucleaseand have employed it in studies of its action on ribonucleic acid. It is possiblethat the enzyme is in the nature of a depolymerase, but available evidencepoints to the probability of a low degree, if any, of polymersation in thecase of ribonucleic acid, although deoxyribonucleic acid probably exists in ahighly polymerised state.K. Makino 33 and J. M. Gulland 34 hold that ribo-nucleic acid shows four primary phosphoric acid dissociable groups and nosecondary phosphoric acid dissociations, and titration experiments by theabove authors confirm this. The liberation of a, fifth acidic group by thecrystalline enzyme is thought to denote the opening of a cyclic structure suchas that envisaged by H. Takaha~hi.~~The preparation of the proteins of green leaveshas involved a number of technical difficulties, and, since the preparationof spinach proteins by T. B. Osborne and A. J. Wakeman36 in 1920, theinvestigations in this field have been carried out largely by A.C. Chibnalland his school. This work is so well known that a reference to his im-portant book3' must suffice here. Amongst recent publications on thesubject, those of J. W. H. Lugg may be mentioned. In an early paper38he deals with the estimation of tyrosine and tryptophan in the hydrolysatesof leaf proteins and suggests means of overcoming previous difficultiesinvolving unsatisfactory results. This was followed 39 by a, series of experi-ments whose main object was to determine the most satisfactory hydrolysisprocedures with a, view to subsequent estimation of tyrosine and tryptophanin the hydrolysates. Hydrolysis in sealed tubes at 100" with alkali or alkalistannite solutions was found to be satisfactory. Two further papers appearedin 1938 4O devoted to the partial analysis of protein preparations from grasses,Proteins.-Leaf proteins.29 J.Qen. Physiol., 1940, 24, 15.31 Ibid., p. 315.33 2. phgsioll. Chem., 1935, 236, 201.s 5 J . Biochem. Japan, 1932, 16, 463.3' " Protein Metabolism in the Plant," Pale Univ. Press, 1939.38 Biochern. J., 1937, 31, 1422.40 Ibid., pp. 2114, 2123.Ibid., p. 203.32 J . Biol. Chem., 1941, 137,34 J . , 1938, 1722.36 J . Biol. Chem., 1920, 42,3e Ibid., 1938, 32, 775.757NORRIS: SOME PLANT PRODUCTS AND ENZYMES. 255including cocksfoot and lucerne. The proteins were prepared by methodslargely elaborated by A. C. Chibnal14143 and co-workers, and in the firstinstance the sulphur distribution was determined, it being shown that thecontents of cystine and methionine were sufficiently high to conform tonormal standards of the nutritional requirements of animals.Secondly, inaddition to the sulphur distribution, the amide, tyrosine and tryptophancontents of leaf proteins of various Chaminern, Leguminosce and Chenopodiaceawere determined. Here again it was shown that in respect of the aboveamino-acids, the leaf proteins compared favourably with other proteins ofthe animal diet. Later,44 various methods of extraction of leaf proteinswere employed, and the samples examined for representativeness. Additionof lipoid solvents to mildly alkaline leaf juices allows most of the granuleprotein to pass into solution, whereas difficulty in avoiding loss of this fractionhad previously been experienced.It was also shown that the presence ofalcohol in the protein solutions near their isoelectric point rendered floccul-ation by acid, and coagulation by heat, more complete. In the latest paperto date, J. W. H. L ~ g g * ~ finds that the amide, tryptophanand tyrosine contentsof proteins of the photosynthesising tissues of some cryptogams are of thesame order of magnitude as those of the leaf proteins of angiosperms of thespermatophyte division ; the tryptophan content of preparations fromPteridium aquilinum is lower than any previously encountered.The basic amino-acids of leaf proteins have been determined by G. R.Tri~tram,~'j who made a critical examination of methods available, andstudied the influence of the presence of carbohydrates on the results obtained.More satisfactory estimation of the three bases in leaf proteins containing12-16% of nitrogen was secured, but where the material was poor in protein,containing less than 8% of protein nitrogen, lysine only could be estimatedreliably.As in the case of the amino-acids determined by J. W. H. Lugg( v J . ) , there appears to be very little variation in content from species tospecies, nor is there much seasonal variation within a single species.A. M. Smith and T. Wang4' also have prepared proteins from grassesand have determined the sulphur distribution in proteins from four species.The amounts of cystine and methionine were substantially the same in allspecies; but it was observed that in proteins from samples that had reachedor passed the flowering stage the amounts present were significantly higher thanin those from young grass, or grass kept short by grazing.General.-Knowledge of the proteins of the latex of Hevea brasiliensis issomewhat scanty, although there are strong indications of their importancein the complicated bio- and physico-chemical mechanism of rubber latexcoagulation.Thus, it has been shown by C. Bondy and H. Freundlich48and by A. R. Kemp and W. G. Straitiff 49 that the stability of latex is41 Biochem. J . , 1921, 15, 60; 1922, 16, 334.*% J . Biol. Chem., 1923, 55, 333; 1924, 61, 303.43 Biochern. J . , 1926, 20, 108; 1933, 27, 1879.44 Ibid., 1939, 33, 110.46 Ibid., 1939, 33, 1271.48 Compt. rend. Lab. Carlsberg, 1938, 22, 89.4 6 Ibid., 1940, 34, 1549.4 7 Ibid., 1941, 35, 404.49 J . Physical Chem., 1940, 44, 78256 BIOOHEML9TRY.dependent upon the proteins present, and the coagulation point of the latexcorresponds to their isoelectric point.G. R. Tristram 50 has isolated a proteinfrom dried latex films and this product has a composition similar in manyrespects to that of the leaf proteins. The similarity is particularly markedin the case of the diamino- and dicarboxylic acids, and suggests a possiblerelationship, which is under investigation, between the proteins of the latexand of the leaves. In a further communication, G. R. Tristram 51 givesanalytical data for a protein prepared from crepe rubber. Close similaritiesbetween this and the latex protein suggest that there is only one proteinin the latter, or that the product obtained from the latex is a mixture of severalproteins.The main bulk of protein is undoubtedly precipitated from thelatex and may be regarded as a representative fraction of the total protein.A loss of tryptophan in the crepe rubber protein is attributed to changesundergone in manufacture. G. S. Whitby and H. Greenberg 52 provide asummary of the arnino-acid composition of latex and latex proteins aspublished to date by previous workers. They have investigated the amino-acids present in the latex of Hevea brasiliensis and have isolated tyrosine,Z-leucine, d-isolewine, d-valine, d-arginine, E-aspartic acid and i-proline.Phenyhhnine is also present but was not isolated.The occurrence of the amino-acid citrulline, discovered by M.Wada 53in the juice of the water melon (Citrullus vulgaris), has suggested to P. S.Krishnan and T. K. Krishnaswamy64 the possibility that the seeds ofthe fruit may be a source of the amino-acid. The proteins and other nitro-genous substances of the seeds were investigated and a crystalline globulinwas prepared. This represented the bulk of the protein, but a glutelin wasalso obtained in pure condition. Both proteins were analysed and bothcitrulline and canavaaine, free or combined, were absent. Canavanine wasoriginally isolated from jack bean by M. Kitagawa and T. Tomita 55 in 1929,and from soya bean meal by M. Kitagawa and S. Monobe 56 in 1933. It hasbeen shown 67, 58 to have the formulaNH,*C( XH)*NH*O*CH,-CH,*CH( NH,)-CO,H.A modified method of preparation is proposed by M.Damodoran and K. G. A.Namyanim 59 whereby increased yields of the amino-acid may be obtainedfrom the seeds of Canuvalia obtzlsifolia.Reference has already been made to recent modifications in the methods ofhydrolysis of proteins, and of the isolation and estimation of the products.In the space remaining, brief reference only may be made to further de-velopments in protein analysis. For example, B. W. Town 6o publishes, incontinuation of his studies 61,62 on the separation of arnino-acids by means60 Biochena. J., 1940, 34, 301. s1 Ibid., 1941, 35, 413.82 Iba., p. 640. 63 Proc. Imp. Acad., Tokyo, 1930, 6, 15.54 Biochem. J., 1939, 33, 1285.S t Proc. Imp. Acad., Tokyo, 1929,5,380.5 6 J . Biochem. Japan, 1933, 18, 333.5' M. Kitagawa et al., J . Agric. Chem. Soc., Japan, 1933, 9, 845.58 J. M. Gnlland and C. J. 0. R. Morris, J., 1935, 763.59 Biochem. J., 1939, 53, 1742.a1 Ibid., 1928, 22, 1083.so Ibid., 1941, 35, 417.6a Ibid., 1936, 30, 1837STEPHENSON AND KREBS : THE UTEISATION OF CARBON DIOXIDE. 257of their copper salts, an investigation of the dicarboxylic acids of gliadin.He reports the isolation of r-glutamic acid, thought to be a true hydrolysisproduct and not an artefact. Some of the methods of hydrolysis and ofestimation of the hydrolysis products adopted by J. W. H. Lugg (21.8.)have been utilised and found satisfactory by D. M. Doty,sS who proposesmore rapid methods for the estimation of some of the amino-acids, includingtyrosine, tryptophan, arginine, histidine and cystine, in the grain of corn(maize).Amongst methods which have been applied almost solely toanimal proteins, but which may be equally capable of application to plantproteins may be mentioned a micro-method for the determination of basicamino-acids based on a preliminary separation of these substances from theother hydrolysis products by electrical transport. A. A. Albane~e,~* theauthor of the method, follows this procedure by separation of arginine asflavianate with subsequent precipitation of the histidine as the mercuricchloride complex, and estimation of lysine from the residual nitrogen.Finally, M. Bergmann and his associates are developing a new principlewhereby the components of a protein hydrolysate are determined by partialprecipitation as salts, the amount remaining in solution being estimatedfrom a known solubility product. Thus, M.Bergmann and W. H. SteinG5estimate glycine by precipitation with potassium trioxalatochromate, andproline similarly with ammonium rhodanilate. The method has beenadapted to a semimicro-scale by M. Bergmann and H. R. Ing,ss the reactionproducts being filtered a t the centrifuge. Glycine has been estimated in thiscase by means of sodium dioxpyridate, and proline as previously indicated.F. W. N.6. 'YHE UT~LISATION OF CARBON DIOXIDE BY HETEROTROPHIC BACTERIABND ANIMAL TISSUES.Carbon dioxide was €or long regarded as a physiologically inert gas exceptin the case of photosynthetic and chemosynthetic organisms.The firstsuggestion to the contrary was the discovery that carbon dioxide facilitatedthe cultivation of the organism of contagious abortion, BruceZZa abortus.'In 1935 it was found that a number of common bacteria failed to developin media from which carbon dioxide was rigorously removed.2 It is nowknown that this gas reacts chemically in various organisms. So far tworeactions can be defined :(1) CO, + 8H = CH, + 2H20( 2 ) C02 + CH,*CO*CO2H = COzH*CH~*CO*COzHIt is highly probable that other reactions occur.63 I n d . E ? L ~ . Chem. (Anal.), 1941, 13, 1696 6 Ibid., 1939, 128, 217.T. Smith, J . Exp- Med., 1924, 40, 219.a G. P. Gladstone, P. Fildes, and G. M. Richardson, B d .J . Zap. Pa&, 1936, 16,For a review of the literature up t o 1938, see Hes, Ann. Perme&., 1938,4,647.J . Biol. Chem., 1940, 134, 467.d B Ibid., 1939, 129, 603.335.REP .-VOL . XXXVIII 258 BIOCHEMISTRY,Early work on reaction (1) is well sunimarised by H. A. Barker.3 Thekey observation was that of SOhx~gen,~ who showed that enrichment culturesfrom soil fermented a mixture of hydrogen and carbon dioxide to methane;this is to be regarded as a reduction of carbon dioxide by molecular hydrogenanalogous to the reduction of sulphate and nitrate catalysed by hydrogenaseon the one hand and by sulphatase and nitratase respectively on the other.This analogy was pointed out by van Niel (quoted by Barker 3), who postulatedthat the production of methane known to occur anmobicallyfrom a richvarietyof substrates was really an oxidation of the substrate with carbon dioxide act-ing as the hydrogen acceptor.Recently Barker and his co-workers have dem-onstrated that in a number of cases this is indeed so ; the difficulty of isolatingmethane bacteria has been largely overcome and each species isolated has beenshown to effect the oxidation of certain groups of compounds by hydrogentransfer to carbon dioxide. It now seems probable that the reductionof carbon dioxide is the sole means by which methane is formed in ferment-ations. Thus Methanobacterium Omelianski, isolated by Barker,4 oxidisesthe following alcohols in the presence of carbon dioxide to their correspond-ing acids : ethanol, n-propanol, n-butanol, sec.-butanol and n-pentanol ;methanol and tert.-butanol are not attacked ; isopropanol and isobutanolare oxidised to acetone and methyl ethyl ketone respectively.The ferment-ation of ethanol in growth experiments on synthetic media with ethanol assole source of carbon occurs closely in accordance with the equation2C2H,*OH + CO, = 2CH3*C0,H + CH,, the slight deficit found in thesubstances on the right side of the equation being due to the fact that about6% of the carbon of the ethanol and 1.5% of the carbon dioxide are used toform cell material.5Where mixed enrichment cultures from mud are used for the oxidationof the alcohols by carbon dioxide a second reaction sets in whereby theacids formed are further oxidised. Two organisms carrying out this secondoxidation have been isolated, Methanococcus Muxei and MethanobacteriumSohngenii ; both ferment acetic and butyric acids but none of the alcohols.The fermentation of acetic acid proceeds according to the equationCH3*C0,H + 2H20 + CO, = 2C0, + CH, + 2H,O.Butyric acid appearsto give rise first to acetic acid, which is then further oxidised as above. Theseexperiments, carried out on 2Mb. Omelianski and Methanosarcine methanica,showed furthermore that cell material as well as methane is produced byreduction of carbon dioxide.6The reduction of carbon dioxide to acetic acid has been described byK. T. Wieringa.' This observer repeated Sohngen's synthesis of metha.nefrom hydrogen and carbon dioxide with crude cultures from mud.Onpasteurising the culture and plating out, he obtained a spore-forming organismby means of which carbon dioxide was reduced by hydrogen, giving noArch. MilcrobioZ., 1936, 1, 7, 404.Idem, J . Biol. Chem., 1941, 137, 153.H. A. Barker, S. Ruben, and M. D. Kamen, Proc. Nut. Acad. Sci., 19 .O, 26, 426.Antonie van Leeuwenhoek, 1936, 8, 263.H. A. Barker, ibid., 1936, ii, 7, 420STEPHENSON AND KREBS THE UTILTSATTON OF CARBON DIOXIDE. 259methane but a quantitative yield of acetic acid. There is no reason, how-ever, to postulate acetic acid as an invariable intermediate in the Sohngenreaction, since there is a case on record in which the reaction has beeneffected by a culture derived from a single cell which was incapable ofproducing methane from any compound with more than one carbon atom.8A special ‘case of carbon dioxide reduction is that due to CZ.acidi u r i ~ i . ~This organism decomposes uric acid, xanthine, hypoxanthine and guanineanaxobically, giving only ammonia, carbon dioxide, and acetic acid :purine, urea, allantoin, uracil and caffeine are unattacked ; adenine,adenosine, guanosine and yeast nucleic acid are attacked slowly. Uricacid and hypoxanthine are decomposed closely in accordance with equations(3) and (4) respectively :The fact that in (4) more than 1 mole of acetic acid was produced froin1 mole of hypoxanthine led to the hypothesis that the decomposition was dueto an oxidoreduction with carbon dioxide, which is itself reduced to aceticacid as in the reaction reported by Wieringa.This was supported by experi-ments in which suspensions of the organism fermented uric acid in the presenceof radioactive 11C02.10 The results showed that the acetic acid produced wasradioactive and that the llC was present in the methyl as well as in the carbonylgroup; also that l1C had passed into cell material. So far the evidencesupports the reduction of carbon dioxide to acetic acid. On the other hand,in experiments in which uric acid was fermented in a rapid stream of hydrogenfree from carbon dioxide the rate of breakdown was the same as in the control ;this evidence is against carbon dioxide acting as the hydrogen acceptorunless its concentration within the cell is sufficient to maintain the optimumrate in spite of its rapid removal from the solution,A second line of investigation relating to carbon dioxide utilisationoriginated from work on the fermentation of propionic acid bacteria.In1936 H. G. Wood and C. H. Werkman,ll studying the fermentation of glycerol~ J Y these organisms, made the unexpected observation that the end-productsof the fermentation-mainly propionic and succinic acids-contained morecarbon than the fermented glycerol. A closer investigation showed that theadditional carbon was derived from calcium carbonate which had been addedto the medium, as is customary, to neutralise acids formed during the ferment-ation. In 1938 Wood and Werkman l2 showed that the quantities of succinicacid formed and carbon dioxide (or carbonate ion) used were approximatelyequimolecular. The experimental data conform with the assumption thatthere are two main reactions when glycerol is fermented, namely,( 5 ) CH,(OH)*CH(OH)*CH,*OH --+ CH3*CH,*C0,H + H,O* M.Stephenson and L. H. Stickland, Biochem. J . , 1933, 27, 1617.(6) CH,( OH)*CH( OH).CH2*OH + C02 -+ CO2H*CH2*CH2*CO,H -+ H2OH. A. Barker and J. V. Beck, J . Bid. Chem., 1941, 41, 3.lo H. A. Barker, S. Ruben, and J. V. Beck, Proc. Nut. Acad. Sci., 1940, 26, 477.l1 Biochein. J., 1936, 30, 4s. l2 Ibid., 1938, 32, 1262260 BIOCH.EMISTRY.Later work lS:l4 has shown that the intermediate stages of reaction (6) areprobably as follows :This scheme is supported by the facts that the carbon dioxide used and thesuccinic acid formed are equimolecular ; l2 that the utilised carbon dioxide ispresent in the carboxyl group of succinic acid l3 (as shown by experimentswith W ) ; that fumaric and malic acids are formed along with succinicacid ; 14 that the reactions postulated readily 0ccur.1~Many micro-organisms form succinic acid when fermenting carbohydrates,glycerol, or pyruvic acid.It is likely that in most cases succinic acid isformed according to reactions (2) and (7). That this is true for Bad. colihas been shown with the help of carbon isotopes.13*14Reaction (2) probably plays a r81e in the synthesis of citric acid, fumaricacid and related substances in mou1ds.l6* 1'That carbon dioxide can be utilised in animal tissues by combiningwith pyruvic acid [reaction (2)] was first suggested by H.A. Krebs andL. V. Eggleston,l* who showed that in pigeon liver pyruvic acid yieldsthe same products as oxaloacetic acid,lg vix., citric acid, a-ketoglutaric acid,mccinic acid, fumaric acid and malic acid. The obvious, and in fact onlysatisfactory, explanation was the assumption that pyruvic acid is firstconverted into oxaloacetic acid, by way of reaction (2); this hypothesiswas supported by the fact that the concentration of carbon dioxide determinedthe rate of conversion of pyruvic acid into the above-named products.Final proof for the occurrence of reaction (2) in pigeon liver was suppliedby E. A. Evans, jun., and L. Slotin,20 who added bicarbonate containing11C and found a-ketoglutaric acid to contain the isotope. This was con-firmed by Wood et aZ.,13 who used the isotope 13C. These authors determinedthe position of the fixed carbon dioxide and found that all the fixed carbon wasin the carboxyl group next to the ketone group. This indicates that citric acidis not an intermediate in the formation of a-ketoglutaric acid, for if the latter acidwas derived from a symmetrical molecule the fixed carbon should be equallydistributed in the two carboxyl groups.. The authors suggest the followingla H.G. Wood, C. H. Werkman, A. Hemingway, and A. D. Nier, J . Biol. Chem.,1940,135, 781 ; 1941,139, 365, 377 ; S. F. Carson and S. Ruben, Proc. Nat. Acad. Sci.,1940, 28, 422; S. F. Carson, J. W. Foster, S. Ruben, and H. A. Barker, ibid., 1941, 27,229.l4 H. A. Kmbs and L. V.Eggleston, Biochem. J., 1941, 35, 676.l6 H. A. Krebs, Nature, 1941, 147, 560.l6 Y. Nishina, S. Endo, and H. Nakayama, Sci. Papers Inst. Phys. Cheni. Res.l7 H. G. Wood and C . H. Werkman, Biochem. J., 1940, 34, 7.Tokyo, 1941, 38, 341.Ibid., p. 1383.See Ann. Reporte, 1937, 34, 418.J . Biol. Chern., 1940, 136, 301; 1941, 141, 439RAISTRICK: METABOLIC PRODUCTS OF THE L O m R 261scheme for the formation of a-ketoglutaric acid : pyruvic acid + oxltloaceticacid + cis-aconitic acid _I, isocitric acid a-ketoglutaric acid + COz.Solomon et a1.21 injected NaHWO, and lactate into fasting rats and foundthe liver glycogen t o contain 11C. This can be explained on the assumptionthat the primary reaction is again reaction (2).Prior to this work S. Ruben and M.D. Kamen 22 had already shown withthe help of isotopes that liver incorporates carbon dioxide or bicarbonateinto organic compounds. These authors, however, did not define the com-pounds containing the isotope; it may have been urea, which has long beensupposed to be formed from carbon dioxide and ammonia; 23 E. A. Evansand L. Slotin, using W 0 2 , have recently supplied final proof for thecorrectness of this ass~rnption.~~+HZ0M. S.H. A. K.7. METABOLIC PRODUCTS OF THE LOWER FuNar.In the short space available it is not possible to review adequatelyprogress in this field since it was last dealt with in these Annual Reports.Interested readers are therefore referred to Annual Review of Biochemistry,1940, 9, 571, for a detailed account to the end of 1939.Attention will beconfined here to work published subsequently.(a) Derivatives of To1uquinone.-Fumigatin was fmt described a8 ametabolic product of Aspergillus fumigatus Fresenius by W. K. Anslowand H. Raistrick.l The molecular constitution, 3-hydroxy-4-methoxy-2 : 6-toluquinoneY ascribed t'o it by these authors has been confirmed bysynthesis .2(b) Derivatives of 2-Methylanthmquinone.--C~~tenarin, present in themycelium of different species of Helminthosporium, particularly Helmintho-sporium catenarium Dre~hsler,~ has now been shown to be 1 : 4 : 5 : 7-tetra-hydro~y-2-methylanthraquinone.~ This conclusion was confirmed bysynthesis. Erythroglaucin, from species in the Aspergillus ghucus series,6has been shown by Anslow and Raistrick 7 to be 1 : 4 : 5-trihydroxy-7-inethoxy-2-methylanthraquinone and was prepared by them in uitro bymethylating cateiiarin with methyl iodide and sodium methoxide in methanolsolution.Cynodontin, 1 : 4 : 5 : 8-tetrahydroxy-2-methylanthraquinone,21 A. K. Solomon, B. Vennesland, F. W. Klemperer, J. 34. Buchanan, and A. B.22 Proc. Nut. Acad. Sci., 1940, 27, 418.23 H. A. Iirebs and K. Kenseleit, 2. physiol. Chem., 1932. 210, 33.24 J . Biol. Chem., 1940. 138, 806.Hastings, J . Biol. Chem., 1941, 140, 171.1 Biochem. J., 1938, 32, 687.W. Raker and H. Raistrick, J., 1941, 670.J. H. V. Charles, H. Ibistrick, (Sir) R. Robinson, and A. R. Todd, Biochem. J . ,W. K. Anslow and H. Raistrick, ibid., 1940, 34, 1124.Idem, ibid., 1941, 35, 1006.J.N. Ashley, H. Raistrick, andT. Richards, ibid., 1939, 33, 1291.Ibid., 1940, 34, 1124.1933, 27, 499; H. Raistrick, (Sir) R. Robinson, and A. R. Todd, ibid., 1934, 28, 569262 BIOCHEMISTRY,from Helminthosporium cynodontis Marignoni,s has also been synthesisedby Anslow and Raistri~k.~H. G. Hind 10 isolated from cultures of Penicillium cnrmino-viohceumBiourge two polyhydroxyanthraquinones, carviolacin, C,H,,O,, andcarviolin, C16H1206, and later 11 showed that carviolin is a monomethylether of o-hydroxyemodin, 4 : 5 : 7-trihydroxy-2-hydroxymethylanthra-quinone, which was itself isolated from cultures of Penicillium cyclopiurnWestling by W. K. Apslow, J. Breen, and H. Raistrick l2 and from Peni-cillium citreo-roseum Dierckx by T. P0~ternak.l~ Posternak l4 showed thatroseopurpurin, which he isolated from Penicillium roseo-purpureurn Dierckx,is the 4-methyl ether of w-hydroxyemodin, i.e., 5 : 7-dihydroxy-4-methoxy-2-hydroxymethylanthraquinone. The present reviewer (unpublished ob-servation) has compared Hind’s carviolin and Posternak’s roseopurpurin,received from their respective discoverers, and has shown that they are oneand the same substance.Penicilliopsin, C,H,,08, the orange crystalline colouring matter ofPenicilliopsis clavariae formis Solrns-Lauba~h,~~ when oxidised in air andsubsequently irradiated , gives chocolate-brown needles of irradiated oxy-penicilliopsin, C30H200s.This substance is closely related to, but notidentical with, hypericin, the photodynamically active colouring matterof St. John’s wort, Hypericum perforaturn. Penicilliopsin, whose constitu-tion is a t present unknown, is a derivative of 2-methylanthraquinone, sinceit gives tetranitroemodin on oxidation with nitric acid.(c) Chlorine-containing MetuboEic Products.-The conversion by mouldsof inorganic chlorides in the culture medium, e.g., potassium chloride, intoorganic chlorine-containing metabolic products is becoming increasinglyrecognised. A survey of the subject was made by P. W. Clutterbuck,S. L. Mukhopadhyay, A. E. Oxford, and H. Raistrick,16 and these workersisolated from the metabolism solution of Caldariomyces ficmago Woronichincrystalline caldariomycin, C5H802C12, the most probable structure for whichis 2 : 2-dichlorocyclopentane-1 : 3-diol. T. P. Curtin and J. Reilly l7isolated from the mycelium of Yenicillium sclerotiorurn van Beyma a yellowcrystalline colouring matter, sclerotiorine, to which the improbable empiricalformula C,oH,,05C1 was assigned. Its molecular structure has not yetbeen determined.(d) Anti-bacterial Substances from Moulds.-The fact that many mouldmetabolic products have marked bacteriostatic or even bactericidal pro-perties is becoming increasingly cvident. A. Fleming showed that cultureH. Raistrick, R. Robinson, and ,4. 11. Todd, Biochem. J . , 1933, 27, 1170.Ibid., 1940, 34, 1546. 10 Ibid., p. 67.l1 Ibid., p. 577. l2 Ibid., p. 159.13 Compt. rend. Xoc. Phys. Hist. mat. GenBve, 1939, 56, 28; T. Posternak and J. P.l4 Helv. Chinz. Acta, 1940, 23, 1046.lG Ibid., p. 664.18 Brit. J . Exp. Path., 1929, 10, 226.Jacob, Helv. Chim. Acta, 1940, 23, 237A. E. Oxford and H. Raistrick, Biochenh. J . , 1940, 34, 790.1 7 Ibid., p. 1419RAISTRIUK : METABOLIC PRODUCTS OF THE LOWER FUNGT. 263filtrates from a strain of Penicillium notaturn Westling contain a substance,penicillin, which is highly bacteriostatic against Gram-positive micro-organisms. Optimum cultural conditions for the formation of penicillinand the fact that it is extractable with ether were established by P. W.Clutterbuck, R. Lovell, and H. Raistrick.19 H. W. Florey et aL20 have recordedstriking successes in the use of penicillin concentrates in clinical trialsand describe the preparation of an intensely active, but a t present impure,barium salt of penicillin. Penicillin is relatively non-toxic to animals.14;. C!. White 21 reports that culture filtrates from strains in the AspergiZEusLfZavusseries contain a bactericidal agent and G . A. Glister z2 has shown that an un-named species of Aspergillus produces a powerful anti-bacterial agent whichis particularly active against Gram-negative organisms. Neither of thesesubstances has a t present been isolated in a pure condition. S. A. Waksmanand H. B. Woodruff 23 have isolated from soil a new species, dctinomycesuntibioticus,24 which produces two crystalline substances, actinomycin Aand actinomycin B. Actinomycin A is intensely bacteriostatic againstGram-positive bacteria, but only moderately so against Gram-negativebacteria. Actinomycin B has littlebacteriostatic action but is strongly bactericidal. Citrinin, CI3Hl4O5, asemi-quinonoid crystalline metabolic product of PeniciZEium citrinurnand penicillic acid, the P-methyl ether of y-hydroxy-y-isopropylidene-tetronic acid, a crystalline metabolic product of PeniciEEium cyclopiumWe~tling,~’ have been shown to be powerful anti-bacterial agents.28 Theiractivity against a wide range of micro-organisms has been determined byA. E. Oxford.29 None of the above-mentioned substances, except possiblyactinomycin A, is as active as penicillin against Gram-positive organisms,t,hough Glister’s substance, penicillic acid, and citrinin arc much more activethan penicillin against Gram-negative organisms.It is extremely toxic to animals.25H. K.J. F. DANIELLI.I,. J. HARRIS.H. A. KREBS.D. M. NEEDHAM.A. NEUBERGER.F. W. NORRIS.H. RAISTRICK.M. STEPHENSON.l9 Biochezn. J., 1932, 26, 1907. *’ ~ ‘ c ~ F I c ~ , 1940, 92, 127.23 Proc. Xoc. Exp. Biol. Med., 1940, 45, 009.2 6 S. A. Waksman, H. Robinson, H. 5. Metzger, and H. B. Woodruff, Proc. SOC. Exp.2 6 A. C. Hetherington and H. Raistrick, Phil. Trans., 1931, B, 220, 209; F. P.27 J. K. Birkinshaw, A. E. Oxford, and H. Raistrick, Biochem. J . , 1936, 30, 394.28 H. Raistrick and a. Smith, Chem. and Ind., 1941, 60, 838: &A. E. Oxford, H.Raistrick, and G. Smith, ibid., 1942, 61, 22.29 Ibid., 1942, 61, 48.2o Lancet, 1940, 239, 226 ; 1941,241, 177.22 Nature, 1941, 148, 470.24 Idem, J . Bact., 1911, 42, 231.Riol. Med., 1941, 47, 261.Coyne, H. Raistrick, and (Sir) R. Robinson, ibid., p. 297