BIOCHEMISTRY.1. INTRODUCTION.DURING the past few years these Reports on biochemistry have usuallyconsisted of articles on special topics of current interest. Now that theChemical Society publishes Quartdy Reviews such articles might perhapsappear more conveniently in this new publication. An attempt will thereforebe made to report on larger fields of biochemistry. As space is limited,however, the subjects will not be covered each year but rather on a triennialbasis. Thus the subjects of hormones, nutrition, and microbiology aredivided into three parts and it is hoped that one part of each will appear eachyear. Although the subject will not be covered so completely as in AnnualReviews of Biochemistry, the Reports should give the main themes of develop-ments in different branches of the subject.E..B.2. ANTIBIOTICS.Introduction.-When the antibiotics were first reviewed in these Reportsseven years ago the discovery of the therapeutic power of penicillin hadbegun to stimulate widespread interest in their properties, and since. that timethey have been the subject of an immense number of publications. Theliterature to the end of 1948 has been discussed elsewhere in some detailand many of the publications in 1949 have also been r e ~ i e w e d . ~ * ~ Thepresent article deals mainly with the salient aspects of recent work.The study of an antibiotic may be carried through a number of more orless distinct stages, concerned with : (1) detection, (2) production, (3) isol-ation, (4) chemical structure and synthesis, ( 5 ) antimicrobial and pharma-cological properties, (6) mode of action, and (7) therapeutic use in animals orman.Most of the research is undoubtedly prompted by the hope of findingnew substances of value to medicine. From this point of view it has provedhighly rewarding, for penicillin, streptomycin, chloromycetin, aureomycin,and terramycin are powerful therapeutic agents, and the list of such substancesis likely to grow. Other aspects of the subject, however, should not beneglected. Only a very small proportion of the antibiotics discovered evercome into the hands of the clinician, but among those that for one reason oranother find no place in medicine are many of considerable interest inchemistry or biology.The ability to produce one or more antibiotics is a rather common propertyof mkro-organisms and the mere detection of antagonism between organismseasily grown in vitro often presents little difficulty.By relatively simpleChain and Florey, Ann. Reports, 1943,40, 180.H . W. Florey, Chain, Heathy, Jennings, Sanders, Abraham and M. E. Florey,Carter and Ford, Ann. Rev. Biochem., 1950,19,487.Herrell, Ann. Rev. Microbiol., 1950, 4, 101.“ Antibiotics,” 1949, Oxford Univ. Press286 BIOCHEMISTRY.methods large numbers of fungi,5 actinomycetes,s* and bacteria have beensurveyed in the laboratories of academic institutions and commercial firms.The detection of antibiotics that are active against animal viruses is moredifficult, but a simple technique has been introduced for finding substancesthat affect the action of bacteriophage^.^Before 1944 antibiotics were normally produced by growing organismsin shallow layers of stationary media.The commercial production of peni-cillin was greatly facilitated when it was found possible to use media in deeptanks under conditions of vigorous stirring and aeration. Deep ferment-ation is now the method of choice for antibiotic production and has beenemployed with fungi imperfecti,1° actinomycetes,ll bacteria,l2 and with abasidiomycete,l5 but it does not always prove succe~sful.~~There is so far little information about the biosynthesis of any antibiotic.The way in which the essential features of the penicillin molecule, for example,are put together is still unknown.15 The preferential formation of a partic-ular kind of penicillin that can be induced by addition, to the culture medium,of compounds containing the side chain of the penicillin concerned l6 appearsto be the only case in which biosynthesis has been influenced in a rationalmanner by the addition of a precursor of known structure, although it hasbeen reported that a strain of R.Zicheniformis can be induced to form alicheniformin-like substance instead of ayfivin (bacitracin) by changing thecarbon : nitrogen ratio of the rnedium,l7 and that glycine and acetate areused in the synthesis of prodigiosin.18 In general the best culture media areonly found by trial and error.Work on the production of penicillin emphasised that the yield of anti-biotic may vary not only with the medium but with the strain of micro-organism used, and that valuable new strains may sometimes be obtainedby mutagenic agents.lg A strain of Streptomyces griseus that had lost itsability to form streptomycin yielded a mutant on treatment with X-raysHervey, Bull.Towey Bot. Club, 1947,74,476; Wilkins, Brit. J . Exp. Path., 1948,29, 364; Lacey, J . Qen. Microbiol., 1950, 4, 122.Waksman and Lechevalier, Science, 1949, 109, 305.7 Kane, Finlay, and Sobin, Ann. N . Y . Acad. Sci., 1950, 53, 226; Cerdos andRosemblit, Rev. Argentina Agon., 1950, 17, 98.8 Fredericq, Compt. rend. SOC. Biol., 1950, 144, 986; Gilliver, Brit. J . Exp. Path.,1949, 30, 214; Gardner, ibid., 1950, 31, 102; Sherwood, Russell, Jay, and Bowman, J .Infect.Dis., 1949, 84, 88.O Asheshov, Strelitz, and Hall, Brit. J . Exp. Path., 1949, 30, 175.lo Peterson, Harvey Lectures, 1946/47, 42, 276; Brown and Peterson, Ind. Eng.Chem., 1950,42, 1769.l2 Newton, Brit. J . Exp. Path., 1949, 30, 306; Stansly, Schlosser, Ananenko, andCook, J . Bact., 1948, 55, 573; Garibaldi and Feeney, I n d . Eng. Chem., 1949, 41, 432;Humfeld, J . Bact., 1947, 54, 689.l1 Bartz, J . Biol. Chem., 1948,172, 445.lS Gill-Carey, Brit. J . Exp. Path., 1950,31, 30.l5 Sus, Annalen, 1950, 569, 153. l4 Gardner,ibid., 1949, 30, 130.l6 Behrens in “ The Chemistry of Penicillin,” 1949, Princeton Univ. Press.l 7 Hills, Belton, and Blatchley, Brit. J . Exp. Path., 1949, 80, 427.lo Backus, Stauffer, and Johnson, J . Amcr. Chem.SOC., 1946,88, 152; Foster, 1949,Hubbard and Rimington, Biochem. J., 1950, 46, 220.U.S.P. 2,458,495ABRAHAM AND NEWTON : ANTIBIOTICS. 287which was a good producer of the drug.2o On the other hand the claim thatthe ability of a strain of B. mesentericus to produce an antibiotic was enhancedby animal passage could not be confirmed.21The antibiotics fall into a large variety of chemical types and any newmethod for purifying organic substances may find some application in theirisolation. Partition chromatography was adapted to the purification of thepenicillins soon after its introduction into biochemistry,22 and was later usedwith the polymyxins 23 and with marcescin.M Chromatography on paper,followed by the determination of the position of the active substances bychemical or microbiological techniques, has been increasingly used for thecharacterisation of antibiotics and the analysis of mixtures.25* 26 Counter-current distribution between solvents 27 has proved particularly useful inthe purification of antibacterial polypeptides not yielding readily to othermethods 28-30 and has shown, for example, that crystalline preparations ofgramicidin may be heterogeneous.An important extension of this procedure,using a carrier (a long-chain acid or base) to bring the substance (a water-soluble base or acid) into an organic phase in which it is not normally soluble,arose out of work on the separation of streptomycin and mannosidostrepto-m ycin .3Only a small proportion of the antibiotics detected have been obtained ina homogeneous state or assigned precise chemical structures.Since anti-biotics are often named when little is known of their properties it may some-times happen that different names are given to the same compound. Someof the names in the literature have thus already fallen into disuse and othersare likely to do so.Antibiotics from Fungi.-Penicillin is still the most interesting andimportant of the antibiotics obtained from fungi. Substances such aspenicillic acid, discovered in early work on the metabolic products of moulds,continue to be isolated from different species.32 A new structure has beenintroduced for patulin, and puberulic and puberulonic acids have been shownto contain the tropolone ring system postulated in 1945.33 Several new2o Waksman and Harris, Proc.SOC. Exp. Biol., 1949, 71, 232.21 Savage and H. W. Florey, Brit. J. Exp. Path., 1950, 31, 14.22 Abraham, W. Baker, Chain, and Robinson in “ The Chemistry of Penicillin,”Bell, Bone, English, Fellows, Howard, Rogers, Shepherd, Winterbottom, Dornbush,1949, Chap. 2, Princeton Univ. Press; Boon and Carrington, op. cit., Chap, 5 .Kushner and SubbaRow, Ann. N.Y. Acad. Sci., 1949, 51, 897.24 Fuller and Horton, J. Gen. Microbiol., 1950, 4, 417.25 R e p a and Murphy, J. Amer. Chem. SOC., 1950,72, 1045.26 Kluener, J. Bact., 1949, 57, 101 ; Karnovsky and Johnson, Analyt. Chem., 1949,21, 1125; Peterson and Reineke, J. Amer. Chem. SOC., 1950, 72, 3598; Catch, Jones,and Wilkinson, Ann. N . Y . Acad. Sci., 1949,51, 917.27 L.C. Craig and D. Craig in “ The Technique of Organic Chemistry,” Vol. 111, p.17 1, Interscience Publ., N.Y., 1950.28 L. C. Craig, Gregory, and Barry, Cold Spring Harbor Symp. Quart. Biol., 1949,14,24.sB Work and Callow, 1950, personal communication.Newton and Abraham, Biochem. J., 1950,47, 257.31 Plaut and McCormack, J. Amer. Chem. SOC., 1949,71,2264; O’Keeffe,Dolliver,and32 Burton, Nature, 1950,165,274. 33 Dewar, ibid., 1945,165,50. Stiller, ibid., p. 2452288 BIOCHEMISTRY.antibiotics have been isolated from basidiomycetes, but with one exceptionlittle is yet known of their structure.Penicillin. There appear to be few outstanding publications on theproduction and chemistry of the penicillins that have not recently beenreviewed.29 3* 34 A satisfactory synthesis is still awaited.Butyric, valeric, hexanoic, and hex-3-enoic acids * can be used as pre-cursors to stimulate the biosynthesis of penicillins containing correspondingn-alkyl side chains by P .chrysogenum Q 176. Biosynthetic n-propyl- andn- butyl-penicillins appear identical with two penicillins formed normally ina synthetic medium.35 5-Chloro- and 5- bromo-2-thienylacetic acids havebeen found to stimulate penicillin prod~ction.~~ New chemical proceduresfor estimating penicillin have been described.37It has been reported that penicillin F may be separated from penicillinG by precipitation as its beryllium salt at pH Penicillin K has beenisolated by continuous counter-current solvent fractionation39A unit has been suggested for penicillinase, the enzyme that inactivatespeni~illin.~O Several compounds, including 2- benz ylglyoxaline and penicill-nmine (P-mercaptovaline) have been found to inhibit the action of penicill-inase.While penicillamine increased the action of penicillin against thepenicillinase-producing B. cereus, however, 2- benzylglyoxaline did not .41The surprising report that after treatment with penicillinase penicillin ispartly reactivated by injection into rabbits would appear to need furtherinvestigation.42Patulin. Strong evidence was recently obtained that this antibiotic hasthe structure (I) instead of the previously accepted structure (II).43 Thishas now been confirmed by the synthesis of patulin in small yield from thelnctol acetate (III)?4 aZloPatulin (11) has also been synthesised.434 “ The Chemistry of Penicillin,” 1949, Princeton Univ.Press ; Wintersteiner andDutcher, Ann. Rev. Biochem., 1949,18, 559.35 Thorn and Johnson, J . Amer. Chem. Soc., 1950,72,2052.36 Ford, Prescott, and Colingsworth, ibid., p. 2109.37 Ortenblad, Acta Chem. Xcand., 1950, 4, 518 ; Knoll, 2. Bakt., Abt. I (Orig.), 1950,155,99; Odo and Hirano, J . Agric. Chm. SOC. Japan, 1950,23,237.3839404 14243238 ;44 *C. Pfizer & Co. Inc., B.P. 633,660/1949.Bartels and Dolliver, J . Amer. Chem. SOC., 1950,72, 11.Levy, Nature, 1950, 166, 740.Behrens and Garrison, Arch. Biochem., 1950, 27, 94.Irrgang and Dornbrack, 2. physiol. Chem., 1950,285, 17.Woodward and Singh, J .Amer. Chem. SOC., 1949, 71, 758 ; Experientia, 1950, 6,Engel, Brzerki, and Plattner, Helv. Chim. Acta, 1949,32, 1166.Woodward and Singh, J . Amer. Chem. SOC., 1950, 72, 1428, 5352.Geneva nomenclature, CO,H = 1ABRAHAM AND NEWTON : ANTIBIOTICS. 289Puberulic and Puberuhic Acids. Although these antibiotics were dis-covered in 1932 there has hitherto been no satisfactory structure for thembased on experimental evidence. Puberulic acid has now been shown toyield aconitic acid on oxidation with alkaline hydrogen peroxide, and hasbeen assigned the structure (IV).45 It is a hydroxystipitatic acid.HO 40 HO //o 0yj-C J&-\\ / / \\ od'=" ' d"" I&H &H 06, yoH o f T O H\-(IV-) (V.1 (VI). 0Puberulonic acid, whose molecular formula has been amended to C,H40,,yields puberulic acid and carbon dioxide when heated in aqueous acid.Puberulonic acid was at first thought to have a structure such as (V),4s butthe anhydride structure (VI) has now been proposed to account for itsultra-violet absorption spectrum and behaviour on t i t r a t i ~ n .~ ~ Structure(VI) is supported by the infia-red absorption spectra of puberulonic acid andrelated 47Baccutine A . This substance has been isolated from the mycelium of aspecies of Fusarium (Gibberelk baccuta;). A possible molecular formula isC,,H,,O,N,.Cordycepin. An antibiotic named cordycepin, with the molecularformula C,,H,,O,N,, has been obtained in crystalline form from Cordycepsmizitcaris (Linn) Link.49The culture fluids of the basidiomycetes,Poria corticola, Poria tenuis, and a fungus from white cedar have been foundto contain two similar antibacterial substances named nemotin and nemotinicacid.s0 These substances cannot be handled out of solution since theydecompose in the solid state. Both show similar absorption spectra withfour high peaks between 230 and 290 mp.I n aqueous solution above pH6 nemotin changes into another neutral substance called nemotin A.Nemotin and nemotinic acid inhibit the growth of Staph. aurew in highdilution but are much less active against Bact. coli and Bact. Friedlunderi.Nemotin is much more active than nemotinic acid as an antifungal agent.This antibiotic has been isolated from the culture fluid ofAgrocybe dura, where it occurs partly in an inactive form that can be activatedby boiling.s1 It yields white crystals which blacken in air but are stable at4" in vacuo.It appears to be neutral orweakly acidic and its ultra-violet absorption spectrum is similar to that ofIt is active against Shph. aureus and a number of fungi?*Nemotin and Nemotinic Acid.Agrocybin.Analysis gave : C, 65.6 ; H, 4.3%.Is Corbett, Johnson, and Todd, J., 1950,6, 147.I6 G. Aulin-Erdtman, Acta Chem. Scand., 1950,4, 1326.4 7 Shepard, Johnson, and Todd, personal communication.40 Cunningham, Manson, and Spring, Nature, 1950,166, 949.50 Anchel, Polatnick, and Kavanagh, Arch. Biochem., 1950, 25, 208; Ksvanagh,Guerillot-Vinet, Guyot, MontBgut, and ROUX, Compt. rend., 1950, 230, 1424.Hervey, and Robbins, PTOC.Nat. Acad. Sci., 1950,36, 1. 51 Idem, ibid., p. 102.REP.-VOL. XLVII. 290 BIOCHEMISTRY.nemotin A. Agrocybin is highly aotive against Stuph. aureus, Bact. mli, andMyco. phlei and is also antifungal, but its activity is greatly reduced byhuman blood. It is highly toxic to mice and liable to cause dermatitis inman.Grri,foZin. An antibiotic, C,,H,,O,, obtained in crystalline form from thesporophores of the basidiomycete Grifola conJEuens has been namedgrifolin.62 The results of hydrogenation and degradations with ozone,potassium permanganate, and lead tetra-acetate provide evidence for thestructure (VII). Grifolin inhibits the growth of Xtuph. aureus and some(~11.1 CMe,:CH*CH,-CMe:CH*CH :CHOCK (OH ) CMeE t OHacid-fast bacteria, but is inactive against grambnegative organisms.Itshows low toxicity in mice.IZZudin. Two antibiotics produced by Clitocybe illudens have been iso-lated in the crystalline state and called illudin M and illudin S. They areneutral and have the molecular formula C15H20O3 and C15H,,0, respectively,They both show considerable activity against mycobacteria, but they arehighly toxic to mice and are liable, like agrocybin, to cause dermatitis inman. 53An antibiotic with quinonoid properties has been iso-lated from the culture fluid of the basidiomycete Zenxites trabea (Pers) Fr.(Lenxites thermophila) and named thermophillin. Its probable formula isC,,H605(OMe), and it shows weak activity against Xtaph. uureus.54This antifungal substance has now been separated into isomericCI- and p-viridin.Both compounds are thought to have the molecularformula C,9H,60,.55AZtemrine. A substance called alternarine has been isolated from theculture fluid of AZternaria sohni in the form of white crystalline needles.s6It is active against a number of gram-positive and gram-negative bacteriaand phytopathogens.Miscellaneous. Antibiotics have also been obtained from Pusariumbostry~oides,~~ Aspergillus japonicw,6a and Marasirnus ureus. 69Antibiotics from Actinowcetes.-Following the discovery of strepto-mycin the ability of the actinomycetes to produce antibiotics has probablybeen examined more extensively than that of any other group of micro-organisms. Many thousands of strains have been surveyed, in some caseswith the particular object of detecting substances active against Mpco.tabercuZosis.6 Three antibiotics-chloromycetin, aureomycin, and terra-mycin-that have emerged from these studies in recent years have provedThermphiZZin.Viriddn.58 Hirata and Nakanishi, J.Biol. Chem., 1950,184, 135.53 Anchel, Hervey and Robbins, Proc. Nut. A&. Sci., 1950, 86, 300.54 Burton, Nature, 1950,166, 570.5 5 Vischer, Howland, and Raudnitz, ibid., 1950, 165, 528.66 Darpoux, Farvre-Amiot, and ROUX, Compt. rend., 1960, 230, 993.5 7 Cajori, Hamilton, Urbanish, and Purshottam, Fed. PTOC., 1950, 9, 158.58 Aketsu, J . Agric. Chem. SOC. Japan, 1950,23,343.E)blom, Mdure, 1950,166, 950ABRAHAM AND NEWTON : ANTIBIOTICS. 291effective in man against infections with a number of organisms insensitive topenicillin ; a fourth, neomycin, is undergoing clinical trial.Chloromycetin,which is remarkable in being a natural compound containing a nitro- and adichloroacetyl group, proved to have a simple constitution and is now readilyobtained synthetically. The structure of the other antibiotics is either notyet known or has not been revealed.The production, chemistry, and use of streptomycin hasbeen described in detail in recent review^.^^ go Dihydrostreptomycin has beenisolated in the form of the crystalline free base.61 Streptidine has beensynthesised from D-glucosamine. 62 Residues from the purification ofstreptomycin have been found to contain a streptomycin derivative that ishighly toxic when injected intravenously into mice.63NH[CH(oH)*st12 The derivative is di-( whydroxystreptomycy1)amine(VIII ; St is streptomycin less CHO), and it has beensynthesised by warming a concentrated solution of streptomycin hydro-chloride containing ammonia.Hydroxystreptomycin.Strephyces griseo-carneue, isolated from aJapanese soil, produces a streptomycin-like antibiotic which has been isolatedas a crystalline helianthate and converted into the trihydr~chloride.~* Itwas distinguished from streptomycin only by paper chromatography. Thenew substance, which also appears to have been obtained from a strain ofStreptomyces found in soil a t Illinois,65 contains an additional oxygen atomin the streptose portion of the molecule; this fragment has the structure(IX), instead of (X) as in streptomycin.Streptomycin.(VIII.)-0-CHI 3 T H HC-O-Neomycin. A basic antibacterial substance obtained from a strain ofStreptomyces related to S.fradiue was named neomycin.6 It was later shownby counter-current distribution to contain a t least three active substances andwas called the neomycin complex.6sOne component of the neomycin complex, neomycin A, has been isolatedas a crystalline p(phydroxypheny1azo)benzenesulphonate. The regener-ated hydrochloride shows only end absorption in the ultra-violet region, aWaksman, “ Streptomycin-Its Nature and Practical Application,” 1949,Williams and Wilkins Co., Baltimore, Maryland ; Brink and Folkers, Adu. EnzymoEogy,1950, 10, 145.Rhodehctmel, McCormick, and Kern, Science, 1950,111, 233.62 Wolfrom, Olin, and Polglase, J .Amer. Chem. SOC., 1950, 72, 1724.63 Solomons and Regna, ibid., p. 2974.e5 Grundy, Schenck, Clarke, Hargie, Richards, and Sylvester, Arch. Biochem.,Benedict, Stodola, Shotwell, Borud, and Lindenfelser, Science, 1950, 112, 77.1950,28,160. e6 Swart, Hutchinson, and Waksman, ibid,, 1949,24, 92292 BIOCHEMISTRY.positive ninhydrin test, and negative glucosamine, maltol, and Sakaguchitests. 67 Other workers obtained crude neomycin whose dominant componentwas different from neomycin A and was called neomycin B.26Neomycin is active against many Gram-positive and Gram-negativeorganisms and especially against mycobacteria. It has a strong bactericidalaction. Resistant organisms have been said to develop less readily to neo-mycin than to streptomycin,6* 68 although this has been disputed in the caseof Myco.tubercul~sis.~~ The substance has a relatively low toxicity, butrecent experience indicates that it can damage the kidneys.70* 71 The LD,,,when given subcutaneously to mice, is 450 mg./kg. It is reported to be moreeffective than streptomycin in suppressing infections in mice with Xtaph.aurew, 8. schottmuleri, and S . t y ~ h i . ~ ~Fradicin. This antibiotic, which is also produced by X. fradiae, is activeagainst fungi but not against bacteria.73Chloramphenicol (Ghloromycetin). An antibiotic was isolated from theculture fluid of a species of Streptomyces and given the trade name “ chloro-mycetin.” 11 Degradation and synthesis showed that this substance was( - )-~-threo-2-dichloroacetamido- 1 -p-nitrophenylpropane- 1 : 3-diol (XI).Itis now known by the trivial name chl~ramphenicol.~~ A cylinder-plate assayand a polarographic procedure for estimating chloramphenicol have beendescribed . tiH NH*CO*CHCl,O , R ’ < ~ > - ~ ~ $ - C H ~ * O H (XI.)Chloramphenicol inhibits the growth of a wide range of pathogens at adilution of a t least 1 in lo6, including members of the genera Streptococcus,Corynebacterium, Escherichia, Pasteurella, Salmonella, Shigellu, and Vibrio,and several fungi. Treatment of infected chick embryos showed that it isalso active against Rickettsiae and some of the larger viruses, including theagents responsible for epidemic typhus, scrub typhus, and lymphogranulomavenereum, but that it is ineffective against most of the smaller viruses.7667 Peck, Hoffhine, Gale, and Folkers, J .Amer. Chem. SOC., 1949, 71, 2590.68 Waksman, Katz, and Lechevalier, J. Lab. Clin. Med., 1950, 36, 93; Weiss andWaksman, Proc. Nut. Acud. Sci., 1950, 36, 293; Jen-Yah Hsie and Bryson, Amer. Rev.Tuberc., 1950,62,286; Waisbon and Spink, Proc. SOC. Exp. Biol., 1950,74,35.68 Yegian and Vanderlinde, Amer. Rev. Tuberc., 1950,61, 483 ; Steenken, Wolinsky,and Bolinger, ibid., 1950, 62, 300.7O Waksman, Brit. Med. J., 1950, 11, 595.7l Karlson, Gainer, and Feldman, Amer. Rev. Tuberc., 1950, 62, 345.7% Waksman, Frankel, and Graessle, J . Buct., 1949, 58, 229.73 Swart, Romano, and Waksman, Proc. SOC. Exp. Biol., 1950, 73, 376.’* Rebstock, Crooks, Controulis, and Bartz, J. Amer. Chem. Soc., 1949, 71, 2458;Controulis, Rebstock, and Crooks, ibid., p. 2463; Long and Troutman, ibid., pp.2469, 2473.7 5 Smith, Landers, and Forgacs, J . Lab. Clin. Med., 1950, 36, 154; Hess, Anulyt.Chem., 1950,22,649.v 6 McLean, Schwab, Hillegas, and Schlingman, J . Clin. Invest., 1949,28,953ABRAHAM AND NEWTON : ANTIBIOTICS. 293If, is said to inhibit the multiplication of staphylococcal phage,77 and toeliminate the cytoplasmic " kappa particles " in killer strains of Para-mecium aurelia so that their power to kill sensitive organisms is lost.78Some bacteria sensitive to chloramphenicol contain enzymes able tobring about reduction of the nitro-group, hydrolysis of the amide linkage,oxidation of the secondary hydroxyl group, and a cleavage of the moleculebetween the first and the second carbon atom of the propanediol chain.79Chloramphenicol is readily absorbed into the blood and the body fluid aftereither parenteral or oral administration.A dose of 50 mg./kg. was welltolerated intravenously by dogs and no serious toxic effects resulted fiomcontinued doses of 200 mg./kg. given orally. The LD,, intraperitoneally inmice is 1300 rng./kgeso The drug is not entirely innocuous to animal tissues,however, and in sufficient amount may cause acute respiratory depression anddamage to the kidneys. Concentrations of 10 pg./ml., less than those attainedin the blood of patients, were found to retard the growth of epithelial cellsand fibroblasts.81 When chloramphenicol is given by mouth to man it isexcreted in the urine partly unchanged and partly as the hydrolysis product( -)-~-threo-2-amino-l -p-nitrophenylpropane-1 : 3-diol (XII), but mostlyas the 3-glucuronide of chloramphenicol.The latter has no antibacterialactivity, but chloramphenicol can be liberated from it by the enzyme p-glucuronidase.82Aureomycin. This is a yellow crystalline antibiotic, with amphotericproperties, isolated from the culture fluid of Streptomyces aureofaciens. Likechloramphenicol it contains non-ionic chlorine, analysis giving : C, 54.6 ;H, 5.3 ; N, 5.8 ; C1,7-2. It is unstable in alkali.83 A fluorometric method 84and rapid biological methods 85 for the assay of aureomycin have recentlybeen described.Aureomycin inhibits the growth of a wide range of Gram-positive and Gram-negative bacteria at dilutions of the order of 1 in lo6 86 andin sufficient concentration may kill the majority of organisms in a culture.87Resistant organisms do not appear to develop readily.88 The substance is7 77 879808 18a81Edlinger and Faguet, Ann. Xnst. Pasteur, 1950,79, 436.Brown, Nature, 1950, 100, 527.Smith and Worrel, Fed. Proc., 1950, 9, 230; Archiv. Biochem., 1950, 28, 1, 232.Long, Bliss, Schoenbach, Chandler, and Bryer, Lancet, 1950,258, 1139.LBpine, Barski, and Maurin, Proc. SOC. Exp. Biol., 1950,73, 252.Glazko, Dill, and Rebstock, J . Biol. Chem., 1950,183, 679.Broschard, Dornbush, Gordon, Hutchings, Kohler, Krupka, Kushner, Lefemine,and Pidacks, Science, 1949, 109, 199.84 Saltzman, J .Lab. Clin. Med., 1950,35, 123.8 5 Schmerson, Proc. SOC. Exp. Biol., 1950, 74, 106; Alture-Werber and Loewe, J .86 Price, Randall, and Welch, Ann. N . Y . Acad. Sci., 1948, 51, 211; Whitlock andLab. Clin. Med., 1950, 35, 660.Tashman, J . Bact., 1950,59,314. a 7 Spicer, J . Lab. Clin. Med., 1950,36, 183.Gezon and Fasan, Proc. SOC. Exp. Biol., 1950,73, 10294 BIOCHEMISTRY.also active against Rickettsia and certain viruses,89 and against E . hiatoty-timW Like chloromycetin, aureomycin is absorbed from the gastro-intestinaltract and has a relatively low toxi~ity.9~192 Its LD,, in mice, when givensubcutaneously, is 30004000 mg. /kg. In high concentrations aureomycindisturbs mitosis in tissue cult~res.~3Terramycin. An amphoteric antibiotic named terramycin has beenisolated in crystalline form from the culture fluid of Streptomyces rimosus.Its probable molecular formula is C22H24-,609N2,2H20.It shows absorptionmaxima at 247,275, and 353 mp., and contains three ionisable groups, forminga hydrochloride and a disodium salt. It is relatively stable in aqueoussolution at pH 1-9 when stored at 5O.94 Terramycin differs from aureo-mycin in containing no chlorine and in being much more stable in aqueoussolution. Terramycin is active in vitro against a variety of Gram-positiveand Gram-negative bacteria and against Rickettsia, and is bactericidal insufficient con~enfration.~~ It has a relatively low toxicity, the LD,, of thehydrochloride being about 800 mg,,’kg.when given subcutaneously in mice.The substance is absorbed from the gastro-intestinal tract. It shows markedchemotherapeutic activity in mice infected with sensitive bacteria.Os# 97This antibiotic was isolated in crystalline form fromcultures of an unidentified species of Streptomyces. It appears to have themolecular formula C28H4,0gN2 and to be a nitrogenous phenoLg8 It isfungicidal,99 insecticidal, and acaricidal.100 A dose of‘ 30 mg.jkg. given bystomach tube to rats proved fatal.lo1A red crystalline antibiotic named actinomycin, whichappeared to have the approximate formula C41H560,1N8, was isolated fromActinomyces antibioticus.lo2 An identical or very similar substance from aspecies of Streptomyces, which has the approximate formula C41H,8011N8, hasAntimycin A .Actinomycin.89 Wong and Cox, Ann.N . Y . Acad. Sci., 1948,51, 290.Hewitt, Wallace, and White, Science, 1950, 112, 144; Watt and Van de Grift,J. Lab. Clin. Med., 1950,36, 741.91 Harned, Cunningham, Clark, Cosgrove, Hine, McCauley, Stokey, Vessey, Yuda,and SubbaRow, Ann. N.Y. Acad. Sci., 1948,61, 182.Q2 Schoenbach, Bryer, and Long, ibid., p. 267.93 Keilova-Rodova, Experientia, 1950, 6, 428.94 Finlay, Hobby, P’an, Regna, Routien, Seeley, Shull, Sobin, Solomons, Vinson, andKane, Science, 1950,111,85 ; Regna and Solomons, Ann. N . Y . A d . Sci., 1950,53,229.9 5 Hobby, Lenert, Pikula, Kiseluk, and Hudders, ibid., p. 266.*6 Herrell, Heilman, Wellman, and Bartholomew, Proc. Mayo Clin., 1950, 25, 183.9 7 Werner, Knight, and McDermott, Proc.SOC. Exp. Biol., 1950, 74, 261; P’an,Reilly, Halley, Richard, Pekich, and Pollets, J . P b r m . Exp. Ther., 1950, 99, 234;Hobby, Dougherty, Lenert, Hudders, and Kiseluk, Proc. SOC. Ezp. Biol., 1950, 73,503; Hobby, Reed, Rinne, Powers, and D’Ambrosia, ibid., p. 511; P’an, Scaduto,and Cullen, Ann. N.Y. Acad. Sci., 1950, 53, 238; Schoenbach, Bryer, and Long,ibid., p. 245 ; Welch, ibid., p. 253.O 8 Dunshee, Leben, Keitt, and Strong, J . Amer. Chem. Soc., 1949, 71, 2436.99 Leben and Keitt, Phytopath., 1948, 38, 899.loo Kid0 and Spyhalski, Science, 1950, 112, 172.101 Ahmad, Schneider, and Strong, Arch. Biochem., 1950, 28, 281.lop Waksman and Tishler, J . Bid. Chem., 1942,142, 619ABRAHAM AND NEWTON : ANTIBIOTICS.296been called actinomycin B or (' antibiotic X-45." lo39 lo4 Actinomycin Byields threonine, L-proline, D-valine, N-methylvaline, and sarcosine onhydrolysis, and appears to be a peptide associated with a quinone system.Actinomycin contains the same amino-acids as actinomycin B.An antibiotic from an actinomycete, which hits the probable formulaC,oH,7O11N7, has been called actinomycin C.lo6 It differs from actinomycinand actinomycin B in containing D-isoleucine or D-alloisoleucine in place ofD-Vahe.l'* 106This is a red crystalline antibiotic, isolated from a strainof Actinomyces, which has the formula C,,H,,O,, and is thought fo contain ahydroxy-quinone system. It prevents the growth of Staph. aurem at adilution of 1 in 1OS.1O6Prwtimmycin.The crude basic antibiotic first described in 1942 lo7has been separated into three active substances, proactinomycin A, B andC, which appear to have the molecular formulae C,,H,,O,N, C,,H,,O,N, andC,,H,lO,N respectively.lo8 It was thought unlikely that any of the threesubstances would be useful clini~alIy.1~~Other antibiotics recently reported to be formed byac tinom y cetes are fungicidin, lo streptocin ,I1 viom y cin, l2 m ycom ycin ,113and an antibiotic resembling xanthomycin.114Antibiotics from Bacteria-Many of the antibiotics obtained frombacteria are polypeptides. Recent work has shown that they often occur aafamilies of closely related peptide~,2~-~~ although in the case of the poly-myxins each pure strain of B. polymym is said to produce a single polypeptideantibiotic.l16 None of these substances has found an established place inmedicine ; the bacitracins, polymyxins, and licheniformins have chemo-therapeutic properties, but their renal toxicity has prevented their generaluse in man.Nevertheless, the detailed structure of the polypeptide anti-biotics is of great interest because of its bearing on the structure of proteins,and the substances continue to be the subject of extensive chemicalinvestigations.llsCrude bacitracin, obtained from a strain of B.Zicheniformis, was fractionated by counter-current distribution, under acidActinorhodin.Miscellaneous.Bucitracin (AyJivin).lo3 Lehr and Berger, Arch. Biochem., 1949, 23, 503; Dalgliesh and Todd, Nature,1949, 184, 830; Abstracts of Communications a t 1st Intern.Congr. Biochem.,Cambridge, 1949, p. 246.lo4 Dalgliesh, Johnson, Todd, and Vining, J., 1950, 2946.lo6 Brockmann and Grubhofer, Nuturwiss., 1949, 12, 376.lo6 Brockmann, Pini, and Plotho, Ber., 1950,83, 161.lo' Gardner and Chain, Brit. J. Exp. Path., 1942, 23, 123.108 Marston, ibid., 1949, 30, 398.118 Hazen and Brown, Science, 1950,112,423.111 Kupferberg, Styles, Singher, and Waksman, J. Bact., 1950, 69, 523.112 Steenken and Wolinsky, A m r . J. Med., 1950,9,633.113 Jenkins, J. Lab. Clin. Med., 1950, 36, 841.114 Mold and Bartz, J. Amr. Chem. SOC., 1950,72, 1847.116 Brownlee, Symposia SOC. Exp. Biol., 1949, No. 111, p. 81.116 Synge, Quart. Reviews, 1949, 3, 246.100 Marston and Florey, ibid., p.407296 BIOCHEMISTRY.conditions, into a major and two minor components.2Q The bulk of theantibacterial activity was accounted for by the major component but analysisof the distribution curve indicated that some of the material in the majorband was not homogeneous. Further information about the nature ofbacitracin has come from studies of the polypeptide antibiotic ayfivin alsoproduced by a strain of B. li~heniformis.~~1 117 Counter-current distributionunder neutral conditions of a mixture of crude bacitracin and ayfivin indicatedthat the two antibiotics contained essentially the same components, and thename ayfivin has consequently been abandoned. The major componentappearing in the distribution under acid conditions could be resolved underneutral conditions into three biologically active components, which werenamed bacitracin A, B, and C 3 0 Bacitracin A was present in the largestamount.Bacitracin C was found to have about the same antibacterialactivity as A, while B was only a third as active. Preliminary investigationsof these substances indicate that they have different toxicities for mice.ll*Licheniformin. Crude licheniformin 119 has been fractionated bycounter-current distribution into three distinct polypeptides (A, B, and C)that appear to have cyclic s t r ~ c t u r e s . ~ ~ A and B contain aspartic acid, serine,glycine, lysine, arginine, proline, valine, and phenylalanine. C contains inaddition glutarnic acid. The three polypeptides have different toxicities forthe mouse, and their toxicity does not parallel their antibacterial activity.They all cause damage to the kidneys.Polymyxins A (aerosporin),B, C, D, and E are similar polypeptides, produced by strains of B.polymyxa,which are active against certain Gram-negative bacteria and dermato-mycetes.3~ lZo* lzl Circulin is formed by a strain of B. circulans.122 All thesesubstances appear to be cyclic polypeptides. On hydrolysis they yieldthreonine, ay-diaminobutyric acid, varying amounts of leucine, phenyl-alanine, and serine, and (+ )-6-methyloctanoic acid.123 The 6-methyl-octanoic acid, which has now been synthesised,l** is thought to be joined tothe rest of the molecule through an ester linkage in circulin and an amidelinkage in polymyxin A and D.An antibiotic named polypeptin has been obtained from astrain of B. circulans as a crystalline ~u1phate.l~~ Counter-current distri-bution indicates that the preparation is about 90% pure.126 Polypeptin isa strongly basic polypeptide of molecular weight about 2000.Paper chro-matograms of hydrolysed material are reported to indicate the presence of anumber of amino-acids, but only ay-diaminobutyric acid has been specificallyPolymyxins (Aerosporin) and Circulin {&I 9).Polypeptin.117 Sharp, Arriagada, Newton, and Abraham, Brit. J . Exp. Path., 1949,30,444.118 H. W. Florey, personal communication.1l0 Callow, Glover, D'Arcy Hart, and Hills, Brit. J . Exp. Path., 1947, 28, 418.120 Ann. N . Y . Acad. Sci., 1949, 51, 855-997.121 Serri, Compt. rend.Soc. Biol., 1949, 143, 362.122 Peterson and Reineke, J . Biol. Chem., 1949,181, 95.123 Wilkinson, Nature, 1949,164, 622.1z5 Howell, J . Biol. Chm., 1950,186, 863.126 L. C. Craig, personal communication to 8. F. Howell.12* Crombie and Harper, J . , 1950, 2685ABRAHAM AND NEWTON : ANTIBIOTICS. 297mentioned. The ultra-violet absorption spectrum of polypeptin sulphateindicates that the substance does not consist only of known amino-acidslinked in a peptide chain. Polypeptin is active against both Gram-positiveand Gram-negative bacteria and against fungi. It is also haemolytic andextremely toxic.Crystalline gramicidin has been resolvedinto at least four components by counter-current di~tributi0n.l~’ X-Rayanalysis of crystals of gramicidin A and B show that they may be both builtup of a repeating unit of molecular weight 3800, which is compatible with theresults of chemical analysis.128 An outstanding problem presented by thecomposition of the gramicidins is how molecules that have neither acidic norbasic properties can be made up only of monoamino-monocarboxylic acidsand ethanolamine.It has been reported that the free OH groups in grami-cidin do not come from the ethanolamine residues. A possible structure inwhich the ethanolamine is involved in an O-peptidyl linkage has beensuggested (XIII), but this structure stillleaves the question of the masked basicGramicidin S is quite distinct from the\CHR~ (xIII.) other gramicidins and contains only five/ different amino-acids.The smallest ob-served unit in crystals of this compound corresponds to a decapeptide. Theantibacterial properties of gramicidin S appear to depend on its cyclicstructure, for various derivatives of an open-chain pentapeptide containingamino-acids of the same configuration and in the same sequence as those inthe antibiotic showed little activity.130Marcescin has been obtained as a, highly active powder fromculture fluids of Serratia m a r ~ s c e n s . l ~ ~ The crude material was found to bea complex which could be separated on a buffered silica-gel column into anacidic and a basic fraction. The antibacterial activity was associated withthe latter, which was shown to be a strongly basic polypeptide of considerablestability. Marcescin is highly active against certain Gram-positive organisms,but it is hzemolytic and very toxic to mice.Nisin. Further studies on nisin, an antibiotic produced by a strain ofStrep. lactis, have indicated that the needles previously observed in purifiedmaterial 132 may not have been true crystals, and that the most active prepar-ations are a mixture of at least two polypeptides.133 The amino-acidsalanine, valine, leucine, and isoleucine were recognised in a hydrolysate ofpurified nisin.Anagar diffusion method for the assay of nisin has been described.l=Gramicidins and Gramicidin 8.R*HC\C/OH // groups unsolved.129HN/ \O*CH2*CH2*N \Marcescin.Cystine and aspartic acid were also probably present.12’ Gregory and Craig, J . Biol. Chem., 1948, 172, 839.Hodgkin, Cold Spring Harbor Symp., 1949, 14, 65.lZ0 Synge, ibid., p.191.130 Harris and Work, Biochem. J . , 1950,48, 582.131 Fuller and Horton, J . Cfen. Microbiol., 1950, 4, 417.132 Berridge, Lancet, 1947, 253, 7 . 133 Idem, Biochem. J . , 1949, 45, 486.Friedman and Beach, Proc. SOC. Gen. Microbiol., 1951,5, V298 BIOCHEMISTRY.lodinin. This antibiotic from Chrombacterium iodinum has finallybeen proved to be the di-N-oxide of 1 : 5-dihydroxyphena~ine.1~6MisceZZuneous. The following antibiotics from bacteria have not yetbeen well characterised. An antibiotic from Bacillus nutto,136 an antibioticfrom B. b r e z ~ i s , ~ ~ ~ various c~licines,~~* diplomycin,139 fungocina,lU Rhizoc-tonia, and Aspergillus factors,141 and ~ t r e p t o t a s i n .~ ~ ~Mode of Action and Acquired Resistance.-The mechanisms by which theantibiotics exert their antibacterial effects are of fundamental interest tobiochemistry ; they are also of practical importance, for a better understand-ing of them may help to place chemotherapy on a more rational basis.Some antibiotics, such as tyr0cidine,l4~ may rupture the surface mem-branes of the bacterial cell or denature cell proteins in an unspecific manner,but there can be no doubt that many others, including those important inmedicine, act in a more subt,le fashion. Possibly these substances interferewith the function of one specific enzyme. Since penicillin is vastly moreeffective against growing than against resting organisms, and the former arealso more susceptible to streptomycin and chl~romycetin,~~~ special import-ance may attach to enzymes concerned with the synthesis of new cell material.The changes in morphology that sometimes occur in the presence of anti-biotics 146 show that cell growth and cell division may be inhibited todifferent degrees.One possible approach to the,subject is to study the fate of the antibioticitself when it comes into contact with sensitive bacteria.This is a difficultprocedure because very small quantities of substance are involved, but itmay be facilitated by the use of antibiotics containing a radioactive atom.146With penicillin containing 35s it has been found that sensitive organisms bindmore of the drug than do resistant ones, and growing organisms more thanresting ones.Sensitive bacteria are able to concentrate penicillin from thesurrounding medium so long as it is present at a higher dilution than thata t which it exerts a maximum bactericidal effect. Since the radioactivity155 Clemo and Daglish, J., 1950, 1481.13b Ishidate, Shibata, and Hagiwara, J . Pharm. Soc. Japan, 1949, 69, 373.lS7 Anzai, Nagaki, Date, Tsukakoshi, Hatori, and Nakamura, Kitisato Arch.E':E":E':E':E':E':E':E. Med., 1948, 21, 23 ; Nagaki and Yajima, ibid., 1948, 20, 28.138 Chabbert, Ann. Inst. Pasteur, 1950, 79, 51 ; Fredericq, Compt. rend. SOC. Biol.,1950, 144, 297, 299, 435, 437, 439, 728, 730; Mondolfo, Bull. SOC. Ital. Biol. sper., 1948,24, 1086.130 Noster, Zentr. Bakt., 1949: 153, 32; Enell, Lingen, and Melin, Acta Paediatr.,Stockh., 1950,39, 251.140 Cer6os, Ministerio de Agr.y Ganed. (Argentina) Pub. Tec. No. 36.141 Michener and Snell, Arch. Biochem., 1949, 22, 208.142 SheTwood, Russell, Jay, and Bowman, J . Infect. Dis., 1949,84, 88.143 Fong and Krueger, J . Ben. Physiol., 1950,33,311.144 Edlinger, Ann. Inst. Pasteur, 1950, 78, 417.145 Fleming, Voureka, Kramer, and Hughes, J. Ben. Microbial., 1950, 4, 257; Leva-diti, Vaisman, and Henry-Eveno, Bull. Acad. Nat. Med., 1950,134, 369; Warbasse andJohnson, J . Bact., 1950, 60, 279.146 Cooper and Rowley, Nature, 1949,163,480; Cooper, Rowley, and Dawson, ibid.,1949,164, 842; Maass and Johnson, J . Bact., 1949,57,415ABRAHAM AND NEWTON : ANTIBIOTICS. 299is not ertsily removed from the cells it appears that penicillin enters intochemical combination with a cell component, which is present in very smallamounts in resting organisms but which increases during g r 0 ~ t h .l ~ ~An alternative approach is to look for enzyme systems in the cell whichare affected by the antibiotic. It is often difficult here to distinguish betweenthe primary action of the drug and one of the innumerable changes that musteventually follow it. The interesting discovery that certain Gram-positiveorganisms are no longer able to .concentrate amino-acids from their environ-ment after contact with penicillin during growth suggested the possibilitythat penicillin acted by preventing amino-acid a~simi1ation.l~~ It does notnow seem probable that this is the first effect of the drug, for other organismsthat are highly sensitive to penicillin do not need pre-formed amino-acidsfor growth.149 Nevertheless, it is not unlikely that the antibacterial effect ofpenicillin is closely related to a disturbance of protein synthesis.Staphy-lococci respiring in glucose and various amino-acids do not increase theirprotein in the normal manner when penicillin is present, but appear insteadto produce extracellular polypeptide.lW The effect of penicillin on a Gram-negative bacterium grown in L-leucine, glycine, L-leucine plus glycine, orL-leucylglycine was thought to indicate that it prevented the incorporationof glycine into a peptide.151 On the other hand, staphylococci growing inthe presence of .penicillin also undergo changes in their nucleotide-nucleicacid balance and it has been suggested that this may really be the primaryeffect of the drug.152 An enzyme system in Staph.aureua concernedwith the catabolism of uridylic and guanylic acids is inhibited bypenicillin .153Streptomycin has been found to affect certain metabolic activities ofresting sensitive bacteria. It inhibits the complete oxidation of stearic acidby resting cells of Bact. coli, apparently by stopping the condensation ofpyruvate with oxaloacetate and thus preventing the entry of pyruvate intothe tricarboxylic acid cycle of the terminal respiration system.154* 156 Theoxidation of higher fatty acids by Myw. tuberculosis Avian Type is inhibitedby streptomycin, but this is reported to take place by a different mechan-ism.165 It does notaffect the respiration or protein breakdown of resting or growing bacteria,but inhibits the action of esterases. Its action on the esterases of animalcells is much weaker than on those of sensitive bacteria and it has been sug-gested that the animal cell wall presents a barrier to the drug.166 Anti-Chloramphenicol also disturbs the metabolism of fats.ld7 Rowley, Cooper, Roberts, and Lester-Smith, Biochem.J., 1950,46, 157.14* Gale and Taylor, J . Gen. Microbiol., 1947, 1, 314.140 Hunter and Baker, Science, 1949,110,423 ; Grelet, Ann. Inst. Pasteur, 1949,77,263.I 5 O Hotchkiss, J . Expt. Med., 1950, 91, 351; Ann. N . Y . Acad. Sci., 1950, 53, 13.151 Simmonds and Fruton, Science, 1950,111, 329.15( Mitchell, Nature, 1949,164,259; Park and Johnson, J .Biol. Chem., 1949,179,585.153 Gros, Beljanski, and Machboeuf, Compt. rend., 1950, 231, 184.154 Umbreit, J . Biol. Chem., 1949, 177, 703; Ann. N . Y . A d . Sci., 1950, 53, 6 .155 Oginsky, Smith, and Solotorovsky, J . Bact., 1950, 69, 29.156 Smith, Worrel, and Swanson, ibid., 1949, 58, 803300 BIOCHEMISTRY.mycin A has proved to be the most powerful inhibitor of succinic dehydro-genase so far discovered, and unlike other inhibitors of this enzyme it isneither competitive nor involved in a reaction with SH groups.l0lSome antibiotics appear to inhibit a process by which the energy ofcellular oxidation is made available for synthesis, showing a resemblance inthis respect to 2 : 4-dinitrophenol.Gramicidin inhibits the uptake ofphosphate by staphylococci without depressing respiration,157 and alsoinhibits phosphorylation in the cyclophorase system. 158 Aureomycininhibits phosphorylation by mitochondria without affecting re~pirati0n.l~~Such effects are thought to be caused by a disturbance of the mechanism bywhich phosphate-bond generation is coupled to oxidative reactions.160The idea that an antibacterial substance may inhibit the utilisation of anessential metabolite of similar structure, which has played so important apart in our understanding of the mode of action of the sulphonamides, hashitherto found little application in studies of the antibacterial action ofantibiotics. It has been found, however, that phenylalanine and certainstructural analogues inhibit the action of chloramphenicol in a non-competitive manner,] 61 and that an antagonist to streptomycin anddihydrostreptomycin is produced during the growth of Pseudomnaspyocyanea.162Bacteria become more resistant to most antibiotics when grown in theirpresence, though the rapidity and extent of the change in sensitivity mayvary enormously.163 Resistance to penicillin, chloromycetin, aureomycin,and terramycin appears to develop gradually, whereas very high resistanceto streptomycin may suddenly be acquired in a single step.164 In additionto acquiring resistance, bacteria may become dependent on streptomycin 165and on chloramphenicol 166 for their growth.The interesting phenomenon of acquired resistance raises two relatedquestions.By what kind of mechanism does resistance develop? and inwhat way do resistant organisms differ from their sensitive ancestors ?It is now widely assumed that resistant organisms are formed by mutationindependently of the antibiotic, but that in its presence they replace themajority of sensitive organisms through the operation of natural selec-ti0n.1~~* 167 The direct evidence for this view comes largely from the applic-15' Hotchkiss, Adu. Enzymology, 1944, 4, 153.lK8 Cross, Taggart, Covo, and Green, J. Biol. Chem., 1949,177, 655.lS0 Loomis, Science, 1950, 111, 474.l 6 0 Loomis and Lipmann, J . Biol. Chem., 1948,173, 807.161 Woolley, Fed. Proc., 1950, 9, 249.163 Gezon, Fasan, and Collins, Proc. SOC. Exp. Biol., 1950, 74, 505; Garrod, Bull.Hyg., 1950, 25, 539; Miller and Bohnhoff, Ann.Rev. Microbiol., 1950, 4, 201 ; Coffey,Schwab, and Ehrlich, J . Infect. Dis., 1950, 87, 142.lti2 Lightbrown, Nature, 1950,166, 356.164 Bryson and Demerec, Ann. N . Y . Acad. Sci., 1950, 53, 283.165 Miller and Bohnhoff, J . Bact., 1947, 54, 467; Foley and Shwachman, J . Gen.Microbiol., 1950, 4, 141.Goche and Finland, Proc. SOC. Exp. Biol., 1950, 74, 824.167 Wyss, Ann. N . Y . Acad. Sci., 1950,53, 183 ; Cavalli and Maccacaro, Nature, 1950,166, 991ABRAHAM AND NEWTON : ANTIBIOTICS. 301ation of an ingenious statistical method 168 to the analysis of the numbers oforganisms that have become resistant to penicillin and s t r e p t ~ m y c i n . ~ ~ ~The interpretation of the results obtained with penicillin, however, has beendisputed,170 and some of the facts about the development of resistance tostreptomycin are thought to remain unexplained by the theory of spontaneousmutati0n.17~The alternative theory that resistance is induced by the drug has beendeveloped in an interesting manner on physico-chemical lines,172 but hasbeen criticised as Lamarkian.173 Although the force of this criticism inrelation to bacteria is far from evident, it would not be surprising if gene-likestructures were involved in some of the enzymic changes that increase theresistance of the bacterial cell to for the presence of specific genes iscertainly required for many biochemical reactions in Neurospora and incertain yea~ts.1'~ However, the acquisition of resistance may sometimesdepend on changes in the amount rather than in the nature of the enzymesystems in the cell, and whether these changes are unconnected with thepresence of the drug itself seems to be still an open q ~ e s t i 0 n .l ~ ~ Boththeories may well play a part in explaining the varied phenomena of acquiredresistance.In some cases bacteria with an acquired resistance to an antibiotic areable to dispense with a biochemical process that is inhibited by the antibioticin the original sensitive strain. As the resistance of Stuph. aurew to penicillinincreased the cells lost their ability to concentrate pre-formed amino-acidsfrom the medium and acquired a mechanism for synthesising the amino-acidsfrom ammonia and g1u~ose.l~~ When Bact.coli acquired resistance tostreptomycin it was no longer able to bring about the rapid oxidation ofstearate that had previously been inhibited by the It is thusreasonable to suppose that the development of alternative metabolic path-ways may be responsible for acquired resistance to these substances.Antibiotics in Medicine.-It is no exaggeration to say that the last fifteenyears have seen a revolution in the treatment of infectious disease. Thechange which began with the introduction of the sulphonamides has becomefar-reaching through the isolation of a variety of antibiotics having systemicchemotherapeutic properties. To-day the majority of bacterial infectionscan usually be treated with some success, and chemotherapy has also beenextended to rickettsia1 and some viral diseases.168 Luria and Delbruck, Genetics, 1943, 28, 491 ; Lea and Coulson, J .Genet., 1948,169 Demerec, J . Bact., 1948,56, 63; J . Clin. Invest., 1949,28, 891.l i 0 Eriksen, Acta Path. Microbiol. Scand., 1949, 26, 269.1 7 2 Hinshelwood, " The Chemical Kinetics of the Bacterial Cell," 1946, Clarendon1 7 4 Clark, Wyss, and Stone, Nature, 1950,166, 340.175 Beadle, Ann. Rev. Physiol., 1948, 10, 17 ; Tatum and Perkins, Ann. Rev. Micro-49, 264.Linz, Ann. Inst. Pasteur, 1950, 78, 105.Press, Oxford; Endeavour, 1949, 8, 151. Lurk, Bact. Reviews, 1947, 11, 1.biol., 1950, 4, 129. 176 Hinshelwood, Nature, 1950,166, 1089.Gale and Rodwell, J . Qen. Microbiol.,, 1949, 3, 127302 BIOCHEMISTRY.The clinical use of the more valuable antibiotics has recently been thcsubject of a number of revie~s.~O1 8op 96* 112* 178 Penicillin remains the drug ofchoice for infections caused by sensitive organisms such &g streptococci,sfaphylococci, pneumococci, neisseria, B.anthracis, and clostridia, and also byspirochaetes. Its very low toxicity makes it almost a perfect chemothera-peutic agent, although reactions due to sensitisation of patients are said tobe increasing.The use of streptomycin has tended to become more restricted, for thissubstance can cause permanent deafness and vestibular damage, and bacteriadevelop resistance to it with remarkable facility. Dihydrostreptomycin hasbeen claimed to be less toxic than streptomycin to the vestibular apparatus,but this is not universally accepted.Streptomycin is highly effective,however, in the treatment of plague and tularemia, and appears to bevaluable in combating the infection that is a significant factor in death fromradiation injury.179 It is still the best drug available for dealing withtuberculosis, and can save the lives of a significant proportion of patients withmiliary or meningeal tuberculosis, diseases that are almost invariably fatalwhen untreated. In the treatment of other forms of tuberculosis the drughas definite limitations. With pulmonary tuberculosis it appears best touse streptomycin together with p-aminosalicylic acid (PAS) .180 Whetherneomycin will prove superior to streptomycin remains to be seen, but since itappears to produce renal damage caution will be necessary in its clinical use.Chloramphenicol and aureomycin are valuable in the treatment of anumber of bacterial diseases that do not respond to penicillin.For example,chloramphenicol is highly effective against typhoid fever, and aureomycinagainst infections with penicillin-resistant staphylococci, while both sub-stances give good results with undulant fever. These antibiotics have alsobeen responsible for the extension of chemotherapy to rickettsia1 diseases,such as typhus, and to certain viral infections, such as psittacosis, lympho-granuloma venereum, and primary atypical pneumonia. No dangeroustoxic reactions appear to follow the use of the drugs, but aureomycin oftenproduces nausea and diarrhoea, and chloramphenicol may cause dry tongueand muscular weakness, Terramycin, which received extensive humantrials in 195O,l8l shows a close resemblance to aureomycin in its therapeuticand pharmacological properties.The polypeptide antibiotics bacitracin and polymyxin exert a chemo-therapeutic action in animals infected with certain Gram-positive and Gram-negative bacteria respectively, and both have been tried on a limited scale178 Garrod, Brit.Med. J., 1950,11, 722; Knight, N.Y. State J . Med., 1950,50, 2173;Herrell, Amr. J . Med. Sci., 1950, 219, 670; Germer, Deut. med. Woch., 1950, 75, 1132;Smadel, Amer. J. Trop. Med., 1950, 30, 357.170 Miller, Hammond, and Tompkins, Science, 1950,111, 719; Hammond and Miller,Ann. N . Y . Acad.Xci., 1950, 58,303.180 Brit. Med. J . , 1949,11, 1615; 1950,11, 1073.181 Yeager, Mansberger, Thomas, and Barnes, Ann. N . Y . Acad. Sci., 1950, 53, 319;Knight, ibid., p. 332 ; Smadel, Jackson, and Ley, ibid., p. 375 ; Kneeland and Melcher,ibid., p. 437; Caldwell, Spies, Wolfe, Lepper, and Dowling, J . Lab. CEin. Med., 1950,36, 747 ; Linsell and Fletcher, Brit. Med. J . , 1950,11, 1190VAN REYNINGEN : BACTERIAL TOXINS. 303in man, but the fact that they can cause serious damage to the kidneytubules has prevented their general use. The use of bacitracin and penicillintogether in caam where the two drugs exert a synergic action, however, isreported to be very effective,l82 and polymyxin B and E have proved valuablein the treatment of infection with Ps.p y o c y a n e ~ . ~ ~ ~Although many of the properties required by a systemic chemotherapeuticagent are well understood, the value of a new antibiotic t o medicine cannotyet be predicted with confidence on the basis of its antimicrobial activityin vitro and its pharmacological behaviour. The effective concentration ofpenicillin in vivo against streptococci and pneumococci is reported to be closeto that which would be expected from its activity in v i t r ~ , ' ~ ~ but no simplerelation is said to be found in the treatment of infection with B. n ~ v y i . ' ~ ~Aureomycin is stated to be five to ten times more effective against Stuph.u w e w in wivo than in vitro,l86 while helvolic acid, which can readily beintroduced into the blood in bacteriostatic concentrations, is unable to controlstaphylococcal infections in a satisfactory manner.187 On the other hand,although aureomycin compared favourably with streptomycin in its activityagainst a strain of Myco.tubercuEosis in oitro, it was quite ineffective againstinfection induced with this organism in mice.ls8 The reason why ohlor-amphenicol is much more effective than aureomycin against typhoid feveris also not apparent from the activity of these antibiotics in vitro.Whether bactericidal activity is essential in a powerful chemotherapeuticagent, or whether a bacteriostatic action may be sufficient, is still uncertain.When tested in vitro the antibiotics that are valuable in medicine usuallykill the majority of a population of sensitive bacteria but leave a smallnumber of survivors.87 How far the natural defence mechanisms of the bodycan contribute to the elimination of infecting organisms, and how far theirefficiency is affected by the various antibiotics, appear to be questions worthyof future study.E. P.A.G. G. F. N.1821831841851861 8 71883. BACTERIAL TOXINS.Two of the more important dates in the history of bacteriology are 1858and 1890, when Roux and Yersin discovered that sterile culture filtrates ofthe diphtheria bacillus contained a toxic substance, and von Behring showedthat the serum of animals injected with sub-lethal doses of this toxincontained a specific antitoxin. Since then many other toxic antigenicsubstances have been found in the culture filtrates of Gram-positive bacteriaJohnson and Meleney, Ann.AN. Y . Acad. Sci., 1950, 53, 42.Pulmki, Baker, Rosenberg, and Connell, J . Clin. Invest., 1949, 28, 1028.Eagle, Fleischrnan, and Musselman, J . Bact., 1950, 59, 625.Kley and Ercoli, Experientia, 1950,6, 153.Klein, Schorr, Tashman, and Hunt, J . Bact., 1950,60, 159.Florey and Jennings, 1946, unpublished.Stoinback, Baker, and Ducn, Proc. SOC. Exp. Biol., 1950, 74, 596304 BIOCHEMISTRY.and in the cells of Gram-negative bacteria, and also in snake venoms andplant seeds. The coagulases, fibrinolysins, hyaluronidases and proteases(and sometimes the haemolysins) listed in Table I are not always classifiedTABLE I.Toxins produced by principal toxin-producing pathogenic Gram-positivebacteria.Diseases associated with these organisms are given in squarebrackets.CZostridium botulinum : Four neurotoxins. [Botulism in man and animals.]CZ. oedematiens : (1) a-toxin, lethal, histotoxic ; (2) /?:toxin (lecithinase), lethal,haemolytic ; (3) y-toxin (lecithinase), lethal, haemolytic ; (4) b-toxin, haemolytic ;(5) c-toxin (lipase), lethal, haemolytic ; (6) I-toxin, haemolytic. [Gas gangrene inman; black disease and bradsot in sheep ; bacillary osteomyelitis in buffaloes.]CZ. septicum : A lethal haemolytic toxin. [Gas gangrene in man; blackleg and braxy insheep.]Cl. tetani : (1) Tetanospasmin, neurotoxic ; (2) tetanolysin, haemolytic. [Tetanus inman and animals.]C1. wekhii : (1) a-toxin (lecithinase), lethal, histotoxic, haemolytic ; (2) p-toxin, lethal;(3) y-toxin, lethal; (4) &toxin, lethal; (5) €-toxin, lethal, histotoxic ; (6) 7-toxin,lethal ( 9 ) ; (7) c-toxin, lethal, histotoxic; (8) &toxin, lethal, haemolytic ; (9) K-toxin(collagenase), lethal, proteolytic ; (10) h-toxin, proteolytic ; (1 1) p-toxin (hyaluroni-dam), spreading factor.[G? gangrene and enteritis necroticans in man; lambdysentery, struck and infectious enterotoxaemia in sheep.]Corynebacterium diphtheriae : A lethal, histotoxic toxin.Staphylococcus aureus : (1) a-toxin, lethal, histotoxic, haemolytic ; (2) p-toxin, lethal,haemolytic ; (3) y-toxin, lethal, hsmolytic ; (4) b-toxin, hsmolytic ; (5) hyaluronidase,spreading factor ; (6) coagulase ; (7) fibrinolysin. [Pyogenic infections in man andanimals .]Streptococcus pyogenes : (1) Dick toxin, lethal, erythrogenic ; (2) Streptolysin-0, lethal,hsmolytic ; (3) streptolysin-S, lethal, haemolytic ; (4) hyaluronidase, spreadingfactor ; (5) streptokinase, fibrinolytic. [Scarlet fever ; tonsillitis, pyogenicinfections, etc., in man.]as toxins, but their exclusion from this category is unwarranted.Theirtoxicity is either unknown or it is intermediate in the range of toxicity ofthe conventional toxins, which stretches from about 0.2 Minimal LethalDoses (per kg., mouse) per mg. (Shigelh shigae endotoxin) to 1.2 x log MLD(per kg., guinea pig) per mg. (Clostridium tetani exotoxin). Nor should theybe excluded merely because their substrates can, in some cases, be definedin more definite anatomical or chemical terms than those of the classicaltoxins.Both groups of substances are biologically active and antigenic,and are not only toxic to the host but also (with the possible exception ofthe neurotoxins) helpful to the parasite in its invasion.Since the beginning of the last war much progress has been made in thestudy of the toxins, as is superficially evident from the Decennial Indices ofChemical L4bstracts ; the Index for 1937-1946 contains 11 columns of entriesunder ‘‘ Toxins ” compared with 3 columns for the previous decade. Thisprogress has recently been reviewed,l* 2* but it continues to receive scantattention in recent textbooks of pathology, bacteriology, and biochemistry.In this review only a few aspects of this subject can be dealt with, and anecessarily over-simplified account of the toxins of Gram-negative bacteria[Diphtheria in man.]1 Pappenheimer, Adu.Protein Chemistry, 1948, 4, 123.9 Pillemer and Robbins, Ann. Review Microbiol., 1949, 3, 265.3 van Heyningen, “ Bacterial Toxins,” Oxford, Blackwell Scient. Publ. Ltd., 1950VAN HEYNINGEN : BACTERIAL TOXINS. 305is included mainly in order that they can be compared with those of theGram-positive bacteria.Lecithinases.-The or-toxin of CZ. welchii, which plays a major role inthe toxaemia of gas gangrene, is a lecithinase.4 In particular it is a lecithin-phosphatase that catalyses the hydrolysis of the bond between phosphoryl-choline and glycerol stearate oleate in lecithin, and therefore it has beencalled lecithinase C.(Lecithinase A splits off the unsaturated fatty acid toleave lysolecithin, and lecithinase B splits off both fatty acids.s The namelecithinase C was once proposed for a hypothetical enzyme that split offcholine,6 but priority should be given to the enzyme whose existence isestablished.) The Cl. welchii lecithinase is.quite specific ; it does not attackkephalin, cerebroside, lysolecithin, a-glycerophosphorylcholine, or p-glycero-phosphate, but it attacks sphingomyelin very slowly.4* '9 It is inactive inthe absence of calcium or magnesium ions and is therefore inhibited byphosphate or citrate buffers. The reaction between toxin and antitoxinalone is unaffected by these ions, but in the presence of substrate it is affectedbecause the toxin-substrate reaction is favoured and consequently theequilibrium of the reaction, toxin + antitoxin toxin-antitoxin isshifted to the 1eft.O Lecithinase C can be quantitatively determined bymeasuring the amount of acid-soluble phosphorus liberated by it fromle~ithin,~ or manometrically by measuring the volume of carbon dioxideliberated by the action of phosphorylcholine on sodium hydrogen carbonate,8or less accurately but with greater convenience by measuring the turbidityit produces in a solution of crude egg-yolk 1ipoprotein.lOLecithinases are also produced by other anaerobic clostridia, wiz., Cl.oedemtiens (p- and y-toxins),ll CZ.haemolyticum (-q-toxin),ll* l 2 q l3 Cl.biferrnentuns.l4 They are probably also produced by Cl. sordellii, chauvoei,sporogenes, centrosporogenes, and tertium.15 The lecithinases of Cl.welchiiand CZ. oedemtiens are antigenically distinct, but Cl. oedemtiens p-toxin andCl. huemolyticum ?-toxin are apparently antigenically identical.12 Thereis some antigenic relationship between the lecithinases of CZ. welchii andCl. bifermentans.14The aerobic sporing bacilli, Bacillus anthracis, B. mycoides, and particularly23. cereus, also produce a lecithinase C, which will hydrolyse kephalin aswell as lecithin and sphingomyelin. Unlike the lecithinases of the anaerobes,its action on free lecithin (but not on lipoprotein lecithin, or on kephalin) isMacfarlane and Knight, Biochem. J . , 1941, 35, 884.Belfanti, Contardi, and Ercoli, Ergebn. Enzymforsch., 1936, 5, 213.Contardi and Ercoli, Biochem.Z . , 1933, 261, 275.Macfarlane, Biochem. J . , 1942, 36, 11 1 ; 1948, 42, 587.Zamecnik, Brewster, and Lipmann, J. Exp. Med., 1947, 85, 381.Oakley and Warrack, J . Path. Bact., 1941, 53, 355; Zamecnik and Lipmann, J.Exp. Med., 1947, 85, 395. lo van Heyningen, Biochem. J . , 1941, 35, 1246.l1 Oekley, Warrack, and Clarke, J . Gen. MicrobioE., 1947, 1, 91.l2 Macfarlane, Biochem. J., 1950, 47, 267.l3 Jasmin, Amer. J . Vet. Res., 1947, 8, 289.l4 E. M. Miles and A. A. Miles, J . Qen. Microbiol., 1947, 1, 385; 1950, 4, 22.l5 Crook, Brit. J . Exp. Path., 1942, 23, 37306 BIOCHEMISTRY.inhibited by normal serum.l6, l7 The hemolytic bacterial lecithinases havea different action from the lecithinases of certain snake venoms.The latterare lecithinases A whose action results in the formation of the hemolyticsubstance, lysolecithin. Neither of the products of hydrolysis by theformer is itself hzemolytic.CZ. wekhii lecithinase has been partially purified, but a quantitativestudy of the toxin-antitoxin flocculation showed that the product could notbe more than 50% pure. Half of the nitrogen of the purified product wasprecipitated by antitoxin, but since it was shown that toxin-antitoxinfloccules could carry down non-specific impurities it could not be assumedthat all the precipitated material was pure toxin-antitoxin.l* This workshowed that quantitative studies of the toxin-antitoxin flocculation re-action l9 can have their pitfalls. The diffusion constant of the toxin, 020, is7-41 x The partly purified toxin had MLD 200 (per kg.,mouse) per mg.The bacterial lecithinases are lethal, hemolytic, and histotoxic, andalthough the fundamental biochemical activity underlying these biologicalactivities has been defined, a complete explanation of their behaviour inbiological systems is still missing.Lecithinases from different species oforganisms can differ in their behaviour in the cells of one animal, and cellsfrom different animals can react differently to an enzyme from one source.The lecithinases of CZ. welchii and CZ. oedematiens have the same action onlecithin in vitro but they differ in their action on the red blood cell. CZ.welchii a-toxin haemolyses sheep red cells readily and horse red cells slowly ;CZ.oedematiens y-toxin hzemolyses sheep cells slowly and horse cells readily ;CZ. oedematiens &toxin haemolyses both species of cells readily.ll Table I1cm.2/sec.18The other bacterial lecithinases have not been purified.TABLE 11.Reactions of different lecithinases with red blood cells from different species.(Adapted from Macfarlane, Biochem. J . , 1960, 47, 270.)Reaction rates.7 A \ Hydrolysis of Hydrolysis ofLecithinme (amounts phospholipids in phospholipids ex-intact cells. tracted from cells.lecithin in vitro). Sheep. Horse. Sheep. Horse. Sheep. Horse.CLwelchiia ............... +++ + +++ + +++ +++Cl. oedematiens y ......... + +++ + +++ +++ +++CL oedematiens ......... +++ +++ +++ +++equipotent in action on H~molysis.shows that the species differences in hemolysis of red cells occur only inthe intact cells; they are not apparent when the lecithinases act on thelecithin in the phospholipids isolated from the cells.The species differencesl6 McGaughey and Chu, J . Qen. Mimobiol., 1938, 2, 234.Chu, ibid., 1949, 3, 255.van Heyningen and Bidwell, Biochem. J., 1948, 42, 130.19 Pappenheimer and Robinson, J . Immunol., 1937, 32, 291VAN HEYNINQEN : BACTERIAL TOXINS. 307therdfore must be concerned with the nature of the intact cells and theaccess that the enzymes have to their substrates in them. Such access couldbe controlled by properties of the enzyme, such as molecular weight, shape,and charge, which would not necessarily affect its action on isolated lecithin.The bacterial lecithinases can also differ in their toxicity to animals.It has been observed that amounts of lecithinase from three different strainsof Cl.bifermentuns that were equipotent in their enzyme activity in vitrowere respectively 9, 60, and 70 times less toxic to mice, and less haemolyticto red cells, than the corresponding amount of Cl. welchii lecithinase. Inthis case there were differences not only between different species oforganisms, but between strains of the same species.14There are differences in species susceptibility to other toxins besides thelecithinases. Thus the guinea-pig is 1000 times more susceptible to diphtheriatoxin than the mouse 1 ; some workers 20 have found that the guinea-pig isG000--8000 times more susceptible to the toxin of Cl.botulinum type B thanthe mouse, others 21 have found only a 3-fold difference ; the ratio of guinea-pig toxicify/rabbit toxicity has been found to vary from 3 to 354 withdifferent samples of toxin from Cl. tetuni.22 The mitis and gravis types ofCorynebacterium diphtheriae cause diseases that differ considerably in severityalthough no differences have yet been observed in their toxins. It isconceivable that while these toxins are equally toxic to guinea-pigs theymay differ in their toxicity to man.Differences in toxicity of toxins having the same biochemical actioncould also be caused by the presence, in the body, of inhibitors, as is the casewith the B. cereus lecithinase. This lecithinase is 4-8 times less toxic tomice than the CZ.wekhii lecithinase because it is inhibited by a constituentof normal serum. In the absence of serum it is just as hEmolytic as theCZ. welchii 1ecithinase.l' The same argument does not apply to the CZ.bifermentam lecithinase because this enzyme is not inhibited by normalserum and it is also less hEmolytic than the CZ. welchii enyme. Enzymeand toxin activators in the body should also be considered, although thesehave not yet been identified. It has been shown that constituents of brothand serum may " potentiate " botulinus and tetanus toxins, and that thispotentiation is not merely a protection of the toxins against denaturation indilute solution .23The hemolysis that is caused by lecithinase acting on the red blood cellfollows the breakdown of cell ph~spholipid,~~ but it is not clear how thebreakdown of lecithin or phospholipid in other tissues of the body results inharmful effects. One means is suggested by the recent discovery that CZ.welchii lecithinase inactivates the magnesium-activated adenosine tri-phosphatase (Mg-ATPase) of muscle.This enzyme, which is distinct from*O Stevenson, Helson, and Reed, Canad. J . Res., 1947, 85, E , 14.21 Lamanna and Glassmann, J. Bacb., 1947,5;4, 576.22 Llewellyn Smith, Bull. Health Organ. League of Nations, 1942143, 10, 104.2s (a) Traub, Hollander, and Friedemann, J . Bact., 1946, 62, 169; (b) Wentzel,Macfarlane, Biochern. J., 1860, 47, 270. Sterne, and Polson, Nature, 1950,166,739308 BIOCHEMISTRY.the myosin-ATPase, has a phospholipid prosthetic group which is essehtialfor its activity and is destroyed by the lecithinase.2sDiphtheria Toxin.-The properties of this toxin, which is the first to havebeen obtained pure,26 are given in Table 111.Recent work on its mode ofTABLE 111.Properties of puri$ied toxins.Diphtheria toxin. Botulinus A toxin. Tetanus toxin.References U 1, 35, b 1, 36, c.Biological propsr ties Lsthal, histo toxic. L9 thal, neurotoxic, Lethal, neurotoxic.haemagglutinat-ing, precipitatesnormal serum.MLD/mg.(per kg. mouse) 3.5 620,000 200,000(per kg. guinea-pig) 3500 1,200,000 1,200,000x 107 (200)Isoelectric point 4.1 5.5 5.1Sedimentation const. 4-6 17.3 4.5Molecular weight 72,000 900.000--1,130,000 67,000Elementary analysisDiffusion const.6.0 cm.2/sec. 2.14 cm.2/sec. -Frictional coeff., f/fo 1-22 1.76 -C, 51-47; H, 6.75; C, 53-73; H, 7.02; N, 15.7; S, 1.04;N, 16.0; S, 0.75; N, 16.29; S, P, 0.065P, <0.03(%)0.437 ; P, 0.052-0.059a, Peterman and Pappenheimer, J. Phys. Chem., 1941, 45, 1 ; Pappenheimer,Lundgren, and Williams, J . Exp. Med., 1940, 71, 247; Pappenheimer, J. Bact., 1942,43, 273.b, Buehler, Borner, Schantz, and Lamanna, J. Bad., 1946, 51, 571; Putnam,Lamanna, and Sharp, J. Biol. Chem., 1946,165, 735.c , Dunn, Camien, and Pillemer, Arch. Biochem., 1949, 22, 374.TABLE IIIA.Properties of purijed toxins.Diphtheriatoxin. Botulinus A toxin.PerAmino-acid composition ,--A-, (-&-, mol.Alanine Arginine - ; 3.8 3.92; 4-62 394; 239Aspartic acid Cysteine -; - 20.26; 0.27 1370; 20Cystine Glutamic -; - 0.53; 15-57 40; 953G1 ycine Histidine - ; 2.4 1.38; 1.03 166; 60Leucine isoLeucine -; - 10.30; 11.94 708; 820Lysine Methionine 5.3; - 7.74; 1.06 477; 64Phenylalanine Proline _.- 1.17; 2.60 64; 203Serine Threonine - a - 4.36; 8.49 374; 642Tryptophan Tyrosine 1.4; 9.5 1.86; 13.50 82; 672Valine - 5.29 406%. %.acidTotanus toxin.Per(-A----, mol. %*- . , 3.36 -; 1315.3 ; - 76; -- ; 10.3 -. , 473-34; 1-15 30; 58.23; 9.36 43; 4810.0 ; 1.78 46; 84.91; - 20; -- ; 5.13 -; 290.91; - 3; -5.39 31production by the organism might provide a clue to its mode of a c t i ~ n . ~ ’ ? ~ ~The production of toxin is always accompanied by the production of extra-2: Kielley and Meyerhof, J.Biol. Chem., 1950, 183, 391.26 Eaton, J. Bact., 1936, 31, 367; Pappenheimer, J. Biol. Chem., 1937, 120, 543.2 7 Idem, ibid., 1947, 167, 251.2 8 Pappenheimer and Hendee, ibid., 1947, 171, 701 ; 1949, 180, 597VAN HEYNINGEN : BACTERIAL TOXINS. 309cellular porphyrin, and the production of both of these is inhibited when theiron content of the culture medium is above an optimum level of about100 pg./l. For every 4 atoms of iron in excess of this level 4 fewer moleculesof porphyrin and 1 fewer of toxin are produced. The excess of iron is takenup by the organism which at the same time acquires a greater concentrationof an intracellular haem protein which is probably identical with cytochromea respiratory pigment which is concerned in the oxidation of succinicacid.When the toxin production has fallen to half the maximum valueobtained in low-iron media the rate of oxidation of succiriic acid has increasedto half the maximum obtained with organisms grown in high-iron media.Since the proportions of iron : porphyrin : protein in cytochrome b are also4 : 4 : 1 it was suggested that the toxin might be the protein moiety ofdiphtherial cytochrome b. In the absence of iron the organism continues tosynthesise the porphyrin and the protein component of cytochrome b, butsince they cannot be built into cytochrome b without iron they are dischargedas waste products. Diphtheria toxin would then be very similar to, but notidentical with, the protein moiety of mammalian cytochrome b, and wouldexert its toxic effect by competitively inhibiting the synthesis of cytochromeb in the tissues of the host.But the iron content of the growth mediumdetermines, not only whether the porphyrin shall be intra- or extra-cellularand bound to protein or free, but what its constitution shall be. Spectro-scopic observations had led some workers 30 t? conclude that the extracellularporphyrin was coproporphyrin (*CH,*CH,*CO,H substituents in positions 2and 4), and others 27 that it was haematoporphyrin [*CH(OH)*CH, substi-tuents]. Recent more detailed examination shows that, it is very likely to bec~proporphyrin.~l The biological conversion of the *CH:CH2 substituentsof the protoporphyrin of the intracellular haem protein into the *CH( OH)*CH,substituents of haematoporphyrin would involve only a hydration, but theconversion of vinyl groups into propionic acid groups is considered to be lesslikely.32 Other experiments also suggest that the intracellular haem proteinand the extracellular coproporphyrin are not directly intraconvertible. Theessential results of this work 33 are summarised in Table IV.The diphtheriabacillus was grown in low- and high-iron media containing glycine labelledwith 15N, and it can be seen that the amount of isotope incorporated into theintracellular haem was more than twice as great as that incorporated intothe coproporphyrin in the toxic filtrate. These results further suggest thatthe coproporphyrin was synthesised later than the ha,em protein, after theisotope content of the glycine had been diminished by randomisation.Thiswork does not disprove the interesting hypothesis that diphtheria toxin isthe protein moiety of diphtherial cytochrome b, but it does suggest that thehypothesis needs elaboration.m Rawlinson and Hale, Biochem. J . , 1949, 45, 247.30 Wadsworth, Crowe, and Smith, Brit. J . Ezp. Path., 1935, 16, 201.31 Gray and Holt, J . Biol. Chem., 1947, 169, 235; Biochem. J . , 1948, 43, 191.aa Watson, Pass, and Schwartz, J . Biol. Chem., 1941, 139, 583.33 Hale, Rawlinson, Gray, Holt, Rimington, and Smith, Brit. J . Exp. Path., 1950,31, 96310 BIOCREWSTRY.The small amount of uroporphyrin that appears in culture filtrates(Table IV) belongs to the isomeric series of aetioporphyrin I , whereas thecoproporphyrin and the intracellular iron-protoporphyrin belong to the 111TABLE IV.Incorporation of 16N into intracellukr h e m and extracellular porphyrins of C .diphtheriae grown in mcdia containing glycine enriched with 15N.(Adapted from Hale et u Z .~ * )16N inCopro- 16N in intra- Uro-Toxin in porphyrin copro- cellular porphyrin 15N in uro-Fe in culture in culture porphyrin haem in culture porphyrinmedium filtrate filtrate (atoms yo (atoms % filtrate (atoms %(pg. I d . ) . (unitslml.) . (pg. /ml.). excess). excess). ( p g . /ml.). excess).0-147 55 1.90 0.51 - 0.04 0.460.753 5 0.16 0.28 1.26 0.02 0.34series. Moreover, Table IV shows that the ratio, units of toxin : pg. ofuroporphyrin, decreases 5.5-fold as the iron content of the medium increases&fold, while the ratio of toxin to coproporphyrin remains unchanged.Thissuggests that the uroporphyrin is not directly concerned with toxinproduction.Neurotoxins.-Toxins that act specifically on the nervous system areproduced by the Gram-positive anaerobes, Cl. botuliwm and GI. tetuni, andby rough and smooth variants of the Gram-negative aerobe, Shigella shigae.The neurotoxins of the anaerobes are 1.5 X lo4 to 3 x lo8 times as toxic asaconitine and are the most toxic substances known. They appear to bequite simple proteins, but we have no idea of their mode of action. Pharma-cological studies 34 suggest that they act on the peripheral motor nerves bypreventing the output of acetylcholine from the nerve endings.Tetanustoxin is thought to act on the central nervous system as well. Their actionis extremely slow, but they appear to be fixed quite rapidly on theirsubstrates. It is conceivable that they may be kinases, i.e., substances thatcatalyse the production of biologically active catalysts from inactin pre-cursors. The pharmacology of Sh. shigae neurotoxin has not yet beenstudied.The four types of CI. botulinum, A, B, C, and D, eachproduce it distinct toxin. The type-A toxin has been purified andcrystallised,36 and its chemical and physical properties are shown in Table 111.It can be calculated that about 20 million molecules of toxin (about 10molecules per nerve cell) will kill a mouse. Unlike all other bacterial toxins,which are effective only when administered parenterally, botulinus type-Atoxin is effective by mouth.Presumably this is because its toxicity is notdestroyed by the proteolytic enzymes of the gut, but the possibility still34 Ambache, J. Physiol., 1949, 108, 127; Ambache, Morgan, and Wright, ibid.,1948, 107, 45; Bvit. J . Exp. Path., 1948, 29, 408; Burgen, Dickens, and Zatman, J.Physiol., 1949, 109, 10.35 Lamanna, Eklund, and McElroy, J. Bact., 1946, 62, 1; Abrams, Kegeles. andHottle, J. Biol. Chem., 1946, 164, 63.Botulinus ToxinsVAN HEYNINGEN : BACTERIAL TOXINS. 31 1remains that the toxin is broken down into smaller toxic fragments. Suchfragments would pass through the wall of the gut more radily than amolecule with a molecular weight of a million.The type-B toxin, which is as toxic as the type-A toxin, has been obtainedin a purified but amorphous condition.21 Its diffusion constant (20") of7.22 x lo-' cm.2/sec.suggests a molecular weight of 60,000, and thereforethat it is less toxic per molecule. It is relatively insoluble in water abovepH 5.0-5.5, and its solubility is not increased by the addition of salt.The type-D toxin has recently been partly p~rified,~3(b) but very fewdetails are yet available. It appears to have the astonishing toxicity of1.3 x 1O1O MLD (per kg., mouse) per mg. This would mean that it isabout 20,000 times as toxic as the type-A toxin, or that 10 mg. would beabout the MLD for the entire human population of the world.The chemical and physical properties of tetanospasmin,the lethal toxin of CZ.tetani, are summarised in Table 111. This toxin waspurified and crystallised36 at the same time as botulinus type-A toxin andis equally toxic. Like diphtheria toxin, its production is inhibited by quitelow concentrations of iron, but some strains of CZ. tetani are apparently notaffected by iron.37 It is unlikely that the toxin is concerned with cyto-chrome b because the organism does not contain cytochromes. Whenkept, the purified toxin becomes less soluble and less toxic without losing itsantigenicity. At the same time its sedimentation constant changes from4.5 to 7 and it has been suggested that an atoxic dimer is formed. Thischange is catalysed by low concentrations of f~rmaldehyde.~~This toxin is unlikely to be as toxic as those of theanaerobes, the best preparation to date having only MLD 20 (per kg.,mouse) per mg.39 It is an intracellular toxin and appears in culture filtratesonly after the organism has autolysed.It is produced by both rough andsmooth variants of Xh. shigae and it has been suggested that it is the toxiccomponent of the toxic somatic antigen (see below).40 The production ofthe toxin is also inhibited by iron.39Ha,emolysins.-Oxygen-ZabiZe Haemolysins. The 0-toxin of Cl. welchii,the tetanolysin of CZ. tetani, the pneumolysin of the pneumococcus, and thestreptolysin 0 of the hzmolytic streptococcus are haemolysins that haveseveral properties in common in spite of their diverse sources. They arereversibly activated by reducing agents, irreversibly inhibited by cholesterol,and serologically related, i.e., any one of them is neutralised by the anti-toxin to any of the others.41 It has been shown that reducing agents arenecessary for the production of streptolysin 0 as well as its activity,42 and itis interesting that ascorbic acid is as effective as, e.g., cysteine in theTetanus Toxin.Shiga Neurotoxin.36 Pillemer, Wittler, Burrell, and Grossberg, J .Exp. Med., 1948, 88, 205.37 Mueller and Miller, J . Immunol., 1943, 47, 15; 1945, 50, 377.as Pillemer and Moore, J . Biol. Chem., 1948, 173, 427.39 Dubos and Geiger, J . Exp. Med., 1946, 84, 143.40 Boroff, J. Bact., 1949, 57, 617 ; Boroff and Macri, ibid., 1949, 58, 387.41 Todd, J . Path. Bact., 1934, 39, 299; Brit. J . Exp. Path., 1941, 22, 172.42 Slade and Knox, J .Bact., 1950, 60, 301312 BIOCHEMISTRY.production of the haemolysin, but completely ineffective for its activation.68None of these toxins has been obtained in a pure state, but streptolysin0 43 and pneumolysin44 have been partly purified. They appear to beproteins. These haemolysins are interesting not only because of theircommon immunological properties but also because their biological activityclosely resembles that of the chemically very dissimilar saponins : (1) Bothtypes of substance are haemolytic, cardiotoxic, and lethal. (2) The hzemolyticactivity of both is inhibited by cholesterol and by a benzene-soluble, heat-stable substance in hzemolysed red blood cells.44+ 45 (3) A single dose ofstreptolysin 0 has no effect on the isolated frog's heart, except that it releasesan inhibitory substance which is heat-stable, non-dialysable, and chloroform-soluble.If the inhibitory substance is washed away the heart is sent intosystolic contracture by a second dose of streptolysin 0. Pneumolysin and0-toxin have the same effect.46 (4) Mice injected with the bacterialhaemolysins, or with saponin, rapidly develop a temporary (non-immune)resistance specifically to lethal doses of both types of s~bstance.~' (5) Micecan be protected against streptolysin 0 by previous injection of cholester01.~~A saponin-like prosthetic group on the oxygen-labile haemolysins wouldnot only explain their biological relationship to the saponiris, but it couldact as a hapten and account for their close serological relationships.Thescanty available evidence does not rule out such a possibility. It must beborne in mind, however, that the saponins are active by virtue of theirsurface-active effects, and that cholesterol inhibits the hamolytic action ofother surface-active agents, such as sodium oleate.Oxygen-stable Haemolysins. There are many other oxygen-stablebacterial haemolysins besides the lecithinases which have been discussed.The haemolysis of red cells by the GI. septicum haemolysin is preceded by aninduction period during which the main reaction takes place, and the cellsswell up and become translucent without lysis. The lysis that then followsis independent of the haemolysin since it still takes place if antitoxin isadded a t the end of the induction period.On the other hand, the spontaneouslysis of toxin-treated cells is inhibited by sucrose, which does not inhibit theprimary reaction between the toxin and the cells.49 This phenomenon isreminiscent of the " hot-cold " haemolysis by CZ. welchii lecithinase. If asuspension of red cells is incubated a t 37" with a low concentration of thishaemolysin no apparent hzmolysis takes place ; but if the suspension is thencooled rapid hEmolysis ensues, and this hamolysis is not inhibited if anti-toxin is added before cooling.50 Hamolysis by streptolysin S (see below) isalso preceded by an induction period.43 Herbert and Todd, Biochem. J . , 1941, 35, 1124.44 Cohen, Halbert, and Perkins, J . Bact., 1942, 43, 607.45 Smythe and Harris, J .Immunol., 1940, 38, 283.46 Bernheimer and Cantoni, J . Exp. Med., 1945, 81, 295; Cantoni and Bernheimer,4 7 Bernheimer and Cantoni, ibid., 1947, 86, 193.48 Hewitt and Todd, J . Path. Bact., 1939, 49, 45.49 Rernheimer, J . Exp. Med., 1944, 80. 309,321, 333; J. Gen. Physiol., 1947,30, 337.50 van Heyningen, Biochem. J., 1941, 85, 1257.ibid., p. 307VAN HEYNINOEN : BACTERIAL TOXINS. 313Some strains of streptococci produce, in addition to streptolysin 0, ahaemolysin called streptolysin S because it was first found when the organismswere grown in serum-broth, or when organisms grown on plain broth wereextracted with serum. It has since been shown that serum can be replacedas an extractant by a crude solution of egg-yolk le~ithovitellin.~~ As atoxigenic factor in growth media serum can be replaced by ribonucleic acid,and even more effectively by an active factor (AF) obtained by digestingribonucleic acid with ribonuclease.Small amounts of maltose (~/20,000) orglucosamine (~/10,000) are also necessary. 52 Streptolysin S is also producedwhen washed " resting '' adult streptococcal cells are suspended in a milieucontaining AF, glucosamine, sodium, potassium, magnesium, and phosphateions, and a reducing agent. The process by which the haemolysin is thusproduced is probably enzymic. By the use of this method a highly activepreparation of streptolysin S was obtained which appeared to contain AFand a hexosamine. The AF in this product did not appear to be essentialfor hzemolytic activity because 40% of the total phosphorus could beconverted into inorganic phosphorus (by treatment with a phosphatase)without loss of activity. A mutant strain that did not produce streptolysinunder these conditions gave rise to an inactive substance with roughly thesame constitution.53The staphylococci produce at least three, and possibly four, oxygen-stable haemolysins. Little is known of their nature or their mode of action.They are differentiated immunologically and by their specificity for the redcells of different species. The f3-toxin is a '' hot-cold " haemolysin, like theCZ. wekhii lecithinase, but it is not known whether the lysis that takes placeon cooling is independent of the haemolysin. Lysis (at 37") of " p-conditioned " cells also takes place if they come in contact with a number ofdifferent agents, vix.: (i) Glycerol.54 (ii) Bacterial l i p a ~ e . ~ ~ (Possibly thelipase acts in this case by producing glycerol. Lipases cause hzemolysis ofnormal cells when sufficient fat is present by producing hemolytic soaps, butin this case no fat was present and normal cells were not hzcmolysed.)(iii) Certain broth constituents.a (iv) Bacterial p r ~ t e a s e . ~ ~ (Possibly theprotease produces substances similar to the broth constituents.) (v) A heat-stable substance produced by Strep. agala~tiae.~~The toxins of a largenumber of Gram-negative bacteria, e.g., the shigellae, salmonellae, neisseriae,brucella, produce roughly the same symptoms, and they appear to beroughly similar complexes.They are identical with the dominant 0 somaticantigens that are located on the surfaces of smooth variants of these bacteria.These antigens consist of polymolecular complexes of phospholipid, poly-Somtic Antigens of Gram-negative Bacteria.5751 Herbert and Todd, Brit. J. Exp. Path., 1944, 25, 242.52 Bernheimer and Rodbart, J. Exp. Med., 1948, 88, 481.53 Bernheimer, ibid., 1949, 90, 373.54 Llewellyn Smith, and Price, J. Path. Bact., 1938, 47, 361.5 5 Christie and Graydon, Austral. J. Exp. Biol. Med. Sci., 1941, 19, 9.56 Munch-Peterson and Christie, J. Path. Bact., 1947, 59, 377.5' For references see ref. 3314 BIOUHEMISTRY.saccharide, and conjugated protein. In their native state it is likely thatthey contain additional loosely- bound lipid and phospholipid, and thatthese complexes are aggregated to form particles of high molecular weight,solutions of which show anisotropy of flow.The polysaccharide-conjugated protein component of the complex issplit into a degraded polysaccharide and an acidic conjugated protein onacid hydrolysis; and into a viscous undegraded polysaccharide and asimple amphoteric protein on alkaline hydrolysis.The toxicity of thecomplex appears to be associated with the undegraded polysaccharide orwith the conjugated protein, according to the method of hydrolysis. Theundegraded polysaccharide is a hapten which combines readily with theconjugated protein to form an antigen which is immunologically indistin-guishable from the original 0 antigen.The conjugated protein will alsocombine with undegraded polysaccharide from heterologous species to formantigens whose specificity is determined by the polysaccharide. Theamphoteric protein will also combine with undegraded polysaccharide butthe resultant complex is not antigenic.The work that has been done on the toxic complexes of Gram-negativebacteria has been mainly concerned with their immunology and very littleis known about the toxic component.W. E. VAN H.4. GENERAL NUTRITION.Poule.--Proteirt and Amino-acids. The chick's tryptophan requirementwas met when the diet contained 0.18% of' L-trypt0phan.l The chick alsoutilised 17--40y0 of the D-tryptophan of an experimental diet.2 The grossand microscopic pathology of tryptophan deficiency was described.g TheL-threonine requirement was 0-45y0 of the diet, D-threonine not beingutilised.4 Raising the protein content of the diet augmented the chick'srequirement for lysine 6 and methionine.6 The chick required 0.6--043% ofDL-phenylalanine in the diet together with a similar amount of L-tyrosine orD- or L-phenylalanine to meet the tyrosine requirement.' The requirementof turkey poults was between 0.75 and 1025% of the diet for glycine,8 about0.5% for methionine, and 0.3% for cy~tine.~ Although methionine couldreplace cystine in the diets of poults, cystine could not adequately replacemethionine.. There was normal feather pigmentation in young poults ondiets containing 1.3% of lysine.1° The leucine requirement of the laying1 Wilkening, Schweigert, Pearson, and Sherwood, J .Nutrit., 1947, 34, 701.* Wilkening and Schweigert, J . BioE. Chem., 1947, 171, 209.3 Brown, Wilkening, and Schweigert, Proc. Soc. Exp. Biol., 1948, 68, 672.4 Grau, J . Nutrit., 1949, 37, 105.6 Almquist, Proc. SOC. Exp. Biol., 1949, 72, 179. ' Grau, J . Biol. Chem., 1947,170, 661.8 Kratzer and Williams, J . Nutrit., 1948, 85, 315,9 Kratzer, Williams, and Marshall, ibid., 1949, 37, 377.Idem, ibid., 1948, 36, 99.10 German, Schweigert, Sherwood, and James, Poultry Sci., 1949, 28, 166DUCKWORTH : GENERAL NUTRITION. 316hen appeared to be between 1-35 and 2.0% of the diet.ll The argininecontent of the diet should be 6% of its protein content.I2The leucine, holeucine , p hen y lalanine, valine , met hionhe, tryptophan ,histidine, arginine, threonine, and lysine contents of light and dark meat,and livers and gizzards of poultry, were reported.13 The arginine, histidine,lysine, and threonine contents of some common feeding stuffs weredetermined.14From a third to a half of native and added lysine was destroyed by auto-claving soya-bean oil meal.Less destruction took place with dry heat.There was only a slight loss of native or added lysine when isolated soya-bean protein was heated but all the lysine was destroyed on heating thiswith sucrose.16 Autoclaving lysine with glucose (" cerelose ") made itunavailable to chicks.16 However, there was no destruction of methionineautoclaved at 120" for 2 hours in 8% aqueous g1uc0se.l~ Refluxing purifiedsoya- bean globulin with aqueous glucose caused partial destruction of lysine,arginine, tryptophan, and histidine.ls Excessive heating (130"/60 minutes)impaired, and moderate heating (100"/30 minutes) improved, the nutritivevalue of soya-bean protein.The former, but not the latter, product wasimproved by adding methionine, cystine and 1~sine.l~ Products of auto-claving for 45 minutes at 4 lb./sq. in. were almost equal: in nutritive value tothose treated for 4 minutes at 15 lb.2O Drastic heating destroyed part ofthe lysine of soya bean and reduced the digestibility of the rest. Theabsorption and utilisation of methionine were also reduced.21 Processingthe meal did not affect the lysine, cystine, or methionine content of eggslaid by poultry fed on the diet.22 Wheat protein was a better source ofarginine, leucine, methionine, and tryptophan than the analysis indicatedbut was markedly deficient in l y ~ i n e .~ ~ The availability of tryptophan inraw soya-bean oil meal was 20%, in fish meal 40% and in casein loo%.%The inferiority of raw to heated soya-bean protein arose in part from thepresence of thermolabile enzyme inhibitors, of which there were at leastthree.26 The inhibitors, which were easily extracted, interfered with theaction of trypsin and erepsin, but not of pepsin or papain, in ~ i t r o . ~ ~ Auto-l1 Cravens, Poultry Sci., 1948, 27, 562.12 Almquist and Merritt, Proc. SOC. Exp. Biol., 1950, 73, 136.l3 Millares and Fellers, J . Amer.Dietetic ASSOC., 1948, 24, 1057.l4 Schweigert, Poultry Sci., 1948, 27, 223.l5 Evans and Butts, J . Biol. Chem., 1948, 175, 15.l6 Stevens and McGinnis, ibid., 1947, 171, 431.l7 Graham, HSU, and McGinnis, Science, 1949, 110, 217.1* Patton, Hill, and Foreman, ibid., 1948, 108, 659.l8 McGinnis and Evans, J . Nutrit., 1947, 34, 725.2 1 Evans and McGinnis, J . Nutrit., 1948, 35, 477.22 Evans, Davidson, and Butts, Poultry Sci., 1950, 29, 104.23 Jeppesen and Grau, ibid., 1948, 27, 588.24 Schweigert, Arch. Biochem., 1948, 19, 265.2Q Borchers, Ham, Sandstedt, Ackerson, Thayer, and Mussehl, Univ. NebTaekuClaudinin, Cravens, Elvehjem, and Halpin, Poultry Sci., 1948, 27, 370.25 Bowman, ibid., 1948, 16, 109.Agric. Exp. Stat. Res. Bull., No. 152, December 1947316 BIOCHEMISTRY.claving at 15-20 Ib.pressure for 20 to 30 minutes destroyed the factors.27~ 28p 29There was no evidence of preferential interference with the liberation ofspecific amino-acids during hydrolysis with ~ a n c r e a t i n . ~ ~ ~ 33 After pre-liminary digestion of raw and heated soya-bean oil med with pepsin therates of in-vitro digestion with pancreatin were the A highlypurified preparation of an antitryptic factor failed to impair the nutritivevalue when added to a normal soya-bean chick ration.31 The amino-acidspresent in the inhibitor have been identified.32If the free gossypol was not above 0.12% of the meal the inciusicn of10% of hydraulic-press cotton-seed meal in the ration did not depress thehatchability of eggs.34 The correlation between the nutritive value ofcotton-seed meals and their content of gossypol and gossypurpurin waspoor ; mechanical removal of pigment glands, hydraulic pressure, andextraction with diethyl ether (but not hexane) removed the toxicDiscoloration of eggs was prevented by isopropanol extraction of cotton-seed meal, but not by hydraulic-pressure treatment .36 Wafer-soaking oflinseed meal destroyed its toxicity for chicks 379 38 and turkeyMeals made from flax seed treated with gaseous ammonia before storagewere non-toxic to chicks.40 There was no trypsin inhibitor in linseed meal.41A new chemical method has been described for determining the digesti-bility of protein by poultry.Good reproducibility was found when resultson normal droppings were compared with those obtained by analyses onpure urine and faeces secured by the artificial-anus technique.42 TheDiakow and the Stotz methods did not yield comparable results.P3 Therewas a good correlation between the evaluations of feed protein by chemicaland growth methods.44The blastoderm of the chick embryo utilisedD-glucose, D-mannose, D-frUCfoSe, ~-galactose, and D-maltose, but not D-arabinose, D-xylose, D-ribose, L-sorbose, sucrose, lactose, trehalose, cellobiose,melifiose, raffinose, melezitose, or glycogen.The minimum concentrationsof the five sugars necessary for embryonic development were respectively :27 Borchers, Ackerson, and Mussehl, Poultry Sci., 1948, 27, 601.28 Desikachar, De, and Subrahmanyan, Ann.Biochem. Ezp. Med., 1948, 8 , 93.29 Ackerson, Borchers, and Mussehl, Univ. Nebraska Agric. E:E:E:E:E:E:E:E:E:E:E:, Stat. Res. Bull.,30 Leiner and Fevold, Arch. Biochem., 1949, 21, 395.31 Borchers, Ackerson, and Mussehl (with A. Moehl), ibid., 1948, 19, 317.32 Work, Biochem. J . , 1948, 42, xlix.33 Ingram, Riesen, Cravens, and Elvehjem, Poultry Sci., 1949, 28, 898.34 Heywang, Denton, and Bird, ibid., p. 610.3 5 Boatner, Altschul, Irving, Pollard, and Schaefer, ibid., 1948, 27, 315.36 Kuiken, Lyman, and Hale, ibid., p. 742.37 Mani, Nikolaiczuk, and Maw, Sci. Agric., 1949, 29, 86.38 MacGregor and McGinnis, Poultry Sci., 1948, 27, 141.a9 Kratzer, ibid., 1949, 28, 618.40 Altschul, Karon, and Schaefer, ibid., 1948, 27, 408.4 1 Mani, Nikolaiczuk, and Maw, Sci.Agric., 1949, 29, 91.42 Ekman, Emanuelson, and Fransson, Kgl. Lantbrukshtigsk. Ann., 1949, 16, 749.48 Hsie, Meld. Norges Landbruksh0gsk., 1948, 28, 399.44 March, Stupich, and Biely, Poultry Sci., 1949, 28, 718.Carbohydrate and Fat.No. 156, June 1948DUCKWORTH : GENERAL NUTRITION. 31720, 20, 50, 200, and 400 mg./100 ml.45 High levels of lactose in the dietrapidly stopped egg production but did not affect the hatchability of theeggs laid. Simultaneous high levels of fat in the diet prevented the effect.46Growth was improved by small additions of cellulose to inferior, but not tosuperior, purified diets, perhaps by permitting increased intestinal synthesisof essential ~ubstances.~' Small additions of sawdust also improved growthon synthetic rations.48 The adverse effect of added fibre in rations wasmore serious in cases where the ration was low in energy.49Phenol, resorcinol, phloroglucinol, and a-naphthol, but not benzoic acid,p-aminobenzoic acid, or chloral hydrate, increased urinary glucuronic acidexcretion.Over 75 yo of injected phenol, resorcinol, quinol, phloroglucinol,and catechol was rapidly excreted as glucuronides and ethereal s ~ l p h a t e s . ~ ~The incidence of spontaneous arteriosclerosis in chickens was reduced byremoving most of the fat and cholesterol from a normal diet.62 It wasintensified in proportion to the amount of cholesterol added to the diet.53Cholesterol-induced arteriosclerosis declined in severity when birds weretransferred to normal or cholesterol-free diet.54 Dosage with desiccatedthyroid reduced the incidence of arteriosclerosis in cholesterol-fed chicks ;dosage with potassium iodide yielded contradictory results.55 Feeding drycholesterol intensified coronary arteriosclerosis but did not increase theincidence of the c ~ n d i t i o n . ~ ~Experiments in which radio-calcium was fed showed that60-75% of the calcium of the egg came from the daily intake, and the restfrom skeletal stores.57 Variations in the uptake of radio-calcium 58 andradio-phosphorus 59 in shell, yolk, and white immediately after dosagedepended on the stage of maturity of the egg component at the time.Calcium deficiency increased the incidence and intensity of black pigment-ation in the down feathers of chicks with brown plumage, and in spite ofhigh vitamin D intake.60 Only 5% of the dietary radio-phosphorus [givenas Ca,(PO,),] appeared in the egg,61 presumably because of the endogenoussupply of phosphorus from resorbing bone.The greater utilisation ofinjected Na,PO, than of glycerophosphate-both labelled with radio-phosphorus-indicated that organic phosphate esters are not used directlyMinerals.4 5 Spratt, J . Exp. Zool., 1949, 110, 273.46 Couch, Barki, Sunde, Cravens, and Elvehjem, J . Nutrit., 1949, 38, 105.4 7 Lepp, Harper, and Elvehjem, Poultry Sci., 1949, 28, 372.4 8 Davis and Briggs, ibid., 1948, 27, 117.48 Robertson, Miller, and Heuser, ibid., p. 736.50 Sperber, Kgl. Lantbrukshogsk. Ann., 1948, 15, 108.5 1 Idem, ibid., 1949, 16, 446.5 2 Horlick, Katz, and Stamler, Amer.Heart J . , 1949, 37, 689.53 Idem, ibid., 1949, 38, 336.5 5 Dauber, Horlick, and Katz, Amer. Heart J., 1949, 38, 25.5 6 Paterson, Slinger, and Gartley, Arch. Pathol., 1948, 45, 306.ci7 Driggers and Comar, Poultry Sci., 1949, 28, 420.5 8 Spinks, Berlie, and O'Neil, Science, 1949, 110, 332.59 Spinks, O'Neil, Jowsey, Lee, and Reade, Canad. J . Res., 1948, 26, D, 163.6o McGinnis and Carver, PouEtry Sci., 1948, 27, 115.61 O'Neil, Jowsey, Lee, Reade, and Spinks, Science, 1948, 107, 295.54 Idem, J . Lab. Clin. Med., 1949, 54, 1427318 BIOCHEMISTRY.in phospholipin synthesis but that they are first hydrolysed and the thenfree phosphate is incorporated into the phospholipin molecule.62 Radio-phosphorus was uniformly distributed in relation to total phosphorus in chickembryos from the eggs of a hen fed with radio-phosphor~s.~~ The changesin ribonucleic and deoxyribonucleic acid phosphorus in the whole embryo 64and in heart and liver 65 during incubation have been determined.Orthophosphates, P-Ca,(PO,),, and mono- dLY, and tri-calcium phosphateswere excellent .sources of phosphorus snd slightly superior to bone meal.Defluorinated rock phosphate and superphosphate were slightly inferior tobone meal.Meta- and pyro-phosphates were of no value, except calciumdihydrogen pyrophosphate.66 Raw rock phosphate (3.3% of fluorine) did notinterfere with growth, maturity, or egg produotion of pullets, or fertility orhatchability of eggs. Although there was high storage of fluorine in thebone there was no increase in the fluorine content of the edible portion of theeggs and second generation chicks grew normally.67 Phytate-phosphoruswas poorly utilised and increased the requirement for vitamin D3.6*,6QCalcium and phosphorus requirements of young White Leghorn cockerelswere 1.07 and 0.65% in the ration respe~tively.~~ The requirements ofBroad-breasted Bronze turkeys appeared to be 1.2% of the ratio for calciumand about l.Oyo for phosph~rus.~~ Young chicks required from 0.38 to0.47% of phosphorus in the ration.68 If the ration was high in phytate-phosphorus it should contain o.4y0 of phosphorus in inorganic form.69Removal of the centre toes of the feet for analysis in vitamin D assayswas suggested. The operation had no effect on growth or calcification ofthe rest of the skeleton.72 Seasonal differences in the ash content of tibiaeof chicks (a source of error in vitamin D assays) were thought to result fromhigher D reserves in the summer- and autumn-hatched chick than in winter-hatched chick~,7~ Crooked keels in birds were not associated with inferiorbody or bone growth.74The clinical and neuropathological effects of magnesium deficiency weredescribed and resembled those in the rat.75 The pathology of potassiumdeficiency has also been described and the requirement found to be 0.24%in the ration ; lower potassium levels were satisfactory if phosphorus supplieswere not minimal.76 The upper safe limit of sodium chloride in the diet was62 Spinks, Lee, and O’Neil, Canad.J . Res., 1949, 27, B , 629.83 O’Neil, Jowsey, Lee, and Spinks, ibid., D, 223.1 3 ~ Davidson and Leslie, Biochem. J . , 1948, 43, xxviii.6 5 Novikoff and Potter, J . Biol. Chem., 1948, 173, 233.8 6 Gillis, Norris, and Heuser, J . Nutrit., 1948, 35, 195.67 Gerry, Carrick, Roberts, and Hauge, Poultry Sci., 1949, 28, 19.6~ Singsen, Storrs Agric. Exp. Stat. Bull., No. 260, 1948.60 Gillis, Norris, and Heuser, Poultry Sci., 1949, 28, 283.70 Mussehland Ackerson, Univ. Nebraska Agric. Exp.Stat. BulL.,No. 386, October 1947.7 1 Motzok and Slinger, Poultry Sci., 1948, 27, 486.72 Stadelman, Boucher, and Callenbach, ibid., 1949, 28, 161.7 3 Hill and Motzok, ibid., 1948, 27, 515.74 Buckner, Insko, Henry, and Wachs, ibid., 1949, 28, 644.75 Bird, J .Nutrit., 1949, 39, 13. 76 Gillis, ibid., 1948, 86, 351DUCKWORTH GENERAL NUTRITION. 3193y0.77 As in the case of fluorine, the resistance of the fowl to bromine ishigh, an effect being noted only on the gonads and without modification ofsecondary sex characteristi~s.7~ Repeated radio-iodine injections causeddestruction of the thyroid inCholine and ethanolaminesupplements to practical rations already containing 0.14 and o*17y0 ofcholine did not improve egg production or egg quality.80 There was no lossof choline in high-protein poultry feeds stored a t 70" F. for up to 6 months.s1Choline and methionine requirements for growing chicks were divided intotwo parts : essential and replaceable. The essential requirement of cholinewas O.lOyo of the diet, and of methionine 0450%.These were consideredto be the basic amounts required for tissue construction. The replaceablerequirements needed for methylation processes were 0.25 yo of methionineand 0.45% of choline chloride. Excess of methionine depressed growthunless glycocyamine was given as a methyl acceptor. Excess of choline upto 1.6% did not depress growthes2 Studies of the replacement of choline bybetaine in incipient perosis were reported.83 Choline and methionine wereto some extent inter~hangeable.~~ Betaine, added to a simplified diet, hada greater supplementary effect than either choline or methi~nine.~~ Ethanol-amine could replace partly the choline of chick rations.86 The fat of turkeycarcases held in cold storage was less liable to rancidity when their rationshad been supplemented with choline or ethan~larnine.~~.88Unidentified chick-growth factors have been found in condensed fishsolubles (made by evaporating the " stickwater " produced during oilextraction of fatty fi~h),~99 91 9 9 2 p 93* 949 100 fish meal,Q59 96 liver prepar-a t i o n ~ , ~ ~ ~ 94 cow dung,@* 99 distiller's by-product^,^^ meat meals,96 casein,Q7Non-vitamin and Unidentiified Dietary Factors.7 7 Barlow, Slinger, and Zimmer, Pou2try Sci., 1948, 27, 542.70 Winchester, Comar, and Davis, Science, 1949 110, 302.*O Gish, Kummerow, and Payne, Poultry Sci., 1949, 93, 305.Cooley and Christiansen, ibid., 1948, 27, 822.82 McKittrick, Arch. Biochem., 1947, 15, 133.84 Gerry, Carrick, and Hauge, Poultry Sci., 1948, 27, 161.8 5 Mishler, Carrick, and Hauge, ibid., 1949, 28, 24.8 6 Kummerow, Weaver, and Honstead, ibid., p.475.8 7 Hite, Kloxin, Kummerow, Vail, and Avery, ibid., p. 244.8 8 Hite, Kloxin, and Kummerow, ibid., p. 249.Borgatti, Arch. Sci. biol., Napoli, 1947, 32, 67.*3 Idem, ibid., 1948, 18, 437.Robblee, Nichol, Cravens, Elvehjem, and Halpin, ibid., 1948, 27, 442.Nichol, Robblee, Cravens, and Elvehjem, J . Biol. Chem., 1949, 177, 631.91 Hnie, Tidsskr. norske Landbmk, 1949, 56, 23.g2 Mishler, Carrick, and Hauge, Poultry Sci., 1948, 27, 263.O3 Robblee, Nichol, Cravens, Elvehjem, and Halpin, J . Biol. Chem., 1948, 173, 117.g4 Nichol, Robblee, Cravens, and Elvehjem, Poultry Sci., 1948, 27, 438.O5 Combs, Heuser, and Norris, ibid., p.238.s6 Wiese, Petersen, and Lampman, ibid., p. 466.O 7 Csonka and Olsen, J . Nutrit., 1949, 39, 485.Schlamb and Winter, Poultry Sci., 1948, 27, 492, 498.Bird, Rubin, and Groschke, J . Biol. Chem., 1948, 174, 611.loo Hill, Poultry Sci., 1948, 27, 536320 BIOCHEMISTRY.and whey.loO Certain of these factors are undoubtedly to be identified withvitamin B12. In some cases confusion has arisen because of failure to depletethe hen's reserves before collecting the eggs for hatching. Purified dietsfor turkey poults were satisfactory for short periods in early life,lol but notfor 2 generations.lo2 Condensed fish solubles provided an essential factorfor poults.lo3 Dietary factors essential for the chick were supplied by oldbuilt-up floor litter.lM The growth-promoting action of cellulose could bereproduced by adding to the diet 10% of sawdust + either O.lyo of lzevulicacid or 0.1 to 0.3% of furfuraldehyde, but not by D-xylose, furamide, or~-cellobiose.~~~ Soil (as a source of B.subtilis factor),lo6 added a t the rateof 1% to a diet deficient in animal-protein factor(s), stimulated growth andreduced mortality.Unidentified hatchability factors have been found in fish meals, l 0 7 ~ lo8cow dung, log and built-up floor litter.l1° Methods of concentrating thefactor(s) in fish meal were reported lo8 and an assessment made of theamounts of fish meal necessary to provide enough of the factor(s) to maintainhatchability. lo7Growth of chickens and turkeys was accelerated by 4-hydroxy-3-nitro-phenyl-, p-hydroxyphenyl-, and phenyl-arsonic acid.lll* lS6* lS7 Aureo-mycin, aureomycin mash (with its antibiotic activity destroyed), strepto-mycin, and succinoylsulphathiazole also stimulated growth.111Thyroprotein supplements in the diet hadlittle effect on growth or feed efficiency of chicks although feathering ratewas improved.1l2 Continuous feeding with thyroprotein delayed birds inreaching 50% egg production.l13 Some workers found no effect on eggproduction,ll** 115, n4* lZ9 others a beneficial effect.l16* ll7~ 118* lZ6 Reportedadvantages of dosage were : delayed moulting and improved health,l16improved shell quality,l14* 124 and higher production in oldSimultaneous dosage with thyroprotein, an estrogen [di(methoxyphenyl)-hexene] and an androgen (in dried cow dung lS5) gave no added advantage.llsFeeding dried pig thyroid checked growth and induced precocious maturity.l*OHormone Administration.101 Scott, Heuser, and Norris, Poultry Sci., 1948, 770.102 Jukes, Stokstad, and Gilbert, ibid., p.434.103 German, Schweigert, Pearson, and Sherwood, ibid., p. 113.104 Kennard and Chamberlin, ibid., p. 240.105 Davis and Briggs, Fed. Proc., 1948, 7, 284.lo8 Stephenson, McGinnis, Graham, and Carver, Poultry Sci., 1948, 27, 827.107 Lindstrom, Petersen, Wiese, and Moore, ibid., 1949, 28, 552.108 Pensack, Bethke, and Kennard, ibid., p. 398.109 Groschke, Rubin, and Bird, ibid., 1948, 27, 302.110 Kennard, Bethke, and Chamberlin, ibid., p. 477.111 Stohtad and Jukes, PTOC.SOC. E:E:E:E:E:E:E:E:E:E:E:. Biol., 1950, 73, 523.112 Wheeler, Hoffmann, and Graham, Poultry Sci., 1948, 27, 103.113 Wheeler and Hoffmann, ibid., p. 509.114 Hoffmann and Wheeler, ibid., p. 609.115 Hutt and Gowe, ibid., p. 286.118 Moore and Rees, Vet. J., 1948, 104, 156.117 Turner and Kempster, Poultry Sci., 1948, 27, 453, 11* Turner, ibid., p. 613DUCKWORTH : GENERAL NUTRITION. 321Chicks from eggs of hens fed with thyroprotein 119* 121* 122* lZ3* 126 orthiouracil l22,1*3 had enlarged thyroids. The length of incubation time ofthese eggs was increased,l231125 in proportion to the dosage of the hen.f1sThiouracil also increased incubation time.lg3 The degree of enlargementof the chick's thyroid varied with the iodine content of the thyroproteingiven to the mother, and not with its thyroxin content.12s Incubation timereturned to normal soon after thyroprotein was left out of the diet.122Injection of incubating eggs of thyroprotein-fed hens with 6 pg. of (&)-thyroxine reduced the chick thyroids to normal size.122 Injection of normaleggs with 1 pg.of thyroxine impaired thyroid demlopment.l27 High levelsof potassium iodide injection of eggs increased thyroid size and extendedincubation time; 128 low levels had no effect.122 Thyroprotein feeding didnot affect fertility or hatchability of eggs.lz9> 132 Semen volume decreasedin thyroprotein-fed cocks,130 and semen quality might 131 or might not I3O beimpaired. It reduced thyroid size in hens, possibly by inhibiting theproduction of pituitary thyrotrophic h0rm0ne.l~~Fast- and slow-growing strains of birds in each of two breeds had theirown optimum levels of thyroid activity for growth, feathering, and feedutilisation.The growth response to thyroprotein was negative in a fast-growing strain and positive in a slow-growing strain of New Hampshirechicks.13* Thyroid activity was higher in birds with high than in those withlow egg production,135 and there was an indication of seasonal variation. 136It is possible that some of the conflict between results in studies of thyro-active materials may have arisen through failure to take such factors intoaccount. Thyroxine secretion was much higher in ducklings thanMoulting could be forced in turkeys by thyr0pr0tein.l~~ Thyroid size washighly variable in turkeys.139Thiouracil alone in the diet reduced growth in cockerels 140 and turkeys,141but when combined with thyroprotein yielded normal growth and superiormarket grade.142 Thiouracil plus diethylstilbcestrol gave the best weight119 Wheeler and Hoffmann, Endocrinology, 1948, 43, 430.lZo Spisni and Sala, Zootec.Vet., 1949, 4, 730.lZ1 Wheeler and Hoffmann, Endocrinology, 1948, 42, 326.lg2 McCartney and Shaffner, ibid., 1949, 45, 396.lZ3 McCartney and Shaffner, Poultry Sci., 1949, 28, 223.lZ5 Wheeler and Hoffmann, Proc. SOC. Exp. Biol., 1949, 72, 250.lZ6 Turner and Kempster, P a l t r y Sci., 1949, 28, 826.lZ7 Booker and Sturkie (with Booker), ibid., p. 147.lZ8 Wheeler and Hoffmann, Endocrinology, 1949, 45, 208.lZ9 McCartney and Shaffner, Poultry Sci., 1950, 29, 67.130 Huston and Wheeler, ibid., 1949, 28, 262.131 Shaffner, ibid., 1948, 27, 527.132 Wheeler and Hoffmann, ibid., p.685.13' Glazener, Shaffner, and Jull, ibid., 1949, 28, 834.135 Booker and Sturkie, ibid., p. 757.13' Hoffmann, ibid., 1950, 29, 109.138 Blakely, Anderson, and MacGregor, ibid., 1949, 28, 757.140 Moreng and Shaffner, ibid., p. 504.142 Henderson, Carver, and Stephenson, ibid., 1948, 27, 667.lZ4 Godfrey, ibid.,p. 867.133 Turner, ibid., p. 155.136 Turner, ibid., 1948, 27, 146.138 Kosin and Wakely,ibid., 1948,27,670.141 Blakely and Anderson, ibid., p. 185.REP.-VOL. XLVII. 322 BIOUHEMISTRY.gains, the highest feed efficiency and the best market grade.f42 Goitrogenswere rapidly absorbed and excreted in birds and mammals so that toxiceffects on consumers would be ~nlike1y.l~~ Thiouracil greatly reducedgrowth in ducklings and caused great enlargement of the thyroid.137Addition of 0.3% of thiouracil to the diet had no effect on fertility of eggsbut reduced production and hatchability ; 0.1 yo additions had no detrimentaleffect .1z9Clhtrogens fed in diets containing 18% of protein did not improve growthbut when added to 14% diets improved skin appearance.When diethyl-stilbestrol, but not dienoestrol diacetate, was given, the liver and abdominalfat contained enough estrogen to cause =estrus phenomena in menopausalwomen eating these tissues.144s148 Implantation of 30 mg. of diethyl-stilboestrol improved the growth of cockerels lP5 but stilboestrol dipropionatefailed.149 Injection of diethylstilbestrol raised plasma lecithin and cephalinmarkedly 146 and increased the incidence of atheroscler~sis.~~~ Treatmentof immature pullets with mtradiol dipropionate produced the changes in theoviduct and blood, similar to those in the normal laying bird, but no increasedcalcium and phosphorus retention.Testosterone had no such effect.Simultaneous dosage with estrogen and androgen produced oviduct andblood changes and increased calcium and phosphorus retentions. 150Di(methoxypheny1)hexene (and chlorotriphenylethylene) increased fatnessof turkeys.l5l* 152 High dosage levels impaired egg production and induced'( leg-weakness," but a t 0.0125% in the diet egg production was irnpr0~ed.l~~Di( methoxypheny1)hexene reduced iodine turnover in the thyroid.154Feed efficiency (bodyweight gain/feed consumption)was higher in fast-growing than in slow-growing strains within a breed, incrossbred than in purebred birds, in outbred than in inbred birds, in NewHampshires than in Barred Plymouth 159* 160 Environmentaltemperature ranges for maximal feed efficiency in chicks have been deter-mined.Maximal feed efficiency characterised maximal growth rates.Variations in humidity from 35 to 75o/b were without effect on growth orefficiency.' Provided that supplies of individual amino-acids wereFt?ed Eficiency.143 Pipes and Turner, Univ. Missouri Agric. Exp. Stat. Res. Bull., No. 422, July 1948.u4 Bird, Off.Rep. Eighth World's Poultry Congr., 1948, p. 131.145 Bos, Tijdschr. Diergeneesk., 1950, 75, 150.1413 Ranney, Entenman, and Cheikoff, J . Biol. Chem., 1949, 180, 307.147 Horlick and Katz, J . Lab. Clin. Med., 1948, 33, 733.14* Gowe, Poultry Sci., 1949, 28, 666.14B Kelly and Roberts, Vet. Rec., 1950, 62, 44.150 Common, Rutledge, and Hale, J. Agric. Sci., 1948, 38, 64.151 Thayer and Davis, Poultry Sci., 1948, 27, 176.lS2 Davis and Thayer, ibid., p. 79.154 Epstein and Wolterink, ibid., 1949, 28, 763.156 Morehouse, ibid., 1949, 28, 375.15' Bird, Groschke, and Rubin, J . Nutrit., 1949, 37, 215.15* McCartney and Jull, Poultry Sci., 1948, 27, 17.l60 Headley, Univ. Nevada Agric. Exp. Stat. Bull., No. 180, July 1948.l a 1 Barott and Pringle, J .Nutrit., 1949, 37, 153.15s Turner, ibid., p. 593.1 5 5 Turner, ibid., 1948, 27, 789.159 Hess and Jull, ibid., p. 24DUCKWORTH : QENERAL .NUTRITION. 323adequate, little advantage in feed eficiency was gained by raising theprotein level in chick rations from 20 to 28%.182 Feed dilution with cellulosebeyond a 10% level decreased feed efficiency.la Poults retained 42.6%,23.4%, and 3103% respectively of the nitrogen, calcium, and phosphorusconsumed during the first eight weeks of life?* Higher feed efficiencieswere found in male than female turkeys.lm By use of the general equationY = C + blxl + bzx2 + b3x3 (Y = total feed consumed; x1 = eggs laid,in g. ; x2 = average body weight ; x3 = change in bodyweight during laying)it was found that 71% of feed consumed was for maintenance, 27% for eggproduction, and 2 yo (non-significant) for weight increase, in pullets with 72 yoegg production.Lower-producing birds used the feed fraction for eggproduction as efficiently as did high-producing birds.lsSblwine.-Protein and Amino-mi&. Addition of 0.4% of DL-tryptophanto a purified diet met the requirement of pigs weighing 50-100 lb.168~1s7The lysine requirement of weanlings was met by 0.6% of L-lysine,ls8 or amaximum of 2 yo of ~ ~ - 1 y s i n e . ~ ~ ~ Hexahomoserine (2-amino-6-mercapto-hexanoic acid) inhibited growth and reduced the red blood-cell count.170The animal-protein needs of bacon pigs were met by giving 7% of fish mealin the diet up to 90-100 lb. liveweight.blinerals. When a ration containing @15y0 of calcium and 1.69% ofphosphorus was fed, the blood-calcium and alkali reserve were lowered, andthe blood- and urine-phosphorus were raised.172 The rachitogenic substancein yeast was heat stable and did not reduce intestinal breakdown of phyticacid.173 Protein deficiency caused a mild normocytic, normochromic anzmiaand a marked reduction in iron-binding capacity of the serum. Irondeficiency caused a severe microcytic, hypochromic anzmia and a reductionin b10od-iron.l~~ In piglets housed in cold conditions and given ironsupplements the anamia was a consequence of liver damage.17s* 176Growth factors (for baby pigs)in liver extract,177 (for weanling pigs) in meat scraps, whey products, andcondensed fish s~lubles,~~* and (for growing and fattening pigs) in condensedThereafter none was needed.171Unidenti$ed and Non-vitamin Factors.Singsen, Poultry Sci., 1949, 28, 713.163 Davis and Briggs, ibid., 1948, 27, 658.164 Ackerson and Mussehl, Univ. Nebraska Agric. Exp. Stat. Res, Bull., No. 151,la5 Joshi, Shaffner, and Jull, Poultry Sci., 1949, 28, 301.lea Beeson, Mertz, and Shelton, J. Animal Sci., 1949, 8, 532.le7 Idem, Science, 1948,107, 599.la8 Loosli, Williams, and Maynard, Fed. PTOC., 1949, 8, 379.16g Metz, Shelton, and Beeson, J. Animal Sci., 1949, 8, 624.1x1 Mertz, Beeson, Waltz, and Gaudry, Proc. SOC. Exp. Biol., 1948, 69, 609.171 Woodman and Evans, J. Agric. Sci., 1948, 38, 354.17a Liegeois and Derivaux, Compt. rend. SOC. Biol., 1949, 143, 126.July 1947.Braude, Henry, and Kon, Brit.J . Nutrit., 1948, 2, 66.Csrtwright and Wintrobe, J . Biol. Chern., 1948, 176, 571.175 Howie, Biggar, Thomson, and Cook, J . Agric. Sci., 1949, 39, 110.176 Naftalin and Howie, J. Path. Bact., 1949, 61, 319.178 Dyer, Krider, and Carroll, J . Animal Sci., 1949, 8, 541.Neumann, Krider, and Johnson, Proc. SOC. Ezp. Biol., 1948, 69, 513324 BIOCHEMISTRY.fish solubles and liquid Baby pigs required aboutO*lyo of choline in the ration.lsO Choline was riot required in baby-pig“ synthetic milk” diets if the methionine content was 1.6% of the drymatter. At half this methionine content 0.1% of choline was needed.lsl“ Synthetic milk ” plus serum or plasma as a colostrum substitute failed tosupport life in new born pigs.lS2 Given “ synthetic milk ” on the third dayof life they grew normally.la3 Aureomycin 18** us and streptomycin lS6stimulated growth in pigs.Hormone Administrution. Thyroprotein increased the growth rate ofpigs.la7* lS8* I n restricted feeding, growth and feed efficiency werereduced thereby.lS7 Improved feed efficiency and growth was associatedwith a higher percentage of fat and lower percentages of moisture, protein,and minerals in the carcase.188 Reduced growth rate and poorer feedefficiency were reported.lWFeeding anti-thyroid drugs gave conflicting results. Some foundthiouracil lgl* lg2 or methylthiouracil 191 improved growth and/or feedefficiency. Others found detrimental effects for these drugs andthiourea.lsgl lg3, lg4 Beyond a slight reduction in skeletal growth and carcase-dressing value there was no effect on carcase quality.lg6 Fresh liver, butnot lean shoulder meat, of methylthiouracil-fed pigs was goitrogenic torats.lgl Combined thyroprotein and stilboestrol feeding improved growthand feed efficiency in fattening pigs.lg6Protein requirements of cattle (andsheep) were factorised in a statistical study of recorded data and the concept“ available protein ” introduced.The available protein is the metabolisableprotein of the feed taxed for the loss of body-nitrogen which its ingestionentails and thus available for covering endogenous losses and the demandsof other anabolic processes.lg7 New estimates of the protein requirements ofgrowing heifers were given lg8* lg9 with discrimination between requirementshave been found.Cattle.-Protein (inchding Urea).170 Geurin, Hoefer, and Beeson, J.Animal Sci., 1950, 9, 94.180 Neumann, Krider, James, and Johnson, .I. Nutrit., 1949, 38, 195.181 Nesheim, Krider, and Johnson, J . Animal Sci., 1949, 8, 627.Bustad, Ham, and Cunha, Arch. Biochem., 1948, 17, 240.183 Lehrer, Moore, Wiese, and Pahnish, J . Animal Sci., 1949, 8, 107.Carpenter, Arch. Biochem., 1950, 27, 469.185 Jukes, Stokstad, Taylor, Cunha, Edwards, and Meadows, ibid., p. 324.Luecke, McMillan, and Thorp, ibid., p. 326.187 Nordfeldt and Hydh, Kgl. Lantbrukshogsk. Ann., 1948, 15, 173.lE8 Perry, Beeson, and Andrews, J . Animal Sci., 1950, 9, 48.Beeson, Andrew$, Perry, and Witz, ibid., 1949, 8, 508.lS0 Vander Noot, Reece, and Skelley, ibid., 1948,7, 84.192 Terrill, Krider, Carroll, and Hamilton, ibid., 1949, 8, 501.lS3 Braude and Cotchin, Brit.J . Nutrit., 1940, 3, 171.lg4 Willman, Asdell, and Loosli, J . Animal Sci., 1949, 8, 191.lS5 Terrill, Hamilton, Krider, and Carroll, ibid., 1950, 9, 58.lS8 Braude, Nature, 1948, 161, 856.lS7 Blaxter and Mitchell, J . Animal Sci., 1948, 7, 351.lQ8 Steensberg, Brit. J . Nutrit., 1947, 1, 139.lg9 Jensen, Steensberg, and Wnther, Forsogslab. Ksbenhauit Beretn. No. 237, 1949.lgl Idem, ibid., 1950,9, 54DUCKWORTH GENERAL NUTRITION. 325for high- and low- quality rations. lg7 The digestible crude protein require-ment per kg. of milk containing 4% of butterfat was 55 to 60 g.200 Dis-crepancies between protein digestibilities of rations computed from tablesand found in experiment may be serious.201 That roughage intakes ofgrazing cattle can be estimated from the nitrogen content of faxes 202 wasdenied .203When suchurea was fed in rations to cattle and sheep, 15N was present in excess ofnormal in various body proteins, indicating the use of urea-nitrogen inprotein synthesis.205* 206 Adding urea to the rations of beef steers improvednitrogen retention and did not affect feed digestibility. There was noammonia in expired or regurgitated air.207 Urea-feeding raised the ammoniaand urea content of the bl00d.~~~1 208There was little or no difference betweencattle and sheep in their capacity to digest common feeds.209* 210* 211 Raisingthe plane of nutrition depressed the digestibility of carbohydrates in linseedoil meal 212 and barley.213 Only excessive barley feeding reduced proteindigestibility.2'2 The protein required for efficient roughage digestion waslow but was increased by additions of The digestibility of mixedrations (protein concentrate, carbohydrate concentrate, and roughage) wasunaffected by altering the nutritive ratio.215The depressing effect of lignin on roughage digestibility difTered markedlybetween species of herbage.216 Digestion in the rumen was studied, usinglignin as an (almost) inert marker.Roughage carbohydrates (except lignin)and protein were rapidly digested during the first 6 hours in the rumen, andcellulose was attacked mainly in the second 6 hours.There was littledigestion after 12 hours and only slight additional digestion in the c i e c ~ m . ~ ~ The use of markers (Cr203 218 and lignin, silica and iron 219) in digestibilitytrials was studied. A statistical analysis was made, of European data, toThe synthesis of urea containing 15N has been described.204Carbohydrate (and Energy).)O0 Ulvesli, Norges Landbruksh0gsk. Foringsforsnk. Beretn., No. 65, 1949.aol Poijiirvi, Maataloust. Aikakausk, 1947, 19, 108.202 Gallup and Briggs, J . Animal Sci., 1948, 7 , 110.403 Forbes, ibid., 1949, 8, 19.204 Leitch and Davidson, Sci. Agric., 1949, 29, 173.*05 Watson, Davidson, and Kennedy, ibid., p. 185.206 Watson, Kennedy, Davidson, Robinson, and Muir, ibid., p. 189.207 Dinning, Briggs, and Gallup, J .Animal Sci., 1949, 8, 24.208 Dinning, Briggs, Gallup, Orr, and Butler, Amer. J . Physiol., 1948, 153, 41.209 Watson, Davidson, Kennedy, Robinson, and Muir, Sci. Agric., 1948, 28, 357.210 Axelsson, Kgl. Lantbrukshogsk. Ann., 1949, 16, 84.211 Idem, Svenslc Jordbruksforsk. Arsbok, 1947.212 Watson, Kennedy, Davidson, Robinson, and Muir, Sci. Agric., 1949, 29, 263.Watson, Davidson, Kennedy, Robinson, and Muir, ibid., p. 400.Burroughs, Gerlaugh, Edgington, and Bethke, J . Animal Sci., 1949, 8, 9.215 Watson, Kennedy, Davidson, Robinson, and Muir, Sci. Agric., 1947, 27, 600.216 Phillips and Loughlin, J . Agric. Res., 1949, 78, 389.a17 Hale, Duncan, and Huffman, J . Nutrit., 1947, 34, 733.a18 Jarl, Kgl. LantbruksMgsk. Ann., 1949, 16, 785.Druce and Willcox, Empire J , Exp.Agric., 1949,17, 188326 BIOCHEMISTRY.relate methane production to dry matter, digestible carbohydrate, anddigestible crude fibre of the ration.220Minerals. Adding calcium carbonate to a calcium-low ration had littleor no effect on the digestibility of the ration.221 Oxalic acid, in paddystraw, hindered the utilisation of dietary calcium and provoked severealkalosis, probably through conversion of oxalic acid into carbonate andhydrogen carbonate in the rumen.222 The giving of calcium supplements,with or without trace elements, to preparturient cows had no effect on thecalcium and phosphorus content of the blood of the cows and their calvesat the time of parturition.223 Supplementing the ration with calcium hadno effect on the total output of lipids in faxes, but the ratio of neutral fatsand sterols to soaps and free fatty acids was altered.224Maximum output of radio-phosphorus in milk was reached 5 hours afterinjection, the level of dosage affecting neither the time required to reachmaximum output nor the magnitude of output.About 20% of the injecteddose was recovered in the milk during 7 days after dosage. Casein labelledwith radio-phosphorus (at about 2 pc. per g.) could be collected during thefirst 3 days after dosage,2z6 Giving 35 g. of phosphorus daily as NaH2P0,,Na,HPO,, or CaHPO, to cows in milk had no effect on faeces consistency orurine pH. Urinary acidity in silage-fed cows was reduced by Na2HP0, andCaCO, supplements.226 Fused Ca3(PO,J2 should not contain over 0.24% offluorine and should be used with care ; over 0.2% of fluorine in the concentrateration was dangerous to dairy cows.227Long-term feeding of growing calves indicated that about 50 mg.ofcobalt daily per 100 Ib. liveweight approached toxic levels, although animalswere variable in their response to dosage Studies with radio-cobalt indicated that adults did not store cobalt and that the main functionof cobalt was in the rumen. However there was liver storage of cobalt inthe fcetus.229 Prepartum dosage with cobalt greatly increased the cobaltcontent of colostrum. Cobalt feeding of calves raised the cobalt content ofall body parts, but particularly of the liver and kidney.230 Oral dosagegave small, and intravenous dosage large, retentions of radio-copper, mostbeing held by the liver.229*231 In lactating cows retentions of manganesewere surprisingly high (154 mg.daily), and independent of intake.2s2Sao Axelsson, Kgl. LantbrukshBgsk. Ann., 1949, 18, 404.221 Mukherjee and Chatterjee, Indian J . Vet. Sci., 1947, 17, 85.222 Talapatra, Ray, and Sen, J . Agric. Sci., 1948, 38, 163.22s Reid, Ward, and Salsbury, J . Nutrit., 1948, 38, 75.224 Ward and Reid, ibid., 1948, 35, 249.225 Kleiber, Smith, and Ralston, PPOC. SOC. Exp. Biol., 1948, 89, 354.22u Axelsson and Kivimae, Kgl. Lantbrukshdgsk. Ann., 1949,18, 101.227 Mather, Pratt, and Holdaway, J . Dairy Sci., 1949, 82, 228.228 Keener, Percival, Morrow, and Ellis, ibid., p. 527.Comar, Proc. Auburn Conf.on the Use of Radioactive Isotopes in AgriculturalResearch, December 18-20, 1947, p. 137.230 Ward, B e ~ e , Webster, Duncan, and Huffman, J . AnimaZ Sci., 1949, 8, 632.'** Reid and Ward, J . Nutrit., 1948, 85, 691.Comar, Davis, and Singer, J . Biol. Chem., 1948, 174, 906DUCKWORTH : GENERAL NUTRITION. 327Hormone Administration. Thyroprotein dosage improved milk andbutterfat production in cows 233,2u* 235,238, 237* 238 except at low dosagelevels.239 Feeding thyroprotein in successive years had less favourableeffects in later lactations.2a A comparison of the effects of oral dosage with(--)-thyroxine and iodocasein indicated either that (-)-thyroxine was moreefficiently used than iodocasein or that estimates of the thyroidal activity ofiodocasein are errone0us.m The energetic efficiency of producing the extramilk was much lower than that of normal milk p r o d u ~ t i o n .~ ~ ~ * ~ ~ l Severeweight losses by cows dosed with thyroprotein in hot weather could not beprevented by giving extra food.242Prolonged thyroprotein dosage of young stock increased body size, withjoint stiffness and osteoporosis.243 Intravenous thyroxine injection inhibitedfattening in non-pregnant non-lactating cows and increased the hardness ofbone.244 Thyroxine injection had an adverse effect on nitrogen and calciumbut not on phosphorus balances of lactating cows.245 The preparation ofiodinated casein containing radio-iodine was described.246 Additions ofthiouracil, nux vomica, and arsenious oxide improved feed efficiency infattening beefSheep.--Protein (incEwling Urea).No advantage was gained by feedingover 10.3% of protein in lamb rations.248 On feeds containing 9.5, 10.4, andl l . O ~ o of protein the daily weight gains of lambs were 0-27,0*30, and 0-30 lb.respectively with daily nitragen retentions of 2.7, 3-4, and 3.6 g.249 Noimprovement was produced in reproductive performance, birth weight oflambs, or subsequent lamb growth 250 by increasing the daily protein intakeof pregnant ewes from 0.15 to 0-31 lb. The protein of rumen bacteria wasabout one-third less digestible than casein but equal to it in biological value(rat a ~ s a y ) . ~ ~ l It was rich in methionine and ~ystine.~62 40% of dietaryzein was converted into microbial protein in the r ~ m e n .~ ~ ~233 Allen, DOW, Logan, and MacKenzie, Sci. Agric., 1948, 28, 340.234 Thomas and Moore, J . Dairy Sci., 1948, 31, 661.*s5 Thomas, Moore, and Sykes, ibid., 1949, 52, 278.236 Opichal, Chumchal, and Kopeckf, Sborn. Ces. Aka4 ZemBd., 1949, 21. 280.2s7 Moustgaard and Thorbek, Forssgslab. Ksbenhuvn Beretn., 1949, No. 240, 1-45.238 Lanik and Isajev, Sborn. Zes. Akad. Zeindd., 1949, 22, 65.239 Swanson and Knodt, J . Dairy Sci., 1949, 32, 257.240 Bailey, Bartlett, and Folley, Nature, 1949, 163, 800.241 Thorbek, Hansen, and Moustgaard, J . Animal Sci., 1948, 7 , 291.242 Gardner and Millen, J . Dairy Sci., 1948, 31, 660.24s Dyrendahl, Thesis, Univ. Stockholm, 1949, p. 116.244 McQuillan, Trikojus, Campbell, and Turner, Brit. J .Exp. Puthol., 1948, 29, 93.245 Owen, Biochem. J . , 1948, 43, 235, 243.246 Courrier, Roche, Deltour, Marois, Michel, and Morel, Bull. SOC. Chim. biol., 1949,a47 Kline, Ensminger, Cunha, Heinemann, and Ham, J . Animal Sci., 1949, 8, 411.248 Klosterman, Thesis, Cornell Univ., 1946.250 Jordan, Klosterman, and Wilson, J . Animal Sci., 1949, 8, 623.261 Reed, Moir, and Underwood, Austral. J . Sci. Res., 1949, 2, B, 304.f5a Johanson, Moir, and Underwood, Nature, 1949, 163, 101.2s3 McDonald, J . Physiol., 1948, 107, 21P.31, 1029.240 Briggs, Thesis, Cornell Univ., 1946328 BIOCHEMISTRY.Urea could replace part of the nitrogen in the rations of lambs weighing50 Ib. or more. I n rations containing 12% of crude “protein ” (totalnitrogen x 6.25) urea could provide half the nitrogen, provided that aquarter of the nitrogen was present as pre-formed protein.254* 255 Rumencontents of sheep given urea as the sole source of nitrogen contained largequantities of 10 “ essential ” amino-acids, in amounts similar to thosefound when casein was given.266 Dietary supplements of thiodiglycollicacid improved nitrogen balances.257 Loss of dietary nitrogen throughabsorption of ammonia from rumen contents was partly compensated for bysalivary urea reaching the r ~ m e n .~ ~ ~ Nitrogen fixation by rumen organismswas demonstrated.259 The rate of reduction of nitrate to nitrite in rumencontents depended on the nature of the diet.260 Preparation of purifieddiets for studies of amino-acid needs of ruminants was described.261Carbohydrate and Fat (including Energy).Formulae for predicting thedigestibility of herbage organic matter from faecal nitrogen,262 and thenitrogen content of herbage grazed from fmal nitrogen,263 were given. Arelation was reported 264 between starch equivalent (S.E.) and crude fibre(2) of herbage, vix., S.E. = 73.56 - 0*62x, but it seems to be tautological :the S.E. is essentially the digestible organic matter less a crude fibrecorrection value proportional to the crude fibre content. Formuls forestimating the digestible crude protein and starch equivalent of grasssilages z65 and dried grass 266 were given.Herbage lignins lost aldehyde groups during digestion, but methoxyland nitrogen contents were unchanged. The nitrogen was considered to bepresent in lignin as part of a heterocyclic structure, and not as protein orprimary or secondary amine.267 This view was rejected, specific amino-acids being identified in lignin.268 Lignins of young clover were morereadily digested and metabolised than those of mature clover, as judgedfrom the proportions of hippuric and benzoic acids voided in urine.269About half of the pentosans of hay were digested in the stomach (mainly therumen), and the remainder in the intestines.270 The digestibilities of pecticacids and pectins were reported.271 Addition of green lucerne or concentrates254 Hamilton, Robinson, and Johnson, J .Animal Sci., 1948, 7, 26.255 Briggs, Gallup, Heller, and Darlow, ibid., p. 35.256 Thomas, Loosli, Ferris, Williams, and Maynard, Fed.PTOC., 1949, 8, 398.2 5 7 Ferrando and Thomas, Bull. SOC. Chim. biol., 1948, 30, 228.2 5 8 McDonald, Biochem. J . , 1948, 42,584.260 Sapiro, Hoflund, Clark, and Quin, Onderstepoort J. Vet. Sci., 1949, 22, 357.Thomas, Loosli, Williams, and Maynard, J . Animal Sci., 1948, 7, 534.262 Lancaster, Nature, 1949, 163, 330.264 Hallsworth, J . Agric. Sci., 1949, 39, 254.285 Dijkstra, Versl. Landbouwk. Onderzoek., 1949, No. 55.10, pp. 15.266 Idem, ibid., 1949, No. 54.11, pp. 48.2 6 7 Bondi and Meyer, Biochem. J . , 1948, 43, 248.268 De Man and De Heus, Rec. Trau. chim., 1950, 69, 271.269 Pazur and Delong, Sci. Agric., 1948, 28, 39.270 Marshall, Brit. J . Nutrit., 1949, 3, 1.3 7 1 Leroy and Michaux, Compt. rend., 1949, 220, 1034.Tbth, Experientia, 1948, 4, 395.263 Raymond, ibid., 1948, 161, 937DUCKWORTH : GENERAL NUTRITION.329improved cellulose digestibility in veldt hay.272 The effect of the nature ofthe ration on the types and numbers of rumen organisms was reported.273The volatile acids of' rumen liquor were acetic acid about 75%, propionicacid 13-15y0, and butyric acid 7-10y0.274 At the height of fermentationafter feeding, acetic acid formed 86-95% of the total volatile acids inblood, but propionic, butyric, and at least one higher acid were also present.276Absorption of acetate ion was prevented by making the rumen contentsalkaline.276Onrefeeding a8fter starvation there was a transitory production of hydrogen,preventable by introducing normal rumen liquor into the rumen of thestarved sheep.Methane production was most rapid during the 4 hoursafter feeding. I n starvation, methane production ceased after 3-5Methane production (ing.) = 2 . 4 1 ~ + 9.80, where x is the hundreds of g. of carbohydrates digested.278Replacing carbohydrate by fat in isocaloric rations did not affect theutilisation of protein and energy, but methane production and dry matterdigestibility were slightly depressed.278The fasting metabolism per kg. W0'73 (W = bodyweight) of sheep was74.5 kcals. and the basal metabolism 59 kcals.,279 or 860 kcals./sq.m./24 hr.280The effects of the plane of nutrition on pregnant ewes, on the growth of theirfoetuses and lambs, and on milk production were reported.281* 282p 283* 284Changes in organ and tissue weights of ewes transferred from super-maintenance to submaintenance rations, and vice versa, were determined.285The influence of plane of nutrition on wool growth and quality was studied.286The daily calcium and phosphorus requirements of adultwethers were suggested to be 6.0 and 1.0-1.5 g.Ureafeeding did not alter the calcium and phosphorus requirements of lambs.288The copper requirement of sheep for the production of wool with satisfactorytextile properties was 5-10 mg. daily. Higher dosages may be bene-ficial.289*290 Attempts to deplete sheep of copper by injections of' BALgave inconsistent results.291 Cobalt-deficient lambs were benefited byNo hydrogen was evolved in the rumen when chopped hay was fed.or a t least reached a very lowMinerals..2722 7321427527727821928088128228328628 728828929029 1Louw, Bodenstein, and Quin, OndeTStepOOTt J .Vet. S C ~ . , 1948, 23, 239.Gall, J . Animal Sci., 1949, 8, 619.Schambye and Phillipson, Nature, 1949, 164, 1094.Reid, ibid., 1950, 165, 448.Pilgrim, Austral. J . Sci. Res., 1948, 1, B , 130.Swift, Bratzler, James, Tillman, and Meek, J . Animal Sci., 1948, 7 , 475.Marston, Austral. J . Sci. Res., 1948, 1, B , 93.Blaxter, J . Agric. Sci., 1948, 38, 207.A. M. Thomson and W. Thomson, Brit. J . Nutrit., 1949, 2, 290.Wallace, J . Agric. Sci., 1948, 38, 93.Idem, ibid., p. 243. 284 Idem, ibid., p. 367.Marston, Austral. J. Sci. Res., 1948, 1, B , 362.Axelsson and Eriksson, KgE.Lantbwksh&gek. Ann., 1949, 16, 71 1.Gallup and Briggs, J . Animal Sci., 1949, 8, 619.Palmer, J . Agric. Sci., 1949, 89, 265.Marston and Lee, Austral. J . Sci. Res., 1948, 1, B , 376.Stewart and Robertson, Biochem. J., 1948, a, xxii.276 Gray, J . Exp. Bid., 1948, 25, 135.Robinson, ibid., p. 345330 BIOCHEMISTRY.injection, but not by oral dosage, with liver extract. Injections of folicacid and vitamin B,, were without effect.292Studies using radioactive thyroprotein showedthat maximum absorption was reached 10 hours after oral dosage, Intra-venously administered thyroprotein disappeared from the blood in 7 ho~rs.29~Thyroprotein improved wool production,29P but not growth or feed efficiencyin fattening lambs.296 It raised the basal metabolism of ewes, the relationbetween dosage (in mg.per kg. W0’73) being linear with an increase of0.128% per mg.280 Overdosages reduced the digestibility of the ration,increased nitrogen losses in proportion to dosage and provoked large lossesof skeletal calcium and phosphorus.296Thiouracil, methylthiouracil, and propylthiouracil had either no effect,296or a detrimental effect,297 on growth and feed efficiency of fattening lambs.In aged ewes fattening was improved by methylthio~racil.~~7 Growth andfeed efficiency of wether lambs were improved by stilbcestrol and testosteroneimplants.298The nutritive value of wheat gluten 300and oat protein 301 was improved by lysine supplements. The biologicalvalue for infants of the proteins of breast milk was higher than those ofproteins of cow’s milk, mainly because of higher digestibility.Hydrolysedliver proteins were intermediate.302 Infants retained 50-90 yo of intraven-ously injected serum protein.303 Increasing calories (as glucose) above normalimproved nitrogen retention in parenteral alimentati~n.~M The averagedaily protein requirement of young women eating a high cereal diet was31.7 6 1.6 g., or 25 g./lOOO kcals. of basal heat, the average protein index(biological value x true digestibility) of the diet being 61.4 24.305Nitrogen retentions of young women were higher when part of the animalprotein intake (meat and milk products) was taken a t each meal than whenrestricted to two of the day’s meals.306Heavy work (mining) did not raise the nitrogen requirement aboveresting levels of 7.0-8.0 g.but productivity declined.307 Casein was aseffective as meat in restoring capacity to work in subjects who had beenHormone Administration.Hexcestrol impaired appetite in ewes.299Man.-Protein and Amino-acids.202 Becker and Smith, J . Animal Sci., 1949, 8, 615.193 Campbell, Andrews, and Christian, ibid., p. 638.294 Labarthe, Bertone, and Washburn, ibid., p. 624.296 Blaxter, J . Agric. Sci., 1948, 38, 1.297 Vander Noot, Reece, and Skelley, J . Animal Sci., 1950, 9, 3.298 Andrews, Beeson, and Harper, ibid., 1949, 8, 578.Z9* Austin, Whitten, Franklin, and Reid, Austral. J . Exp. Biol. Med. Sci., 1947,25,343.300 Hoffman and McNeil, J . Nutrit., 1949, 38, 331.302 Rossier and Beauvillain, Compt.rend. SOC. Biol., 1949, 143, 976.3O3 Pliickthun, 2. Kinderheilk., 1949, 66, 496.304 Ellison, McCleery, Zollinger, and Case, Surgery, 1949, 26, 374.305 Bricker, Shively, Smith, Mitchell, and Hamilton, J . Nutrit., 1949, 87, 163.306 Leverton and Gram, ibid., 1949, 89, 67.307 Kraut and Lehmann, Biochem. Z., 1949,319, 228.Barrick, Beeson, Andrews, and Harper, ibid., p. 243.Kuether and Myem, ibid., 1948, 35, 651DUCKWORTH : UENERAL NUTRITION. 33 1on low-protein diets, but meat extracts were ineffective.308 Reducingdietary protein from 90-95 g. (46 g. of animal protein) to 76 g. (19 g. ofanimal protein) daily did not affect output in heavy workers, but reducingintakes from 80-90 g. ( 3 0 4 0 g. of animal protein) to 70 g. (10 g.of animalprotein) did.309Wholemeal bread, water-extracted bran, and the aqueous extract ofbran raised fecal output of nitrogen above normal, but filter paper didThe increase was bacterial nitr~gen.~ll A metabolic fecal protein (namedfecanin) was isolated from infant stools and found to be rich in essentialamino-acids. Its amino-acid content was unaffected by the nature of thedietary protein .Tentative minimum daily requirements of adults for L-tryptophan(0.25 g.), L-phenylalanine (1.10 g.), L-lysine (0.80 g.), L-threonine (0-50 g.),L-valine (0.80 g.), L-methionine (1.10 g.), L-leucine (1.10 g,), and L-isoleucine(0.70 g.) were determined. Histidine was not essential, D- and L-methioninewere equally effective, and D-phenylalanine was partly utilised.Dietscontaining casein, hydrolysed casein, and pure amino-acids were found toraise the caloric requirement for nitrogen equilibrium in these experiments.313About 200 mg. of L-tryptophan daily met adult requirement and acetyl-L-tryptophan had about the same nutritive value. D-Tryptophan and acetyl-D-tryptophan were ineffective.314 Comparable results were found forinfants.316 The sulphur-amino-acid needs of infants were met by 85 mg.of L-methionine plus 15 mg. of L-cystine, or 65 mg. of L-methionine plus50 mg. of L-cystine per kg. bodyweight daily.31s The infant's daily need forL-isoleucine was 90 mg./kge317After rapid intravenous injection of 50 g. of amino-acids (withoutglucose) 9.5% of the a-amino-nitrogen appeared in the urine, with from 1 to16% of individual amino-acids, and a total of 4.3% of the 10 " essential "a m i n o - a c i d ~ .~ ~ ~ The distribution of amino-acids in the urine did not reflectthe pattern in the blood or injected solution.319 When DL-methionhe wasinjected intravenously only the D-isomer appeared in the urine.322 Amino-acid-nitrogen wastage was greater with injected enzymic hydrolysates ofcasein than with acid hydrolysates or amino-acid mi~tures.~~OMales eating diets providing 1 g. of protein/kg. of bodyweight dailyexcreted leucine 25.8, isoleucine 17.5, valine 2043, threonine 60, arginineKraut, Lehmann, and Szakall, Biochem. Z., 1949, 319, 247.Lehmann and Michaelis, ibid., 1949, 520, 99.slo Fournier, Bull. SOC. Chim. biol., 1949, 31, 407.81a Albanese, Davia, Lein, and Smetak, J .Biol. Chem., 1948, 176, 1189.s13 Rom, Fed. Proc., 1949, 8, 546.314 Baldwin and Berg, J . Nutrit., 1949, 89, 203.s16 Albanese, Davis, and Lein, J. Biol. Chem., 1948, 172, 39.316 Albanese, Holt, Davis, Snyderman, Lein, and Smetak, J . Nutrit., 1949, 37, 611,817 Idem, ibid., 1948, 35, 177.s18 Eckhardt and Davideon, J. CEin. Inve~t., 1948, 27, 727.slQ Harper, Proc. SOC. Exp. Biol., 1949, 72, 184.s*O Smyth, Levey, and Lasichak, J . Clin. Inveet.. 19.48, 27, 412.sll Idem, ibid., p. 411332 BIOCHEMISTRY.25.8, histidine 263.3, lysine 100, and methionine 8.3 mg. daily in the urine, thetotal being 2.46% of that ingested.321 Only methionine levels in the bloodfluctuated in relation to the amino-acid pattern of the diet.Adding DL-methionine to the diet caused a rise in free and combined non-precipitablemethionine and lysine in plasma. Urinary outputs were low (about 265%of intake), with only histidine and threonine fluctuating slightly in relationto intake.323 Urinary excretion of amino-acids was largely independent ofprotein intake (0-150 g. daily).324 D- and L-Methionine were absorbed a tthe same rate, but more rapidly from solutions than from tablets.325 Faecaloutput of amino-acids bore little relation to dietary intake.326The digestibilities of rape-seedand cotton-seed oils were 99.0 and 96.5% respectively, 51-6 g. being eatendaily. Butter, olive oil, lard,and margarine failed to raise urinary excretion of oxalicThe total specific dynamic action (S.D.A.) of a 993-kcal.high-protein testmeal during 16 hours post prandium was 17.0y0 of its energy content. Thecorresponding value for a high-carbohydrate meal was 9.6%. Suchincrements were of little practical importance to workers in hot or coldenvironments.329 Work reduced and shortened the S.D.A. of food,particularly if done before aEnergy expenditures (including basal metabolism) of boys a t quiet playwere 3.1 (age 7-8 years), 2.6 (9-11) and 2.1 (12-14) kcals./kg. of body-weight/hr. and when cycling were 6.6, 5-1, and 4.5 kcals. respectively.331Energy expenditures of boys (9-1 1) sitting listening, sitting singing, standingsinging, standing drawing, and dressing and undressing were 2-07, 2-23,2-35, 3.19, and 4-29 kcals./kg./hr.respectively. Corresponding values forgirls (9-11) were 1.80, 2.06, 2.13, 2.62, and 4 ~ 0 4 . ~ ~ ~ ~ ~ ~ ~ The average basalmetabolic rates (B.M.R.) of the children ranged from 1.44 to 1.50k ~ a l s . / k g . / h r . ~ ~ ~ The B.M.R. of girls and young women living in subtropicalconditions and a t an altitude of 2400 ft. declined from 36.4 (14 years) to31.2 (18 years) kcals./sq.m./hr. with little change from 18 to 23 years ~ f a g e . ~ ~ Basal heat production of healthy lean Brazilian adult males was not pro-portional to the body surface. The relation between basal heat productionand body weight ( W ) was found to be : Kcals. per hr. = 2.038 W083 (coldCarbohydrate and Fat (including Energy).There was no effect on nitrogen balance.327Sz1 Sheffner, Kirsner, and Palmer, J.Biol. Chem., 1948, 175, 107.322 Kinsell, Harper, Barton, Hutchin, and Hess, J. Clin. Invest., 1948, 27, 677.323 Kirsner, Sheffner, and Palmer, ibid., 1949, 28, 716.3z4 Eckhardt and Davidson, J. Biol. Chem., 1949, 177, 687.326 Harper and Uyeyama, Proc. SOC. Exp. Biol., 1948, 68, 296.326 Sheffner, Kirsner, and Palmer, J. Biol. Chem., 1948, 176, 89.327 Deuel, Johnson, Calbert, Gardner, and Thomas, J. Nutrit., 1949, 38, 369.328 Kabelitz, Klin. Woch., 1943, 22, 439.3eo Glickman, Mitchell, Lambert, and Keeton, J. Nutrit., 1948, 36, 41.s30 Wachholder, P$iigers Arch., 1949, 251, 485.331 Taylor, Lamb, Robertson, and MacLeod, J. Nutrit., 1948, 35, 51 1.332 Taylor, Pye, Caldwell, and Sostman, ibid., 1949, 88, 1.33s Idem, ibid., 1948, 36, 123.334 Thompson, Cox, and Ridgway, ibid., p.50DUCKWORTH 1 GENERAL NUTRITION. 333climate value is WoSs). For better proportioned subjects the relation, kcals.per hr. = 2.334 wasEstimates of energy needs for h o u s e w ~ r k , ~ ~ ~ ~ 3379 339 sedentary occu-p a t i o n ~ ; ~ ~ ~ light,337 medium,337 and heavy 337* 338 work have been given.Resting-fasting metabolism (Erhaltungsumsatz) was increased or decreasedby raising or lowering food intakes. Errors may be introduced in estimatesof energy needs for work if the resting-fasting rate is assumed to beconstant .340Comparison of bone meal and dried skimmed milk as sourcesof calcium for adults yielded inconclusive results.341 High utilisation ofwhey-calcium was found.342 Organic and inorganic, soluble and insoluble,calcium salts were all absorbed equally well, as judged by blood-calciumlevels. Magnesium favoured calcium a b s o r p t i ~ n .~ ~ Negative calciumbalances were found when crude or refined coconut oil was fed to adults.344Wholemeal bread and oatmeal provoked strongly negative calcium balancesin subjects living almost exclusively on these cereal pr0ducts.~~~9 346 Evidencewas presented to indicate that subjects became adapted to high phytate-phosphorus diets in time, and that calcium balances improved.347 Anoptimum pH of 5.2 was found for faecal p h y t a ~ e . ~ ~ ~ 80% of the calcium inrectal suppositories was absorbed and at a rate comparable with that ofintramuscularly injected calcium. The tone of the uterus was the criterion.34sAdaptation in the adult allowed calcium equilibrium to be maintained onintakes of about 0-5 g. daily.360 Older women (52-74 years) required 1-07 g.of calcium daily for equilibrium.351Gastric reductions of Fe+++ to Fe++ of sO-90~o were found in stomachcontents after eating bread, meats, and fruit.Milk and eggs gave irregularresults.352 More Fe+++ than Fe++ was dialysable from gastric juice betweenneutrality and pH 3.5. Mucin was less important than other gastric juicecomponents in forming stable iron complexes.353 Iron absorption wasMinerah.335 Galvao, J. Appl. Physiol., 1948, 1, 385.336 Droese, Kofranyi, Kraut, and Wildemann, Arbeitsphysiologie, 1949, 14, 63.337 Lshmann, Muller, and Spitzer, ibid., p.16G.338 Kraut, Bauer, Droese, Spitzer, and Wildemann, ibid., p. 147.330 De Langen, Nederland. Tijdschr. Geneesk., 1949, 93, 3247.340 Wachholder, PJliigers Arch., 1948, 250, 534.341 Drake, Jackson, Tisdall, Johnstone, and Hurst, J. Nutrit., 1949, 37, 369.34a De and Som, Indian J. Vet. Sci., 1948, 18, 241.343 Lafontaine, Compt. rend. Xoc. Biol., 1948, 142, 1089.344 De and Karkun, Indian J. Dairy Sci., 1949, 2, 114.345 McCance and Walsham, Brit. J. Nutrit., 1948, 2, 26.346 McCance and Glaser, ibid., p. 221.347 Walker, Fox, and Irving, Biochem. J., 1948, 42, 452.348 Courtois and PBrez, Bull. SOC. Chim. biol., 1949, 31, 1373.34B Sauter, Schweiz. Med. Woch., 1948, 78, 404.350 Kraut and Wecker, Biochem. Z . , 1948, 318, 495.3.51 Roberts, Kerr, and Ohlson, J .Amer. Dietectic ASSOC., 1948, 24, 292.352 Bergeim and Kirch, J. Biol. Chem., 1949, 177, 591.353 Lederer, Aata pntro-enterol. belg., 1949, 12. 233334 BIOUHEMISTBY.improved by adding beef to the diet of women.sM Urinary output of ironwas reduced by dosage with sodium hydrogen carbonate and increased byammonium chloride.356 Iron needs of girls (13-14 years) were 12-13 mg.daily.356 The needs of women (18 years) were between 7 and 10.4 mg.daily, the higher level being more satisfactory for repairing menstruallosses. Beef promoted iron retention.367* 358 Positive iron and copperbalances were found in women (17-22 years) on iron intakes 64-13.6 mg.,and copper intakes of 6.5-13.0 mg. daily. The amounts of iron and copperretained were similar.36Q The so-called ‘‘ physiological ” anEmia ofpregnancy was caused at least in part by inadequate iron reserves.36o Theiron reserve of the adult male was estimated at 600 mg.361 Iron losses insweat were low, probably not exceeding 0.02-0*06 mg.daily.362Addition of sodium citrate to a diet low in sodium chloride increasedsodium but not chloride output. Urinary potassium output was increasedby sodium citrate but not by sodium When lithium chloridewas used in food as a taste substitute for sodium chloride toxic symptomsdid not appear until serum-lithium levels rose to 1.0 m. e q ~ i v . ~ 6 ~ Radio-sodium studies indicated that the sodium transfer across the placenta duringthe twelfth week of pregnancy was 160 times, and during the fortieth week1100 times the foetal retenti~n.~~b After feeding of potassium citrate tochildren, renal clearances of potassium tended to vary inversely with theplasma-potassium level.In the adult given potassium dihydrogen phosphateor potassium citrate the plasma-potassium levels fell and clearances rose.Clearance with little change in plasma-potassium followed ingestion ofpotassium ~hloride.~6~Radio-iodine was not collected by the foetal thyroid before the fourteenthweek of pregnan~y.~67 The anti-thyroid activity of a large range of foodswas tested, by using radio-iodine. The inhibitory effect was greater invegetable than in animal products, swede, turnips, beets, cabbage, lettuce,and spinach being most potent.368J. D.854 Johnston, Frenchman, and Boroughe, J.Nutrit., 1948, 55, 453.5s5 Barer and Fowler, J. Lab. Clin. Med., 1949, 54, 932.366 Schlaphoff and Johnston, J. Nutrit., 1949, 59, 67.357 Johnston, Frenchman, and Boroughs, ibid., 1949, 38, 479.s58 Frenchman and Johnston, J. Amer. Dietetic ABSOC., 1949, 25, 217.350 Holt and Scoular, J. Nutrit., 1948, 35, 717.a60 Wills, Hill, Bingham, Miall, and Wrigley, Brit. J. Nutrit., 1947, 1, 126.361 Hynes, J. Clin. Pathol., 1949, 2, 99.a62 Johnston and Hagan, Fed. Proc., 1949, 8, 387.364 Talbott, Arch. Int. Med., 1950, 85, 1.365 Flexner, Cowie, Hellman, Wilde, and Vosburgh, Amer. J. Obstet. Gynecot., 1948,366 Wilson, Arch. Dis. Childhood, 1948, 25, 176.367 Chapman, Corner, Robinson, and Evans, J. Clin. Endocrinol., 1948, 8, 717..568 Greer and Astwood, Endocrinology, 1948, 45, 105Leaf, Couter, and Newburgh, J.Clin. Invest., 1949, 28, 1082.55, 469HERBERT 3356. OXIDISING) ENZYMES.The number of papers on oxidising enzymes published during the last threeyears has been so large that it has not been possible to refer to all of themwithin the space provided ; where a choice has had to be made, papers on theless well-defined enzymes have been omitted. The report deals primarilywith the properties of enzymes, and not with their physiological functions orchanges in various pathological or nutritional conditions. Of the referencescited, 63% are from American laboratories, 12% from British or Common-wealth laboratories, and 12% from Sweden. Clearly the study of thisimportant field is being neglected in this country.Books and Review Articles.-Second editions have appeared of Sumnerand Somers’s useful book,l and the collaborative work on respiratoryenzymes edited by H.A. Lardy; the latter has been almost completelyrewritten. Lemberg and Legge’s book on tetrapyrrole compounds (3182references) contains detailed accounts of a11 the iron-porphyrin enzymes andwill be invaluable to workers in this field; three excellent review articles onhaemoproteins have also appeared. 4 Dixon’s book contains (besides muchother matter of interest) an account of the author’s theoretical treatment ofphosphorylation reactions according to a “ scale of phosphorylationintensitx ” or rP scale, analogous to the rH scale or reducing intensity.AnEnglish translation of Warburg’s “ Schwermetallen ” has now appeared.The new edition of W. A. Waters’s book on free radicals has a long chapteron biochemical and enzymic mechanisms ; three review articles on mechan-isms and kinetics of enzymic reactions have also appeared.8 Review articleshave appeared on lipo~idase,~ the enzymes of snake venom (includingL-amino-acid oxidase) , l o and the citric acid cycle.llGeneral Trends.-The bulk of the work done during the review periodhas proceeded along well-tried lines, but some new trends are emerging, ofwhich the following appear to be significant : (a) The detailed physico-chemical study of pure enzymes by the methods of the protein chemist hasbeen elaborated. ( b ) A new approach to enzyme kinetics has been initiatedby the work of Britton Chance, described in the section below on haemo-proteins.This treatment, combined with the physico-chemical studies,seems most likely to provide an answer to the fundamental problem of allenzyme studies, namely, the problem of how enzymes work. (c) Increasingattention is being paid to insoluble enzyme complexes (“ succinoxidase,”l “ Chemistry and Methods of Enzymes,” Academic Press Inc., 1947.“ Respiratory Enzymes,” Burgess Publ. Co., 1949.“ Haematin Compounds and Bile Pigments,” 1949.Theorell, Adv. Enzymol., 1947, 7 , 265; Granick, ibid., p. 305; Wyman, Adv.Warburg, “ Heavy Metal Prosthetic Groups and Enzyme Action,” Clarendon Press,Protein Chem., 1948, 4, 410.Oxford, 1949.“Multi-enzyme Systems,” Camb.Univ. Press, 1948.’ “ Chemistry of Free Radicals,” Clarendon Press, Oxford, 1948.Michaelis, Adv. Enzymol., 1949, 9, 1 ; Stearn, ibid., p. 25; LuValle and Goddard,* Bergstrom and Holman, Adv. Enzymol., 1945,8,426. Quart. Ref. Biol., 1948,23, 197.lo Zeller, ibid., p. 459. l1 Martins and Lynen, ibid., 1950, 10, 167336 BIOCHEMISTRY." cyclophorase," etc.), and their relationships to mitochondria and otherformed elements of the cell.Methods.-Increasing use is being made of spectrophotometric techniquefor following the course of enzyme-catalysed reactions ; there are few enzymeswhich cannot be studied by this method. A disadvantage of the techniqueis the difficulty of adequate temperature control in most commercialspectrometers.Essenti-ally this is a micro-modification of the original instrument of Hartridge andRoughton, reduced t o a scale which permits complete measurements to bemade with 0.2 ml.of solution. Monochromatic illumination with highlystabilised light sources, photocells, and high-gain amplifiers, permits theautomatic recording of minute spectral changes (log I,/I - lo4). Reactionswith half-times of a few milliseconds can be followed by the " rapid-flow "technique, and slower reactions by a " stopped-flow " method.Some advances in methods for isolation and purification of' enzymes havebeen recorded, though this subject remains more a skilled form of cookerythan a science. Low-temperature fractionation with organic solvents isbeing increasingly used; the classical researches of the Harvard school inthe application of this technique to plasma-protein fractionation are reviewedby Ed~a1l.l~ So far, ethanol and methanol have been used almost exclusivelyfor this purpose, and the experiments of Dixon and Askonas l5 which givecomparative data for a number of solvents are therefore particularly welcome.Svensson l6 has reviewed preparative-scale electrophoresis methods.Animproved version l7 has been described of Kirkwood's apparatus whichfractionates protein mixtures by a combination of electrophoretic transportand vertical convective transport; components move from an upper to alower reservoir at rates depending on their electrophoretic mobilities. I nan interesting apparatus described by Svensson and Brattsten,l* the mixtureflows downwards through the space between two vertical glass plates, packedwith " Celite " to avoid convection, while a horizontal electric field is main-tained between the ends of the cell. Both these methods are in the develop-mental stage, but the latter in particular, on account of its simplicity, affordspromise for the future.Two-phase partition methods, so successful in otherfields, have not hitherto been applied t o protein separation owing to thedifficulty of finding non-miscible solvent pairs which will both dissolve pro-teins. Herbert and Pinsent l9 used the two phases which are formed in certainconcentration regions with three-component systems of the type water +salt + water-miscible solvent, in this case water-ammonium sulphate-ethanol ; the method was successfully applied in the isolation of crystallinebacterial catalase.More work needs to be done before it can be said whetherI n a different category is Chance's rapid-flow instrument, 12- l3l2 Chance, Acta Chem. Scand., 1947, 1, 236.13 Idem, Biochem. J . , 1950, 46, 387.1 5 1st Intern. Congr. Biochem., Cambridge, Aug. 1949 ; Askonas, Biochem. J . , 1951,1 7 Cann, Brown, and Kirkwood, J . Amer. Chem. SOC., 1949,71, 1609.18 Arkiv Kemi, 1949,1,401.l4 Adv. Protein Chem., 1947, 3, 384.l6 Adv. Protein Chem., 1948, 4, 251.lS Biochem. J., 1948, 43, 193.in the pressHERBERT : OXIDISING ENZYMES. 337this will prove a generally applicable method on the preparative scale, butMartin and Porter 2o have shown that it may be applied to the partitionchromatography of proteins on a analytical scale, using columns packedwith (‘ Hyflo.” By this technique, using water-ammonium sulphate-( ( Cellosolve ” mixtures, they have shown that crystalline and electro-phoretically homogeneous ribonuclease contains two enzymically activecomponents.Adsorption chromatography of proteins has been little prac-tised, since the best protein absorbents are gels whose physical propertiesare unsuitable for columns. Tiselius, however, has used strong salt solutionsto promote adsorption of proteins on paper.21 Mitchell, Gordon, andHaskins 22 separated the adenosine deaminase, amylase, and phosphatase ofa takadiastase preparation on a “ chromatopile ” (a column made from astack of filter papers), using an ammonium sulphate solution of graduallydecreasing concentration.Ion-exchange chromatography on a colourlesscarboxylic acid resin of a new type has been used to purify cytochr~me-c.~~The separation and purification of enzymes associated with insolubleparticles has always been a baffling problem and has succeeded in only a fewcases. A recent note by Morton 24 describes the use of n-butanol to obtaintrue solution into distilled water, or buffer solutions, of a wide range ofenzymes from insoluble particles, apparently by removal of lipid and othermaterials. If this proves a general method for dealing with insoluble enzymesit will be invaluable to workers in this difficult field.Coemymes.-[Abbreviations : Diphosphopyridine nucleotide (coenzyme1) is referred to in general as DPN; where the reference is specifically to itsoxidised or reduced form, DPN+ or DPNH is used respectively.TPN,TPN+ , and TPNH are used similarly for triphosphopyridine nucleotide(coenzyme 2), and NMN, NMN+, and NMNH for nicotinamide mono-nucleotide. AMP, ADP, and ATP are used for adenosine mono-, di-, andtri-phosphates, FAD for flavine adenine dinucleotide, FMN for flavinemononucleotide, and DPT for diphosphothiamine.]New or improved methods have been described for the preparation ofDPN 2s and TPN.26 Results reported by Slater 27 indicate that LePage’swidely used assay method 28 for DPN may give spuriously high results withcrude DPN preparations ; these may contain substances which give an absorp-tion band a t 340 mp.on reduction with sodium dithionite but are enzymicallyinactive ; the Reporter’s unpublished observations confirm this. Reductionwith sodium borohydride has been advocated for assay but this2o Biochem. J., 1951, in press.21 Tiselius, Arkiv Kemi, Min., Geol., 1948, 26, B, No. 1 ; Chem. Eng. News, 1949,22 J . Biol. Chem., 1949, 180, 1071.23 Paleus and Neilands, Actu Chem. Scund., 1950,4, 1024.a4 Nature, 1950,166, 1093.25 Hogeboom and Barry, J . Biol. Chem., 1948, 176, 935; Clark, Dounce, and Stotz,28 LePage and Mueller, ibid., 1949, 180, 975,z a .I. Biol. Chem.. 1947, 168, 623,27, 1041 ; Shepherd and Tiselius, Discuss. Paraday SOC., 1949, No. 6.ibid., 1949, 181, 459.Biochem. J . , 1950, 40, 484.29 Mathews.ibid., 1948, 176. 229338 BIOCHEMISTRY.produces similar absorbing but unreactive products. Enzymic reductionmethods would seem preferable, both for assay and for preparation of DPNHor TPNH ; convenient techniques using alcohol deh ydrogenase are describedby Bonnichsen30 and Ra~ker.~l Enzymic methods have also been used toobtain accurate values for the molar extinction coefficients of DPNH andTPNH.32Biochemists working with crude tissue extracts have often been botheredby the presence of enzymes catalysing the breakdown or interconversion ofDPN and TPN. There are several of these enzymes, and Kornberg and hiswssociates have characterised and partly purified four of them :(1) An enzyme 33 obtained in partly purified form from yeast and livercatalyses the reversible reaction :"y:'} + ATP 3 DzP' } + pyrophosphate (i)"H DPNHThe reaction proceeds readily in either direction (equilibrium constantK = 0.45). This enzyme appears to be specific for all four reactants.(2) A second also present in yeast, catalyses a similar reversiblereaction with flavine mononucleotide (FMN), which reacts with ATP to giveFAD :M A + FMN + ATP - FAD + pyrophosphate.. . (ii)(3) An enzyme 35 of the phosphokinase type, also found in yeast, catalysesthe synthesis of TPN+ from DPN+ by direct phosphorylation with ATP;DPNH reacts similarly."?:+}+ ATP y; ':+}+ ADP . . (iii)DPNH TPNHThis reaction throws important light on the structure of TPN, indicatingthat it is not a triphosphoric acid derivative, but a pyrophosphate like DPNwith the third phosphate group attached to some other part of the molecule(see below).(4) A " nucleotide pyrophosphatase ", obtained in highly purified formfrom potatoesY36 cleaves DPN+ or DPNH at the pyrophosphate linkage,forming AMP and the oxidised or reduced form of nicotinamide mononucleo-tide (NMN+ or NMNH)."z?'} + H,O --j "EN') + A M P . . . (iv)DPNH NMNHTPN, FAD, DPT, ATP, and probably ADP are also attacked, though atThe same enzymeJo Acta Chem. Scand., 1950, 4, 714.se Horecker and Rornberg, ibid., 1948,175, 385.as Kornberg, ibid., 1950,182, 779.35 Kornberg, ibid., p, 805,slower rates, all being split a t the pyrophosphate linkage.s1 J . Biol. Chem., 1950,182, 313.Schrecker and Kornberg, ibid., p.795.Bornberg and Pricer., ibid., p. 763HERBERT : OXIDISING ENZYMES. 339is believed to attack all these compounds; crude extracts also contain en-zymes specifically attacking ATP and DPT. The products formed by theaction of this enzyme on TPN are NMN, and an adenosine diphosphate whichis not identical with ordinary ADP and is not a pyrophosphate compound;i.e., its two phosphate groups are attached to adenosine a t different points.37One of these is attached to C(5) of the ribose, and is cleaved by a specificadenosine4 phosphatase (isolated from potatoes) ; the monophosphateresulting is identical with the adenylic acid a recently isolated by Carter 38and Cohn 39 from yeast nucleic acid, which is probably adenosine2phosphate.The adenosine diphosphate arising from TPN is thereforeadenosine4 : 5 diphosphate, and the structure of TPN is probably :CHHi:-j OHHV*OOj~-OHH9-1 HV*OHHCOHi OH$!*OH IHF 9- QH HV CH2-O-~-O-~-O-CH20 0This work is an outstanding example of the use of enzymes in the elucidationof chemical structure.Mehler et a1.40 have tested thecoenzyme specificity of a number of dehydrogenases. The ratioactivity with DPN was found to be 135-220 for lactic dehydrogenase activity with TPN(three sources), 25-34 for malic dehydrogenase (two sources), and about 1for glutamic dehydrogenase (liver). isoCitric acid and glyceraldehyde-3 phos-phate dehydrogenases were completely specific for TPN and DPN respectively.Lactic and malic dehydrogenases had previously been thought to be com-pletely specific ; possibly other “ specific ” dehydrogenases might prove tobe more or less unspecific if tested equally rigorously.DPN Dehydrogenases.-AZcohoZ dehydrogenase of yeast has been crystal-lised by Racker 31 by a simple method giving good yields, which should makeit a readily available enzyme.The effect of pH was studied on the equili-brium :The true equilibrium constant of the reaction is obviously K =[CH,*CHO][DPNH][H+]/[C,H,*OH][DPN+], and K has the constant value1.15 x 10-l1 over a wide pH range. The equilibrium constant is usvallyCoenzyme Specificity of Dehydrogenuses.C2H,*OH + DPN+ CH,*CHO + DPNH + H+ (v)s7 Kornberg and Pricer, J Biol. Chem., 1960, 186, 657.s8 J . Amer. Chem. SOC., 1050, 72, 1466.?e Ibid., p.1471. 40 J . Biol. Chem., 1948,174, 96340 BIOCHEMISTRY.(wrongly) written as K' = [CH,*CHO][DPNH]/[C,H5*OH][DPN+], and isobviously pH-dependent; in fact a rise in pH of 1 unit causes a 10 foldincrease in the value of K'. Similar relations were found for lactic dehydro-genase, and should apply generally. At a fairly alkaline pH, in the presenceof excess of alcohol, reaction (v) goes almost completely from left to right, andadded DPN+ is quantitatively reduced to DPNH which may be determinedspectrophotometrically ; this makes a convenient assay method for DPN.The enzyme may also be used for the preparation of DPNH from DPN'.Bonnichsen 41 has crystallised the alcohol dehydrogenase of horse liver ;the apparently pure enzyme has, curiously, a turnover number for DPN verymuch lower than that of the yeast enzyme.He has also described enzymicmethods for assay of DPN and preparation of DPNH.30Racker 42 has discovered and purified to a considerable extent the pre-viously unknown acetczldeh yde dehydrogenase, which occurs in liver andcatalyses the virtually irreversible reaction :CH,*CHO + H20 + DPN' -+ CH,*C02H + DPNH + H+ (vi)A mixture of this enzyme with alcohol dehydrogenase behaves as an" aldehyde mutase," catalysing the dismutation of acetaldehyde accordingto the equation :2CH3*CH0 + H20 --+ CH,*CH,*OH + CH,*CO,H (vii)which results from adding equations (v) and (vi). There would seem to benow no grounds for postulating the existence in liver of a distinct aldehydemutase enzyme.Crystalline D-glyceraldehyde 3-phosphate dehydrogenuse of rabbit skeletalmuscle was isolated by the Cori's and their associate^.^^ It is electrophoretic-ally homogeneous.Accurate values are given for its catalytic activity, andits amino-acid content has also been determined.u This enzyme has a num-ber of unusual properties. The crystalline enzyme as isolated contains onemole of firmly bound DPN per 50,000 g. of protein ;45 this is not removed bydialysis or repeated recrystallisation, though it can be removed by treatmentwith phosphatase or " Norit." The DPN-free protein retains its activityafter these treatments, but will no longer crystallise unless DPN is added;the crystals then formed contain the original amount of bound DPN. Thisbound DPN is reduced, with appearance of an absorption band at 340 mp.,when glyceraldehyde-3 phosphate + phosphate (or arsenate) is added.Toobserve this spectrophotometrically, enzyme concentrations of 2 - 4 mg. /ml.are needed, the reaction being then too fast for its kinetics to be followed;if glyceraldehyde is used as substrate, however, the reaction is slow enoughBonnichsen and Wassen, Arch. Biochern., 1948, 18, 361 ; Bonnichsen, Acta Chem.43 G . T. Cori, M. W. Slein, and C. F. Cori, ibid., 1948,173, 605.44 Velick and Ronzoni, ibid., p. 627.Scand., 1950, 4, 715. 42 J. Biol. Chem., 1949,177, 883.4 5 Taylor et d,* ibid., p. 619.* References with four or more co-authors are cited bv the name of the first authoronlyHERBERT : OXIDISING ENZYMES. 341to be followed.reaction then occurs : 46glyceraldehyde + enzyme-DPN+ -+ glyceric acid +one molecule each of substrate and DPN reacting for each molecule ofprotein.The kinetics show that neither DPN+ nor DPNH can dissociate toa detectable extent, which means a dissociation constant of a t most lo-'-probably much less. If DPN+ is added to the DPN-enzyme the added DPN+is also reduced and the overall rate increases ; when the DPN+ concentrationis large compared to that of the enzyme, the reaction is :glyceraldahyde + DPN+ + glyceric acid + DPNH + H+ (ix)The enzyme here acts catalytically, and the kinetics indicate that DPN+and DPNH form reversible compounds with the enzyme, with equal dissoci-ation constants of 4 x The simplest hypothesis covering theseapparently contradictory facts is that the enzyme has two binding sites forDPN, one with comparatively low affinity (dissociation oonstant 4 xand the other with very much higher affinity.That DPN bound at thelatter site does dissociate to a finite (though very small) extent is presumedbecause ( a ) bound DPN exchanges with added DPN labelled with 32P, and( b ) bound DPNH is oxidised on addition of lactic dehydrogenase andpyruvate. These interesting observations are referred to again under" cyclophorase " (see below). Another interesting property of this enzymeis described by Rapkine et ~ 1 . ~ ~ Crystalline preparations lose activity onstorage a t room temperature or 0" ; such partly inactivated " aged " speci-mens are re-activated to a considerable extent by being heated for shortperiods at 55-65".The percentage reactivation by heat increases with ageand extent of previous inactivation, though the original activity of thefreshly crystallised enzyme is never regained in full. Heat-activation isincreased by addition of inert protein, formed on ageing. To explain theseunique phenomena the hypothesis is advanced that heat-activation " is aresult of the interaction between mercapto groups of the inactive protein anddisulphide groups of the active protein."L-a-GZym-ophosphk dehydrogenase of rabbit skeletal muscle has beenisolated by fractional crystallisation of Myogen A preparation^.^^ Itcatalyses the reaction :a-glycerophosphate + DPN+ =+In the presence of arsenate the following stoicheiomtricenzyme-DPNH + H+ (viii)enzymedihydroxyacetone phosphate + DPNH + H+ (x)The equilibrium position lies far to the right (K' = 1.4 x lo4 at 20" andpH 7 ) ; in the presence of excess of DPNH, dihydroxyacetone phosphate iscompletely reduced, and it may be assayed by this system.O 6 C.F. Cori, S. F. Velick, and G. T. Cori, Biochim. Biophys. Acta, 1950, 4, 160. '' Rapkine, Shugar, and Siminovitch, Arch. Biochem., 1950, 86, 33,u Baranowski, J . Biol. Chem., 1949, 180, 636342 BIOOHEMISTRY.The substrate specificity of crystalline lactic dehydrogenase has beenstudied by following spectrophotometrically the oxidation of DPNH byvarious keto-a~ids.~~ a-Ketobutyric acid is reduced nearly as fast aspyruvic acid, but for higher a-keto-acids the rate falls off rapidly with chainlength.The straight-chain ay-diketo-acids (from C, to Cll) are also reduced,all at about the same rate, equal to about one-tenth of the rate for pyruvicacid; only the a-keto-group is reduced.The formic dehydrogenase of green peas has been partly purified.s0 Itcatalyses the reaction ;As expected from the calculated free energy change (AFo = -6310 cals.),reaction (xi) is virtually irreversible, and when it was carried out in the pres-ence of NaH14C0, only minute amounts of 14C were incorporated into theformate. It is therefore unlikely that reversal of reaction (xi) can be asignificant pathway of C0,-fixation. The enzyme should be useful for DPNassay, since it is completely specific and reduction of DPN+ goes to comple-tion.Similar enzymes are present in small amounts in liver and kidney.Glucose dehydrogenase of lamb liver has been partly purified 6 1 p s2 and itscarrier systems studied.61* s3 Papers on this topic, discussing whether ornot cytochrome-c, diaphorase, etc., are involved in the electron transportbetween glucose and oxygen, have appeared at intervals ever since thediscovery of the enzyme (cf. Harrison et The impression is given bysome authors that the problem is in some way specifically connected withglucose dehydrogenase, whereas it applies to all DPN-dehydrogenases ; the realproblem (discussed in a later section) is the mechanism of aerobic oxidationof DPNH, irrespective of the particular dehydrogenase system reducingDPN+.A heat-stable malic dehydrogenase has been obtained by Militzer et aLS6from a thermophilic organism of the Bacillus group; the dehydrogenase inmesophilic bacilli is not heat-stable.A soluble liver enzyme has been obtained by Sweat et al.66 which inthe presence of DPN oxidises the >CH*OH group of testosterone to >CO,giving androstenedione.Further purification of the enzyme is needed todetermine if it is a simple DPN-dehydrogenase; if so, it will be the firstdehydrogenase known to act on a sterol.TPN Dehydmgenases.-isoCitric acid dehgdrogenuse of pig heart has beenstudied extensively by Ochoa and his associates. The previously unknownsubstance oxalosuccinic acid was synthesised s7 (it was also independently4o Meister, J .Biol. Chem., 1950, 184, 117.50 Mathews and Vennesland, ibid., 1950, 186, 667.51 Eichel and Wainio, ibid., 1948,175, 155.s4 Brunelli and Wainio, ibid., 1949, 177, 75.65 Renvall, Acta Chem. S a n d . , 1950, 4, 738.54 Harrison and Hawthorne, Biochem. J . , 1939, 33, 1573.6 6 Arch. Biochem., 1949, 24, 75.5 6 Sweat, Samuels, and Lumry, J . Biol. Chem., 1950,186, 75.Ochoa, ibid., 1948,174, 115.H*CO,-+ DPN+ j CO,+ DPNH . . . (xiHEBBERT : OXIDISING ENZYMES. 343synthesised by Lynen et ~ 1 . ~ ~ ) and found 50 to be the primary oxidationproduct of isocitric dehydrogenase (eqn. xii). It is decomposed by a secondenzyme " oxalosuccinic decarboxylase " 8o .into a-ketoglutaric acid (see xiii).isocitrate + TPN+ + oxalosuccinate + TPNH + H+ (xii)Mn++oxalosuccinate + H,O -.....a-ketoglutarate + HC0,- (xiii)bBoth reactions are reversible, but only the second needs Mn++. While(xi;) proceeds readily in either direction, the equilibrium of (xiii) lies far to theright ; nevertheless it was possible to reverse both reactions, bringing abouta fixation of carbon dioxide in isocitric acid, by coupling the above systemwith glucose-6 phosphate dehydrogenase and its substrate ; this systemperforms the function of keeping all the TPN present in the reduced state.69This reaction may be of importance in C0,-fixation; the left-to-right reac-tions (xii) and (xiii) are certainly important steps in the citric acid cycle.While the enzymes catalysing (xii) and (xiii) have been given different names,it is by no means certain that they are in fact different enzymes.Theyalways occur together and attempts to separate them have so far beenunsuccessful ; on purification the ratio of their activities is not altered.s1Their instability, however, has so far prevented extensive purification.Ochoa suggests that this may be a case of a single enzyme with a doublefunction, as has been suggested for the " malic enzyme" (see below).Enzymes catalysing reactions (xii) and (xiii) have been found by Venneslandand her co-workers in pigeon liver 62 and in plants,Gs and C0,-fixation by thesystem has been demonstrated with 14C0,. Here again, it is uncertainwhether one or two enzymes are concerned.A 6-phosphogluconic acid dehydrogeme has been obtained in soluble formin rat liver extracts,M and separated by ammonium sulphate fractionationfrom glucose and glucose-6 phosphate dehydrogenases.Partial purificationof a similar enzyme from yeast has also been reported.s6The " malic enzyme " is the name given by Ochoa et aZ.401 66 to an enzymeor enzyme system of pigeon liver that catalyses the reversible overallreaction :M d malate" + TPN+ - pyruvate- + CO, + TPNH (xiv)The equilibrium of this reaction lies to the right, but it has been possible toreverse it, with a resulting net fixation of carbon dioxide; this reaction,rather than (xii) and (xiii), is thought by Ochoa et at?. to be a major pathwayof C0,-fixation. 66 Reaction (xiv) involves a simultaneous oxidation and6 859606 16863646666Lynen and Schere, Annalen, 1948,560, 163.Ochoa, J .Biol. Chem., 1948,174, 133.Ochos and Weisz-Tabori, ibid., p. 123.Grafflin and Ochoa, Biochirn. Biophys. Acta, 1950, 4, 205.Grisolia and Vennesland, J . Biol. Chem., 1947, 170, 461.Ceithaml and Vennesland, ibid., 1947,178, 133.Dickens and Glock, Natwe, 1950,166, 33.Horecker, Fed. PYOC., 1950, 9, 186.Ochoa, Mehler, and Kornberg, J . BioE. Chem., 1948,174,979344 BIOCHEMISTRY.decarboxylation of malate. All enzyme preparations which catalyse it willalso decarboxylate oxaloacetic acid (eqn. xv) :Mn++ oxaloacetate‘ + H+ --+ pyruvate- + C02 . . (xv)Malic dehydrogenase, though a DPN-enzyme, also reacts fairly rapidly withTPN according to eqn. (xvi) :and this reaction followed by (xv) would give the overall reaction (xiv).However, the (( malic enzyme ” seems not to be a simple mixture of malicdehydrogenase and oxaloacetic decarboxylase, but a single enzyme catalysingboth reactions (xiv) and (xv).The ratio of these two activities remainsconstant on extensive purification, and the enzyme in the absence of Mn++does not have the properties of a malic dehydrogenase.66 Moreover,artificial mixtures of pure malic dehydrogenase and purified oxaloaceticdecarboxylase of Micrococcus lysodeikticus 671 68 [which catalyses reaction(xvi) only] do not have all the properties of the (‘ malic enzyme,” 4°-669 68which seems, therefore, to be a “ double-barrelled ” enzyme of a new type.A similar ( ( malic enzyme,” but utilising DPN instead of TPN, has beenfound in Lactobacillus arabino~us.~~ Vennesland and her co-workers 70* 71* 72have described apparently similar systems in parsley root and other plants,though it is not so certain in these cases that a single enzyme is concerned.In addition to reactions (xiv) and (xv), the plant enzyme systems catalysereaction (~vi),~O which the “ malic enzyme ” of pigeon liver does not ; how-ever, the malic dehydrogenase and oxaloacetic decarboxylase activities arealways closely associated.72Flavoprotein Enzymes.-L-Bmino-acid Oxidase of Snake Venom (“ Ophio-amino-acid Oxidme ”).Zeller’s review 10 covers the literature on this enzymeto 1948. Since then the enzyme has been isolated in a pure state frommocassin venom by Singer and K e a r n e ~ .~ ~ Homogeneity is indicated byultra-centrifuge, electrophoresis, and solubility data. The prosthetic groupis FAD,74 and analysis indicates one mole of FAD per 62,000 g. of protein;the molecular weight from ultra-centrifuge data is also 62,000. The sameauthors have described an interesting type of inhibition of the enzyme bymultivalent anions such as phosphate and arsenate. 75Some new amino-acid oxidases frommoulds have recently been described. These have not been purified andtheir chemical nature remains unknown ; they are included in this section onflavoprotein enzymes on grounds of analogy only.This organism6’ Herbert, Biochem. J . , 1950, 47, i.6 8 Idem, SOC. Exp. Biol. Symp. Carbon Dioxide Fixation, 1950.&@ Korkes and Ochoa, J .B i d . Chem., 1948,176,463.70 Vennesland, Gollub, and Speck, ibid., 1949,178, 301.7 1 Vennesland, a i d . , p. 591.72 Conn, Vennesland, and Kraemer, Arch. Biochem., 1949,23, 179.73 Ibid., 1950, 29, 190.7 5 Kearney and Singer, ibid., 1949, 21, 242.malate- + TPN oxaloacetate + TPNH + H+ (xvi)Amino-acid Oxidases from Moulds.(a) D - and L-Amino-acid oxidases of Neurospora crassa.74 Singer and Kearney, ibid., 1950, 27, 348HERBERT : OXIDISING ENZYMES. 345was previously known to produce a D-amino-acid ~ x i d a s e . ~ ~ It has nowbeen found that some strains produce an L-amino-acid oxidase but not theD-enzyme, while other strains produce both enzymes.77 The L-enzyme, butnot the D-enzyme, is excreted in considerable amounts into the culturemedium.(b) D- and L-Amino-acid oxidases of Penicillium qnd Aspergillus s p c k s .L-Amino-acid oxidases were discovered by Knight 78 in a number of Peni-cillium and Aspergillus species. Their properties were all very similar, butdiffer somewhat (e.g., in specificity) from the Neurospora enzyme; they arenot excreted into the culture medium, remaining firmly attached to themycelium.More recentl~,7~ D-amino-acid oxidases were found in eightPenicilliurn strains and in Aspergillus niger. These also are not excretedinto the culture medium, but active cell-free extracts could be obtained bygrinding the mycelium with sand. Bender and Krebs 8o have studied therate of oxidation of 48 amino-acids, among them 27 not occurring naturally,by the D-amino-acid oxidases of sheep kidney and Neurospora crassa and theL-amino-acid oxidases of Neurospora crassa and cobra venom.Considerabledifferences in specificity and relative rates of oxidation were found.D-Aspartic acid oxidase differs from the above amino-acid oxidases, butresembles glycine oxidase, in being specific for a single amino-acid. It hasbeen obtained in soluble form in liver or kidney extracts,81 but is distinctfrom Krebs’s classical D-amino-acid oxidase. It has not been greatly purified,but was identified as a flavoprotein by resolution into the apoenzyme, whichis inactive unless supplemented with FAD ; FMN is ineffective.82(The Reportersuggests that the use of the name “notatin” for this enzyme should beabandoned in favour of “ glucose oxidase,” which besides being descriptiveand conforming to normal enzyme nomenclature, has clear historicalpriority.) Keilin and Hartree 83 have made a detailed study of the propertiesof this enzyme, purified according to the method of Coulthard et aLs4 Theprosthetic group was identified as FAD, and direct analysis (corrected forca.8% of impurity in the enzyme) gave one mole of FAD per 75,000 g.protein. The molecular weight by ultra-centrifuge measurements is 152,000 ;hence the enzyme molecule contains two molecules of FAD. The specificitytowards a large number of compounds was tested; a few sugars and somemethylated glucoses are oxidised at very low rates, but to all intents andpurposes the enzyme may be considered specific for glucose.This makes itan invaluable reagent for glucose analysis; Keilin and Hartree 85 have alsoshown how it may conveniently be used to follow manometrically the actionof other enzymes which liberate glucose from its derivatives (e.g., glucosidases,Glucose oxidase of Penicillium notatum (“ notatin ”).78 Horowitz, J . Biol. Chem., 1944,154, 141.7 7 Bender, Krebs, and Horowitz, Biochem. J . , 1949,45, xxi. ’* J . Bact., 1948, 55, 401.Emerson, Puziss, and Knight, Arch. Biochem., 1950, 25, 299.Biochem. J . , 1950, 46, 210.Biochem. J . , 1948,42, 221.81 Stilletal., J. Biol. Chem., 1949, 179, 831.84 Ibid., 1945, 39, 24.82 Still and Sperling, ibid., 1950, 182, 585.85 Ibid., 1948, 42, 230346 BIOCHEMISTRY.maltase, invertase, glucose-phosphatases). Bentley and Neuberger e6used water enriched with H,lSO to study the reaction mechanism of theenzyme.The oxygen atoms of the hydrogen peroxide produced werederived entirely from molecular oxygen, without any detectable admixtureof oxygen derived from water. Polarimetric studies also showed theinteresting fact that the primary oxidation product is not gluconic acid but8-gluconolactone, which is hydrolysed to gluconic acid by a non-enzymicmechanism. The overall action of the enzyme must therefore be formulatedas : --+ H,O,HO&H 11 1 o + o , =The enzyme oxidases p-glucose 1.3 times as fast as a-glucose; suggestive,though not conclusive, evidence was obtained that the enzyme catalysesthe mutarotation of glucose, and that or-glucose is converted enzymicallyinto the p-form before being oxidised.TPN-Cytochrome c rediictuse is an important enzyme isolated from pigliver by Hore~ker.~’ In tissue extracts it is associated with small particles(probably mitochondria, cf.ref. 177), and digestion with trypsin was necessaryto bring it into solution. The pure enzyme contains one mole of FAD per68,000 g. of protein, differing from the corresponding yeast enzyme whoseprosthetic group is FMN. When resolved into the apoenzyme, however,this will combine to form active enzymes with both FAD and FMN, andcuriously, the “ artificial ’’ FMN-enzyme is the more active. The corres-ponding yeast apoenzyme also forms an “ artificial ” enzyme with FAD,and this is less active than the natural FMN-enzyme.The enzyme is reducedspecifically by TPN; the reduced enzyme is oxidised >50 times faster byferricytochrome-c than by oxygen.Xunthine oxiduse has been isolated from milk by an improved methodwhich gives preparations three times more active than any previouslydescribed, but still containing a little lactoperoxidase.88 Ferricyto-chrome-c is reduced by the enzyme with either aldehydes or purines assubstrates ; reduction is slow anaerobically but is accelerated by oxygen.The mechanism of this effect is not clear. Kalckar et aLe9 reported the oxid-ation of xanthopterin to leucopterin by a partly purified milk enzyme; this“ pterin oxidase ” and also xanthine oxidase were strongly inhibited bypteroylglutamic acid. Lowry et aLgO showed that this was due to traces ofan inhibitor, 2-amino-6-formyl-4-hydroxypteridine, in the pteroylglutamicacid preparations ; this substance also inhibits xanthine oxidation byxanthine oxidase.Purified preparations of the latter oxidise 2-amino-4-hydroxypteridine to isoxanthopterin as well as oxidising xanthopterin ;“ pterin oxidase ” is probably identical with xanthine oxidase. This is8’1 J . Biol. Chem., 1950, 183, 593. 86 Biochem. J., 1949, 45, 584.8 8 Horecker and Heppel, ibid., 1949, 178, 683.sB Kalckar and Klenow, ibid., 1948, 172, 349.Bo Lowry, Bessey, and Crawford, ibicl., 1949,180, 389, 399HERBERT : OXIDISING ENZYMES. 347confirmed by Kalckar et aZ.,91 who find that 6-formylpteridine is also a stronginhibitor of both xanthine and xanthopterin oxidation. Krebs and Norris 92confirm the probable identity of xanthine and pterin oxidases.Xanthopterinis oxidised more slowly than xanthine, but has a much higher affinity forthe enzyme ; it thus acts as a competitive inhibitor of xanthine oxidation.Copper-protein Enzymes.-PhewICt8eS. The hitherto obscure relationbetween the enzymes oxidising monophenols (“ tyrosinase,” “ monopheno-lase,” “ cresolase ”) and those oxidising o-dihydric phenols (“ catecholase,”“ polyphenol oxidase ”) is considerably clarified by two papers from Nelson’slaboratory. Mushroom tyrosinase can be separated into fractions of highand relatively low catecholase-to-cresolase activity ratios.gs Several prepar-ations with different activity ratios were homogeneous on electrophoresisand ultra-centrifugation but had different electrophoretic mobilities andcopper contents.94 The preparations with relatively high catecholaseactivity had properties differing from those of the enzyme as it occurs inmushroom juice.It is suggested94 that natural mushroom tyrosinase is asingle protein complex possessing both catecholase and cresolase activitiesand containing 4 copper atoms; the cresolase activity is associated withparts of the molecule which are split off by the purification procedure usedfor obtaining “ high catecholase ” preparations, The simultaneous oxid-ation of o-dihydric phenols is assumed necessary for the ozfidation of mono-phenols. It remains to be seen whether this hypothesis applies to otherphenolases.It is significant that catecholases with no monophenolaseactivity have been obtained, but preparations with only monophenolaseactivity have never been reported. Eiger and Dawsong5 have isolatedsweet-potato phenolase, which only oxidises o-dihydric phenols ; potatoslices oxidise monophenols readily, but this activity is lost as soon as thecell is destroyed. Kendalg6 also thinks that mushroom tyrosinase is asingle enzyme with different centres oxidising mono- and di-hydric phenols,but believes the two activities to be independent ; i.e., o-dihydric phenols arenot necessary for monophenol oxidation,Mammalian tyrosinase of mouse melonoma possesses both activities, butit is associated with insoluble cytoplasmic particles and no purification hasyet been possible.97 Schacter 98 describes a catecholase in human serumwhich oxidises only dihydric phenols. Studies have appeared on the pig-ments produced by secondary reactions when oxidation of phenols occurs inthe presence of certain amino-acids and amines,98*99 on the oxidation ofnumerous substituted phenols by tyrosinase,100 and on the oxidative in-activation of poison-ivy allergens by lac case. 101 A micro-spectr opho t o -91 J .Biol. Chem., 1948,174, 771.93 Mallette et al., ibid., 1948,16, 283.95 Ibid., 1948, 21, 181.97 Lerner et al., J . Biol. Chem., 1949, 178, 185; DuBuy et al., J . Nut. Canc. Inst.,Jackson and Kendal, Biochem. J . , 1949,44, 477 ; James et al., ibid., 1948,43, 626.92 Arch. Biochem., 1949,24, 49.94 Mallette and Dawson, ibid., 1949, 23, 29.g6 Biochem.J . , 1949, 44, 442.s8 J . Biol. Chem., 1950,184, 697. 1949, 9, 325.loo Beevers and James, ibid., p. 636; Cushing, J . Amer. Chem. Soc., 1948,70, 1184.lol Sizer, Arch. Biochem., 1949,20, 103348 BIOCHEMISTRY.metric method for catecholase assay using dichlorophenol-indophenol ashydrogen-acceptor is reported.lo2Dodds Io3 has partly purified the ascorbic acidoxidase of cucumber juice, and has compared its catalytic activity towards anumber of compounds with that of inorganic copper. Of the compoundstested, Cu++ ion only catalysed the oxidation of enediols, all at roughlysimilar rates ; the oxidase was more specific, only oxidising enediols with anadjacent carbonyl group and ring system, and these were attacked at widelydiffering rates.‘( Caerulosin,” a blue protein with a haemocyanin-typeabsorption spectrum, has a molecular weight of 151,000 and contains 8 copperatoms per molecule.104 It has been isolated from human and pig serum andaccounts for most of the serum-copper, but so far has not been found topossess any enzymic activity ; it is not identical with haemocuprein.Iron-porphyrin Proteins.-Catalase. (a) New catalases. Crystallinecatalases have now been isolated from nine different sources. They are allproteins of molecular weight ca. 230,000 containing 4 haematin groups permolecule ; these may all be protohaematin, or a varying proportion, in someliver catalases, may be “ bile pigment haematin ’’ (verdohaematin).Bonnichsen 105 crystallised catalase from horse erythrocytes and horse liver ;the former contained no bile pigment haematin while the latter containedca. 0.2y0 (18% of the total haematin).The protein components of the twoenzymes, however, were immunologically identical and had the same amino-acid contents ; they were immunologically distinct from human- bloodcatalase. Bonnichsen also isolated crystalline catalases from human erythro-c y t e ~ , ~ ~ ~ human liver,lo6 guinea-pig liver,lo7 and horse kidney.lo6 None ofthese contained any bile pigment haematin, which is therefore not an invari-able component of liver catalases ; Bonnichsen lo’ believes it to be an artefactarising during isolation. Catalases containing bile pigment haematin have anincreased end-absorption in the red spectral region, and a decreased absorp-tion at 405 mp., compared with catalases containing only protohaematin.lo7Bonnichsen’s preparation from human erythrocytes Io5 appeared to containonly 0.83% of total haematin, corresponding to only 3 haematin groups permolecule, but the same enzyme crystallised by a different method by Herbertand Pinsent 108 had 4 haematin groups per molecule.Herbert and Pinsent l9isolated a crystalline catalase from a bacterium, Micrococcus Zysodeikticus,which has an extremely high catalase content ( 1--2yo of the total dry weight).A two-phase partition method (cf. ‘( Methods ” section) was successfullyused in the isolation of this enzyme, which contained 4 protohaematins permolecule and no bile pigment haematin; its sedimentation constant in theultra-centrifuge log was the same as that of human-erythrocyte catalase, andits molecular weight is 230,000.It is an unfortunate fact that mostOther Coppek-proteins.(b) Activities and assay methods.lo2 Smith and Stotz, J .Biol. Chem., 1949,179,856.lo( Holmberg and Laurell, Acta Chem. Scand., 1948, 2, 550.lo5 Arch. Biochem., 1947, 12, 83.lo6 Acta Chem. Scand., 1947, 1, 114.lo8 Biochem. J . , 1048, 43, 203.loS Arch. Biochem., 1948,18,51.Ibid., 1948, 2, 561.Cecil and Ogston, ibid.,p. 205HERBERT : OXIDISING ENZYMES. 349catalase assays carried out before 1947 have been more or less unsatisfactory.The best known method is that of von Euler and Josephson,llo and catalasoactivities measured by this method have been expressed as ( ( Katalase-fahigkeit ” (Kat.f.). It involves exceedingly dilute enzyme solutions andthere is considerable enzyme destruction during the assay period, shown by aprogressive fall in the observed values of the first-order velocity constant, k ,whose initial value has t o be determined by an uncertain extrapolation tozero time.Furthermore, Kat.f. is defined in a confusing manner which hasled many workers to make mistakes in its meaning (cf. ref. 3, p. 411).Bonnichsen, Chance, and Theorell ll1 have devised a satisfactory assaymethod which depends simply on using high catalase concentrations ; allthe hydrogen peroxide is then decomposed before any appreciable enzymedestruction occurs, and the value of k remains constant throughout the reac-tion.These authors used a rapid titration method over short (ca. 15 scc.)time intervals; Lord Rothschild 112 used the same principles in a rapidmanometric method, employing a Barcroft- type manometer specially modi-fied for readings over short time intervals. Chance 113 used the Beckmanspectrophotometer to follow catalase activity by measuring the decrease inabsorption due to hydrogen peroxide at 215 mp. ; by modifying its electroniccircuits and coupling it t o a pen-recorder the instrument could be madeautomatic, and reaction half-times of 5 seconds or less could be accuratelymeasured.l14 By use of the rapid titration method, the kinetics of catalatichydrogen peroxide decomposition were found ll1 to follow the equation-dS/dt = kES, where S, E , are substrate and enzyme concentrations inmoles/l., t is time in seconds, and k is a characteristic velocity constant ofthe enzyme.The authors advocate the use of k, expressed in the properunits (1. mole-l sec.-l) instead of Kat.f. for the designation of catalase activi-ties, and there is much to be said for this. As the above equation implies,the decomposition of hydrogen peroxide is always a first-order reaction. Theusual (‘ saturation ” of the enzyme with change to a zero-order reaction at-high substrate concentrations is not observed with catalase, which has no(‘ Michaelis constant ” in the ordinary sense; these kinetics appear to beunique, and are a consequence of the unusual mode of action of the enzyme(see below). Other unusual properties of the enzyme are the very lowtemperature coefficient (activation energy only ca.1700 cals.) and the constantactivity over the pH range 3-5-8 ; the higher temperature coefficients andapparent pH optimum found by earlier workers, like the apparent (‘ Michaeliseonstant,” areal1 artefacts caused by enzyme destruction under unsuitableassayconditions. Feinstein 115 has described a catalase assay method using per-borate as substrate ; the catalase apparently attacks, not the perborate itself,but the hydrogen peroxide arising from its spontaneous decomposition. TheReporter sees no advantages in this method, which introduces a new variableinto the system and does not avoid the important factorof enzyme destruction.110 Annalen, 1927, 452, 158.*12 J .Ecp. Biol., 1950, 26, 399.114 Chance and Herbert, Biochem. J., 1950,46,40!2.1 1 1 Acta Chem. Scand., 1947, 1, 685.113 J . Biol. Chem., 1949, 179, 1299.115 J . Biol. Chem., 1949,180,1197350 BIOCHEMISTRY.When assayed by reliable methods, all erythrocyte, liver, and kidneycatalases containing 4 protohaematin groups per molecule have aboutthe same activity (k = 3.5 x lo7 l.mole-lsec.-l; Kat.f. = 60,000).19~107Liver catalases containing verdohaematin have lower activities per molecule,but roughly the same activity per molecule of profohmmatin. Since allthese catalases are of mammalian origin their equal activities are perhapsnot unexpected ; bacterial catalase, however, has ca.50% greater activity(k = 5.3 x lo7; Kat.f. = 97,000).19 It would be interesting to have datafor plant, invertebrate, and fungal catalases.(c) Kinetics and .mode of action. A series of important papers byChance 12, 13* 113- Il4* ll6? 117* 118 has greatly increased our knowledge of thisenzyme. Combination of the spectrophotometric rapid-flow apparatus 12with a platinum micro-electrode l3 allowed changes in oxygen, hydrogenperoxide, free catalase, and catalase-substrate complexes to be automaticallyrecorded during millisecond periods. The main conclusions are as follows :Catalase combines with hydrogen peroxide to form a green primary com-pound,12 “ catalase-H,O, complex I,” in which H,O, replaces the OH groupnormally attached to the catalase iron atoms (equation xvii).ComplexI then reacts with a second molecule of hydrogen peroxide with theformation of oxygen and the regeneration of free catalase (“ catalatic ”reaction, equation xviii) :L.,_ Fe*OH + H,O, ----- Fe*OOH + H,O . . . (xvii)Fe-OOH + H,O, -$ Fe-OH + H,O + 0,. . (xviii)k:kThe overall rate of destruction of hydrogen peroxide is proportional tothe steady-state concentration of complex I. The fraction of the totalenzyme present as complex I depends only on the velocity constants of itsformation (k,) and breakdown (k4), and is independent of the hydrogenperoxide concentration; hence the unique kinetics of catalase and the lackof any Michaelis saturation effect (see previous section). For erythrocytecatalase the steady-state ratio Fe*ooH ~ is almost exactly 0.25, i.e., on the total Feaverage one of the four haematins in each molecule is bound to peroxide.ll6This is a purely statistical effect, however, for with bacterial catalase 1.6 ofthe 4 haematins are bound to peroxide in the steady state.l14 This isbecause reaction (xvii) is faster with the bacterial enzyme ; hence the steady-state concentration of complex I is higher, and the overall activity is greater.Results with the bacterial enzyme 114 support the view that all four haematinsreact independently, disproving an earlier suggestion 113 that combination of asingle haematin with hydrogen peroxide gave the molecule “ special proper-ties.” Bacterial and mammalian catalases display other interesting differ-ences which suggest that the haematins of the former are more deeply“ buried ” in the protein molecule.114116 Chance, J .Biol. Chem., 1949, 179, 1311.117 Idem, ibid., p. 1331,1341 ; 180,865,947; 1950,182,643. 118 Idem,ibid.,p. 649HERBERT : OXIDISINC ENZYMES. 35 1The Fe*OOH-complex I will also react with a number of compounds 11*including methanol, ethanol, propanol, formaldehyde, formate, and nitrite,llgwhich are oxidised (“ peroxidatic ” reaction) :Fe-OOH + CH,*CH,-OH -% Fe-OH + CH,*CHO + H,O (xix)The catalatic reaction (xviii) is 104-105 times as fast as the peroxidaticreaction (xix); hence oxidation of ethanol, etc., only occurs when theconcentration of free hydrogen peroxide is very small. This can be achievedby continuous generation of hydrogen peroxide in very low concentration byglucose oxidase-glucose-0, or similar systems,l14 when “ coupled oxidation ”of ethanol occurs, as discovered by Keilin.Chance’s work provides a quanti-tative explanation of coupled oxidations, and emphasises the fundamentalsimilarity of catalatic and peroxidatic action. No valency changes of theiron atoms are postulated in his theory of catalase action. In experimentswith methaemoglobin as a “ catalase model,” however, Keilin and Hartree 120showed that initial formation of a MetHb*OOH complex is followed by itsreduction to haemoglobin during oxygen evolution ; they suggest a similarreduction of catalase-Fe in the catalatic reaction (xviii).If complex I remains for long in contact with hydrogen peroxide it isslowly transformed to a red “ complex 11,” which is catalytically inactive.17Formation of complex I1 is responsible for the inactivation of catalaseunder the conditions of Kat.f.determinations l3 (see previous section).George,121 using a manometric technique, describes an initial burst ofoxygen-evolution (a-activity) soon falling to an almost steady state (p-activity). This also may be caused by formation of a secondary complex.The inactivation of catalase by ascorbic acid is almost certainly due toformation of complex IT, produced by the hydrogen peroxide arising fromautoxidation of ascorbic acid.Catalase combines similarly with methyl and ethyl hydrogen peroxidesto form green primary complexes formulated as Fe*OOR (R = CH, or C,H,),which slowly change to red secondary complexes.117 The primary complexesreact with hydrogen peroxide or alcohols in the same way as the primaryFeoOOH complex : 117Fe*OOR + H,O, --+ Fe*OH + R*OH .. . . (cf. xviii)Fe*OOR + CH,*CH,OH + Fe*OH + R*OH + CH,*CHO (cf. xix)Chance 113 has studied the combination of catalase with cyanide;inhibitions by azide and hydroxylamine were studied by Foulkes andLemberg ,123Peroxidaim.-The kinetics of horse-radish peroxidase have been studiedin a series of papers by Chance, 124-126 and are rather similar to those ofllg Heppel and Porterfield, J . Biol. Chem., 1949,178, 549.120 Nature, 1950, 166, 513.lZ2 Foulkes and Lemberg, Austral. J . Exp. Biol. Med. Sci., 1948,26,307.lZs Idem, Enzymologia, 1949, 13, 302.la4 Arch. Biochem., 1949, 21, 416; 22, 224; 24, 389, 410.lZ5 Chance, J .Amer. Ch,em. Soc., 1950,72, 1577.lZ1 Biochem. J . , 1949, 44, 197.la6 Idem, Fed. Proc., 1950, 9, 160352 BIOCHEMISTRY.catalase ; the sequence of reactions is as follows (Fe-OH represents the ironatom of peroxidase with its OH group, and A*H, an acceptor * such asascorbic acid or pyrogallol) :Fe-OH + H,O, 2% Fe*OOH (I) + H20 . (xx)Fe*OOH (I) -% Fe*OOH (11) . . . (xxi)Fe*OOH (11) + A*H2 -2% Fe*OH + H20 + A . (xxii)The primary complex I is green and spectrally similar to catalase complexI. It rapidly changes to a pale red complex TI, which reacts with the acceptor,oxidising it and regenerating free peroxidase. Similar complexes Fe*OOR (Iand 11) are formed with alkyl hydrogen peroxides, the latter reacting similarly,but more slowly, with acceptors as in (xxii). Peroxidase complex 11 thusdoes not correspond to catalase complex 11 (which is inactive) and theirspectra are different.On long contact with hydrogen peroxide in absence ofan acceptor, I1 slowly changes to an inactive complex 111, which is brightred and has a similar spectrum to the inactive catalase complex 11. Onedetail which remains unexplained is that the change I --+ I1 (xxi), thougha first-order reaction, is accelerated by acceptors. Lactoperoxidase behavesin an essentially similar manner,125 which is interesting since it has a differentprosthetic group (an unidentified green haematin which Lemberg thinks maybe monoazahaematin). Another interesting finding 126 is that both theseperoxidases and also myeloperoxidase will oxidise ferro-cytochrome-c a tabout one-tenth of the rate of its oxidation by the yeast enzyme cytochrome-cperoxidase ; re-investigation of the specificity of the latter enzyme seemscalled for,Theorell et ~ 1 .1 2 7 have extended previous work on " artificial " peroxidasesfrom horse-radish peroxidase, made by splitting off its protohaematingroups and coupling the apo-enzyme with other iron-porphyrins. Several ofthe products are enzymically active, including the compound with mono-azahaematin. The prosthetic group of myeloperoxidase is a green haematin,different from that of lactoperoxidase. According to Lemberg and Purdom 12*i t closely resembles the as yet unidentified prosthetic group of choleglobin ;it is definitely not verdohaematin, and use of the name " verdoperoxidase ''for this enzyme should be abandoned.Smith et ~ 1 . l ~ ~ have developed acolorimetric assay method for peroxidase using dichlorphenol-indophenol.Ettori l30 has converted the pyrogallol method into a manometric procedureby measuring carbon dioxide output instead of purpurogallin formation.Cytochrome-c.-Cytochrome-c preparations, obtained from beef, pig, andchicken hearts by the trichloroacetic acid method, contained 0-37-38% of12' Theorell and Maebly, Acta Chem. Scund., 1950, 4, 422.lZ8 Abstr. 1st Intern. Congr. Biochem., 1949, p. 348.lag Smith, Robinson, and Stotz, J . Biol. Chem., 1949,179, 881.130 Biochem. J . , 1949, 44, 35.* In Chance's nomenclature which is used here, the " substrate " is the molecule(HOOK or ROOH) which combines with the enzyme, and the " acceptor " is themolecule (AH,) which reacts with the enzyme-substrate compoundHERBERT : OXIDISING ENZYMES.353iron and showed on electrophoresis a second component which was colourless,iron-free, and enzymically inactive.13f Since it migrates independentlyof cytochrome-c over the pH range 3.9-1 1.9, it is presumably an impurity andnot a dissociated part of the cytochrome-c molecule. The iron contents ofthe pure cytochrome-c components varied from 0.42 to 0.45 yo for the differentspecies ; these variations are considered significant .132 Horse cytochrome-chad an initial iron content of 0.45% and was homogeneous electrophoretically.The pure cytochromes of the different species had slightly different mobilitiesand could be separated by electrophoresis.Paleus and Neilands 23 have alsoseparated a colourless impurity from cytochrome-c on an ion-exchangecolumn. Paul 133 has made observations on the stability of cytochrome-con drying and at extreme pH values, and describes a succinoxidase prepar-ation of low cytochrome-c content, suitable for the biological assay ofcytochrome-c.Haematin-c is attached to its protein by thio-ether linkages with cysteine,and is not split off by treatment with hydrochloric acid and acetone; itmay be split, however, by treating cytochrome-c with silver ~u1phate.l~~This introduces a hydroxyl group into the side-chain, giving a haemato-haematin-c which may be converted into a haematoporphyrin-c ; the exactstructure of these has not yet been determined.Tsou 135 has obtained amodified cytochrome-c by peptic digestion, which does not remove theprosthetic group.Ferricytochrome-c combines with azide in a 1 : 1 molecular ratio to forma spectroscopically distinct complex; the effect of pH on the dissociationconstant of the complex shows that combination takes place with azideanion.136 Unlike the cyanide-ferricytochrome-c complex, which is formedvery slowly but has a low dissociation constant, the azide complex is formedrapidly, but the dissociation constant is so high ( 0 . 1 5 ~ . ) that negligibleamounts of it are formed with the low azide concentrations used for mostinhibition studies.Two new studies are reported on the oxidation-reduction potential ofcytochrome-c and its variation with pH; these are not in complete agree-ment with each other, or with accepted views on the dissociating groups ofcytochr~me-c.~~** 139 Reference has already been made to the reduction ofcytochrome-c by xanthine oxidase 88 and its oxidation by peroxidases; 126it is not known whether these reactions are of physiological significance.New C*chromes.-A new pigment “cytochrome-e” with a band at550 mp.(between the b and the c bands) has been observed by Keilin andHartree 139 in heart-muscle succinoxidase preparations ; owing to its lowconcentration it only becomes visible on cooling to liquid-air temperatures.131 Tirt and Reiss, J.Biol. Chem., 1950, 182, 385.13* Idern, ibid., p. 397.134 Paul, ibid., 1949, 3, 1178.13* Horecker and Stannard, J. BioE. Chem., 1948,172, 589.13’ Paul, Arch. Biochem., 1947,12, 441.13* Rodkey and Ball, J. Biol. Chem., 1950, 182, 17.139 Keilin and Hartree, Nature, 1949, 164, 254.133 Acta Chem. Scand., 1948, 2, 531, 557.135 Nature, 1949, 164, 1134.REP .-VOL . XLVII . 354 BIOCHEMISTRY.It is widely distributed in animal tissues, yeasts, and bacteria. The " cyto-chrome-b, " band which seemingly replaces bands b and c in certain bacteria isresolved a t liquid-air temperatures into b, c, and e bands, with the lastgreatly predominating. This cytochrome has not yet been obtained insolution, and its function is still unknown. Hill 140 and Davenport 141 haveobtained in soluble form, and purified to a considerable extent, a new pig-ment " cytochrome-f " from green plants.Its oxidation-reduction potentialis considerably higher than that of other cytochromes, so much so that it israther difficult to obtain it in the oxidised form. Its possible connection withphotosynthetic oxygen-production is an intriguing possibility.Electron Transport between Substrates and Oxygen.-The scheme belowattempts to summarise present views on this subject, the arrows indicatingpaths of electron transport ; the detailed pathways are discussed in turn.succinate substrate substrateL..--._ ----........--.-.--- TPN-succinic DPN TPN dehydrogenase; $ E;irogenase + dehydrogenase4 I 1 _ - - - - - - _ .J... ____.-_-.._._.__._-...___...__-.. _-----------. J.TPN-cytochrome-creductase1 cytochrome-b- - - - - - - - _.____.__.--..--- --._--_- .______---- __----..--._.-, factorI + Ic ;':---------- .1: cytochrome-cmethylene-blue: cytochrome-a ;14,i cytochrome-a, i0 2In the above scheme, thecomponents within the dotted line are all present in bound form (attachedto insoluble submicroscopic particles) in standard heart-muscle succinoxidasepreparations. So far only cytochrome-c, diaphorase, and TPN-cytochrome-creductase have been separated from the particles in soluble form and purified.Numerous claims to have brought cytochrome oxidase into true solution,and separated it into different factors are refuted in two important papersby Keilin and Hartree.142* 143 Most of such " purified " preparations have alower Qo, than the starting-material, and reported activation by variousComponents of the Xuccinoxidase Sydem.Hill, SOC.Exp. Biol. Symp. Carbon Dioxide Fixation, 1950.Ibid., 1949, 44, 205.1 4 1 Davenport, Nature, in the press.142 Biochern. J., 1947, 41, 500, 502HERBERT : OXIDISING ENZYMES. 355factors could be reproduced by adding indifferent proteins, or by precipitat-ing gelatinous calcium or aluminium phosphates in the preparation. (Re-ported “activation” by these ions only occurs in a phosphate buffer.)Preparations partly inactivated by freezing or drying could be reactivatedby the same means, a n t addition of serum proteins protects the preparationsagainst inactivation on drying.l@ It is considered that the catalysts in thecolloidal particles, as in the intact cells, are more or less rigidly held togetherin a framework or mosaic which ensures their mutual accessibility.Forexample, endogenous cytochrome-c in the preparations is reduced muchfaster by succinate than is added cytochrome-~,~~~ but it is reduced moreslowly by chemical reducing agents such as ascorbic acid; 14p i t would seemto be “ built in ” to the colloidal framework. Disruption of the mosaiclowers the overall activity without necessarily destroying or removing anyindividual catalyst ; flocculent precipitates of foreign materials can reactivateby providing new surfaces for re-orientation. Some such “ mosaic ”concept is now fairly generally accepted, and has been further extendedby Green to his “ cyclophorase ” preparations (see below).Kidneysuccinoxidase is essentially similar to the heart-muscle preparation ; it wasformerly thought to have a different cytochrome system (“ cytochrome-b, ”in place of b and c) but this was not ~0nfirmed.l~~Electron Transport from Succinate to Cytochrome-c. It is well known thatcytochrome-c is 8 link between the succinic dehydrogenase and cytochromeoxidase systems. Important contributions by Slater 145* 1469 14’ indicatethat : (i) cytochrome-b is an intermediary carrier between succinate andcytochrome-c, and between succinate and dyes such as methylene blue; (ii)a new respiratory catalyst, inactivated by “ BAL ” (2 : S-dimercaptopro-panol) is an intermediary carrier between cytochrome-b and cytochrome-c,but not between cytochrome-b and methylene blue; this is the “ BAL-labile factor ” in the scheme on p. 354.An unknown haematin compoundaccounts for m. 20% of the total haematin of heart-muscle preparations,145and this is destroyed by BAL treatment.14’ It is tempting to identify thiscompound both with Slater’s factor and with Keilin and Hartree’s newly-discovered cyto~hrome-e,~~~ but there is no real evidence for this so far.Studies on diphtherial and ox-heart succinoxidase preparations ledPappenheimer and Hendee 14* to suggest that succinic dehydrogenase andcytochrome-b are identical. There appears to be no evidence contradictingthis, and Slater 146 found a correlation between the dehydrogenase and cyto-chrome-b contents of different heart and kidney preparations ; actual proofof identity is, however, lacking.* Wainio et ~1.14~ claim the preparation ofcytochrome-b in “ soluble ” form and separated from other components ofu4 Borei, Biochem.J., 1950, 47, 227. 145 Ibid., 1949, 45, 1.14’ Ibid., p. 14. Ibid., p. 8.Pappenheimer and Hendee, J . Biol. Chm., 1949,180,597.Eichel, Wainio, and Person, ibid., 1950, 183, 89.* [Added in proof.] Tsou (Biochem. J., 1951, in the press) has found that potassiumcyanide slowly inactivates succinic dehydrogenase without apparently affecting cyto-chrome-b, and concludes that they are not identical356 BIOCHEMISTRY.the cytochrome system by fractionation with deoxycholate, but they give nodata on its succinic dehydrogenase activity.(The criterion of “ solubility ”adopted is non-sedimentation in 1 hour a t 25,000 g., which does not seemadequate to the Reporter to prove true solubility, in view of the well-knownpeptizing action of bile salts.)Warburg (ref. 6, p.70) describes improved measurements showing that the cytochrome-c turn-over in living yeast cells is fast enough to account for ca, 95% of the totaloxygen uptake. In other words, effectively all electron transport betweensubstrates and oxygen passes over cytochrome-c. The electron-transferroute for TPN systems has been made fairly clear by the isolation of TPN-cytochrome-c reductase (see scheme on p. 354). The route for DPN systemsis not so well understood.Straub’s diaphorase oxidises DPNH but does notreduce cytochrome-c. Either (i) it plays no part in electron transfer fromDPPU’H to cytochrome-c, or (ii) it is a transformation artefact derived from ahypothetical “ DPN-cytochrome-c reductase ” similar to 1 the TPN-cytochrome-c reductase, its ability to reduce cytochrome-c having beendestroyed in the isolation process, or (iii) it reacts with cytochrome-c via anintermediary electron-carrier. Slater 27* 150 brings evidence to show thatalternative (iii) is correct, and that the intermediary carrier involved is thesame “ BAL-labile factor ” as that involved in the reduction of cytochrome-cby cytochrome-b (see scheme on p. 354); in other words, the hypothetical“ DPN-cytochrome-c reductase ” is equated with diaphorase-plus-factor.He also confirms that aerobic oxidation of DPNH by heart-muscle prepar-ations proceeds via cytochromes c, a, and a,, and shows that the rate ofoxidation of DPNH by diaphorase in the preparation (with methylene-blueas H-acceptor) is 3-4 times as fast as the overall oxidation of DPNH byoxygen.A brief note by Heppel lS1 describes the preparation of (‘ DPN-cyto-chrome-c reductase ” of ox liver in soluble form, removal from insolubleparticles being effected by digestion with steapsin (cf.ref. 87). Hogeboomand Schneider have also obtained soluble preparations by ultrasonic treatmentof rat-liver rnit0~hondria.l~~ Until these preparations have been purified it isimpossible to say whether or not this work fits Slater’s views.Electron Transport between Cytochrome-c and Oxygen. Slater 27 describesexperiments supporting the scheme on p.354, and confirming Keilin’sobservations on cytochrome-a, and its CO-compound and the probableidentity of a, with cytochrome oxidase; he also shows that cytochrome-bis not concerned in this part of the system. Wainio et aZ.1499153 claim thepreparation of cytochrome oxidase in “ soluble ” form by fractionation ofheart-muscle preparations with deoxycholate. The reduced pigment hadbands at 440 and 601 mp., and was separated almost completely from cyto-chrome-b and -c. These workers do not admit the existence of a separatecytochrome-a ; it appears to the Reporter, however, that the pigment whoseElectron Transport between Coenzymes and Oxygek150 Biochem.J . , 1950,46,499.152 Nature, 1950,166, 302.151 Fed. Proc., 1949, 8, 205.153 J . Biol. Chrm., 1949,173, 145HERBERT : OXIDISING ENZYMES. 357. spectra they describe could in fact be a mixture of cytochrome-a and-a3. Hogeboom and Schneider 152 obtained a small fraction of the cyto-chrome oxidase of rat liver mitochondria in soluble form by ultrasonic treat-ment. In their preparations, which are not sedimented a t 148,000 g., theoxidase would appear to be in true solution; the QO,, however, is only abouthalf that of the mitochondria.Rawlinson and Hale 154 found that the haematins extracted with acidacetone from Corynebact. diphtheriae and from heart-muscle preparationsconsisted in both cases of protohaematin (derived from cytochrome-b),and " haematin-a " (probably derived from cytochrome-a + -a3).Thelatter is a dichroic haem and contains a t least one aldehyde group. Lemberget ~ 1 . l ~ ~ compared the properties of haematin-a and porphyrin-a with varioussynthetic porphyrin derivatives ; the one showing closest resemblance wasoxyrhodoporphyrin.Stannard and Horecker 156 studied the inhibition of cytochrome oxidaseby cyanide and azide a t different pH values, and conclude that the oxidasecombines exclusively with undissociated hydrocyanic and hydrazoic acids.Ferricytochrome-c, methaemoglobin, and metmyoglobin on the other handcombine exclusively with the cyanide and azide ions. These workers deter-mine cytochrome-oxidase activity by following the aerobic oxidation ofreduced cytochrome-c spectrophotometrically.Smith and Stotz 15' describea colorimetric method with dichlorphenol-indophenol, while Slater 158 usesa manometric method with ascorbic acid as substratb.Coupling of Phosphorylution with Oxidations. During the oxidation ofmany intermediary metabolites by tissue homogenates and particulatepreparations in the presence of inorganic phosphate and a, phosphate acceptorsuch as AMP or ADP, about three atoms of inorganic phosphate may beesterified for each atom of oxygen consumed. The esterified phosphateappears as ATP, if precautions are taken to prevent its breakdown. Themechanism of this process remains largely obscure. I n Green's " cyclo-phorase " preparations, not only is orthophosphate esterified during theoxidation of all intermediary compounds of the citric acid cycle, but thepresence of phosphate increases their rates of 0xidati0n.l~~ In the absenceof an acceptor, orthophosphate disappearing is transformed into inorganicpyrophosphate. It is suggested that phosphorylation is linked to theprimary oxidation of the substrates and results in the formation of reducedcoenzyme pyrophosphates, which can either transfer phosphate to anacceptor system (such as glucose + hexokinase) via adenylic acid, or in itsabsence break down to inorganic pyrophosphate.Studies with 32P showedthat cyclophorase contains a very labile form of phosphate (" gel P "),possibly identical with the labile coenzyme pyrophosphates.160Friedkin and Lehninger,lG1 using rat-liver mitochondria, found thatlS4 Biochem.J . , 1949, 45, 247. ls5 Abstr. 1st Intern. Congr. Biochern., 1 9 4 9 , ~ . 351.lS6 J . BioLChem., 1948,172,599. 15' Ibid., 1949, 179, 891.Biochem. J . , 1949, 44, 305. 159 Cross et al., J . Biol. Chem., 1949, 177, 655.l 6 0 Green et al., Arch. Biochem., 1949, 24, 359; Albaum, ibid., p. 375; Tepley, ibid.,161 J . Biol. Chem., 1949,178, 611. p. 383358 BIOCHEMISTRY.esterification of orthophosphate into adenine nucleotides accompanies theaerobic oxidation of DPNH ; studies of the oxidation of P-hydroxybutyrate 162suggested that phosphate uptake is coupled with oxidation of DPNH viathe cytochrome system, and not with primary oxidation of the substrate assuggested by Green. In the Reporter’s view it is still too early to decidebetween these possibilities, which may not be mutually exclusive. Thelocation of the phosphorylation reaction has been further narrowed bySlater’s recent work,163 in which cytochrome-c was used as h a 1 acceptor ofelectrons from the substrate, functioning of cytochrome oxidase beingprevented by cyanide or anaerobiosis.Oxidation of cc-ketoglutarate by aheart-muscle preparation gave about the same phosphate uptake (P : 0>2 : 1) whether oxygen or cytochrome-c was the final electron-acceptor.Enzyme Complexes and Cell StrUctnre.-Keilin’s concept of the succin-oxidase system as a colloidal complex or mosaic of enzymes has already beenmentioned. Recent work has extended this concept to a wider range ofenzymes, while parallel cytochemical studies have located these enzymecomplexes in definite structural elements of the cell, notably the mitochondria.Methodologically, the subject has been approached from two angles.Someworkers (e.g., Green, Lehninger) have set out to isolate particular enzymecomplexes in a functionally unaltered state, while others (e,g., Claude, Dounce,Schneider) have set out to isolate cell structures such as nuclei or mito-chondria in a morphologically unaltered state. These two approaches haveon the whole given fairly similar results.Green (ref. 2, chapter X) has reviewed hisresearches on this system. Cyclophorase preparations l6* are obtained fromrabbit kidney or liver by differential centrifugation after the tissues have beenminced in 0.9 yo potassium chloride solution in a Waring blendor ; the productis a viscous gel containing much nucleic acid.Fresh kidney preparationswithout any additions but phosphate buffer catalyse the complete oxidationto carbon dioxide and water of (a) all the substrates of the citric acid cycle,(b) the fatty acids from acetic to tridecanoic and derivatives such asacetoacetic acid 165 and (c) certain amino-acids.ls6 Their activity rapidlyfalls but is restored for group ( a ) substrates by adding Mgf+ and AMP orATP; group (b) and (c) substrates require in addition a small amount of agroup (a) substrate to be added as a co-oxidant or “ sparker,” indicating thattheir oxidation is linked with the citric acid cycle.167The individual dehydrogenases of cyclophorase preparations (e .g., lactic,malic, isocitric) have different properties from the previously known solubleforms of these enzymes.They do not require the addition of DPN or TPN,which are present in cyclophorase preparations in firmly bound form, notremoved by washing; also their functioning requires phosphate and isaccompanied by phosphate esterification. In Green’s view, the dehydro-162 Friedkin and Lehninger, J . Biol. Chem., 1949,178,625. lES Nature, 1950,166,982.16* Green, Loomis, and Auerbach, J. Biol. Chem., 1948,172, 399.1 e 5 Grafflin and Green, ibid., 1948, 176, 95; Atchley, ibid., p. 123.1 e 6 Taggart and Krakur, ibid., 1949,177,641 ; Still, Buell, and Green, Arch. Biochem.,le7 Knox, Noyce, and Auerbach, J .Biol. Chem., 1948, 176, 117.The Cyclophorase System.1950, 26, 406, 413HERBERT : OXIDISING ENZYMES. 359genases exist in cyclophorase preparations as “ pyridinoproteins ” withfirmly bound DPN or TPN prosthetic groups, and in this state possesscertain firoperties that are lacking in the soluble dehydrogenases.168 Onsubjecting cyclophoraRe preparations to various treatments the bound co-enzymes are split off,168 and such preparations need to be supplemented withDPN or TPN, which untreated cyclophorase does not, and the dehydrogenasesthen behave as dissociating pyridinoproteins. On more drastic treatmentmost of the dehydrogenases are released in soluble form, and are thenapparently identical with the known soluble dehydrogenases. To whatextent the classical dehydrogenases must be regarded as transformationartefacts is not yet clear, but is obviously of great importance.In thisconnection the fact that glyceraldehyde-3 phosphate dehydrogenase has beencrystallised in the form of a protein containing firmly bound DPN 46 is ofconsiderable interest. Green’s conception is that all the enzymes in thecyclophorase system, as in the intact cell, occur as an organised mosaic ofproteins with non-dissociating prosthetic groups.Intracelluhr Locution of Respiratory Enzymes. This field has been fairlyrecently reviewed by Schneider (ref. 2, Chapter XIV) and by Claude; 169only a few selected topics will therefore be discussed. The technique thathas led to’the major advances of the last five years or so is that of isolatingparticulate fractions from disrupted cells by differential centrifugation.Cells are broken mechanically, usually with a Potter-Elvej hem homogeniseror a Waring blendor ; the resulting suspension can then be separated by theapplication of increasing centrifugal fields into successive fractions consistingof nuclei, mitochondria, submicroscopic particles or “ microsomes,” and asupernatant liquid containing the soluble cell components.The initial celldisruption must not be too violent or fragments of broken-up nuclei andmitochondria will appear in other fractions. The suspending fluid is alsoimportant. Hogeboom et uZ.l7O found that the use of salt solutions causesswelling and agglutination of mitochondria which then appear as “ largegranules ” in the nuclear fraction.If the tissue is homogenised in 30%sucrose solution, however, the mitochondria retain their normal morphologyand vital staining characteristics and do not agglutinate. In isotonic (8.5%)sucrose the mitochondria appear spherical instead of rod-shaped, but theirother properties remain unchanged, and this medium is more convenient.Analysis of such fractions shows that the glycolytic enzymes appear almostentirely in the soluble form in the final supernatant liquid,170 while the mainoxidation systems are concentrated in the mitochondria and to a lesser extentin the “ microsomes,” only m. 5% appearing in the nuclei. Thus, up to 80%of the total succinoxidase appears in the mitochondria,170* 171 and also mostof the enzyme systems responsible for the citric acid cycle 172* 173 and for the168 Huennekens and Green, Arch.Biochem., 1950, 27, 418, 428.169 Adv. Protein Ghem., 1949, 5, 423.170 Hogeboom, Schneider, and Pallade, J . Biol. Chem., 1948, 172, 619.171 LePage and Schneider, ibid., 1948, 176, 1021.Kennedy and Lehninger, ibid., 1948,179, 957.173 Schneider and Potter, ibid., 1949, 177, 893360 BIOCHEMISTRY.oxidation of fatty 17* Cytochrome oxidase,171 cytochrorne-c,176DPN-cytochrome-c reductase, 176 and TPN-cytochrome-c reductase 1 7 7 havealso been found to be concentrated in the mitochondria, which seem indeedto be the main seat of intracellular oxidations. Ribonucleic acid is found inall the fractions, but deoxyribonucleic acid appears only in the nuclei.Green’s “ cyclophorase ” preparations are not cytologically homogeneous,containing a considerable proportion of nuclei and nuclear fragments, andsome “ microsomes.” However, Harman 178 found that on fractionatingsuch preparations by differential centrifugation in sucrose solutions, almostall the cyclophorase activity was obtained in the mitochondrial fraction.The ‘‘ microsomes ” contained succinoxidase and most of the individualenzymes of the cyclophorase system, but could not bring about completeoxidation of citric acid cycle intermediates or esterification of inorganicphosphate.Most workers have found that enzyme systems concentrated inthe mitochondria are found also to some extent in the “ microsomes ” ; itseems possible that an appreciable proportion of the “ microsomes ” are infact disintegrated mitochondria. Prolonged homogenising in the Waringblendor leads to break-up of mitochondrial or cyclophorase preparations, andtransfer of oxidase activity to the “ microsome ” fraction and the super-natant At the same time the prosthetic groups of the non-dissociating dehydrogenases of the cyclophorase system are split off, so thatthey appear as ordinary dissociating dehydr0gena~es.l~~The manner in which the various enzyme systems are integrated in themitochondrial structure is unknown.Cytological studies 180 have apparentlyshown mitochondria to possess well-defined membranes, and a large portionof their total nitrogen represents soluble proteins that are released when themembranes are disrupted by ultrasonic treatment.ls2 Green,e.g.s 168 however,does not accept this view, and Harman 178 believes that mitochondria have agel-like structure, without a limiting membrane.The point is of considerableimportance in connection with Green’s conception of the cyclophorasedehydrogenases as “ non-dissociating pyridino-proteins,” but it cannot beconsidered settled.D. H.6. HORMONES OF THE ANTERIOR-PXTUI!l’ARY GLAND.This subject has not been reviewed in these Reports since 1940, so it mayThreeSix well-authenticated hormones are known to be secreted by the anterior-often be necessary to include work done as much as ten years ago.comprehensive reviews have more recently appeared elsewhere. l* 2r174 Schneider, J.Biol. Chem., 1948, 176, 259.1 7 5 Schneider, Claude, and Hogeboom, ibid., 1948,172, 451.176 Hogeboom, ibid., 1949, 177, 847.177 Hogeboom and Schneider, ibid., 1950,186, 417.178 Harman, Exp. Cell Research, 1950,1,382,394.180 Dalton et al., J. Nat. Cancer Inst., 1949, 9, 439.178 Still and Kaplan, ibid., p. 403.White, Phyeiol. Reviews, 1946, 26, 574.3 Li and Evans, in “The Hormones,” Vol. I, 1948 (G. Pincus and K. V.Chow, Adv. Protein Chem., 1944,1, 163.Thimann ed.), New York, Chap. 14FOLLEY : HORMONES OF THE ANTERIOR-PITUITARY GLAND. 361pituitary (A.P.) gland; there may be others but none has yet been satis-factorily characterised. The known A.P. hormones can conveniently beclassified as gonad-stimulating hormones or gonadotrophins, and “ meta-bolic ” hormones.The first group comprises the follicle-stimulatinghormone (FSH, thylakentrin), the luteinising or interstitial-cell-stimulatinghormone (LH, ICSH, metakentrin), and the lactogenic hormone (prolactin,mammotrophin, luteotrophin) ; the second comprises the growth hormone(GH, somatotrophin), the adrenocorticotrophic hormone or hormone complex(ACTH), and thyrotrophin. All appear to be proteins or protein derivatives ;prolactin, ICSH, GH, and possibly FSH have now been prepared fromanterior-pituitary tissue as proteins satisfying the currently accepted physico-chemical criteria of molecular homogeneity and in addition exhibiting ahigh degree of biological purity.Follicle-stimulating Hormone (FSH).-Several reviews of the chemistry ofFSH have been p~blished.l-~Puri3cation. FSH, together with ICSH, may easily be extracted fromanterior-pituitary tissue, fresh or acetone-dried, by dilute acid or alkali,saline or aqueous alcohol.The richest sources are human or horse pituitaries,but the most abundant and therefore the best and usual sources in practiceare sheep and pig glands ; the FSH activity of ox glands is low. The chiefproblem has been to free FSH from ICSH and this has mostly been accom-plished by utilising the greater solubility of FSH under most conditions.Thus it is the only known A.P. protein-hormone soluble in half-saturatedammonium sulphate solution. Only recently has FSH been obtainedas a pure protein ; earlier procedures gave preparations which, thoughsensibly free from ICSH and perhaps other A.P.hormones, containedinactive contaminants or, though relatively highly purified, were con-taminated with traces of ICSH.Procedures based on ammonium sulphate fractionation were used byFevold who first employed aqueous pyridine extraction of the tissuefollowed by adsorption of the gonadotrophins on benzoic acid, and later 7extraction with dilute aqueous ammonia. Fraenkel-Conrat et aL8 extractedacetone-dried sheep pituitaries with 40 yo alcohol and precipitated thegonadotrophins by increasing the alcohol concentration. Further purific-ation by ammonium sulphate fractionation gave a product showing someICSH contamination. Greep et aL9 obtained a product free from ICSH fromfresh pig pituitaries by a method based on the fact that FSH is soluble inacetate buffer (pH 444) containing 20.5y0 of sodium sulphate while ICSHis insoluble.This preparation however proved to be molecularly poly-disperse.1° The fact that FSH is more resistant to tryptic digestion thanChow, in “The Chemistry and Physiology of Hormones,” Amer. Assoc. Adv.Sci., 1944, p. 26.Endocrinology, 1939, 24, 435.Fevold, Lee, Hisaw, and Cohn, ibid., 1940, 26, 999.Li, Vitaminsand Hormones, 1949, 7 , 224.* H. Fraenkel-Conrat, Simpson, and Evans, Ann. Fac. Med., Montevideo, 1940,25, 617. Greep, van Dyke, and Chow, J. Biol. Chem., 1940,133,289.lo Chow, Ann. N . Y . Acad. Xci., 1943,43, 309362 BIOCHEMISTRY.ICSH has been utilised l1 to obtain preparations of FSH often apparentlyfree from ICSH ; these preparations contain inactive contaminants butare reported to be free from a toxic substance, present in earlier preparations l2and causing local reactions, and also to be free from lactogenic and thyro-trophic hormones.Li et ~ 1 .~ 1 ~ ~ have prepared FSH behaving as a single substance in theultra-centrifuge and on electrophoresis (solubility studies were not done)by extraction of sheep pituitaries with calcium hydroxide solution, followedby alternate fractionation with ammonium sulphate and cold aqueousethanol. This preparation in a total dose of 0-05 mg. initiated ovarianfollicular development in hypophysectomised rats, but in a total dose of2.0 mg. over four days showed no ICSH, ACTH, GH, or thyrotrophicactivities. Koenig and King l4 have recently described a method ofextracting FSH (with ICSH and prolactin) from sheep pituitaries, novelin that the extraction is carried out with solutions of high ionic strengtha t the pH of minimum solubility of the gonadotrophins.The method issuit able for large- s cale use.Chemical Properties. So far most data have been obtained with(slightly) impure preparations ; amplification of the few results for thepure protein The isoelectric point of puresheep hormone has been given as pH 4.5; its molecular weight estimatedfrom a single ultra-centrifugal run is 70,000.5 The hormone is in generalvery soluble in aqueous solutions (e.g., ref. 12) and is not precipitated by0.6 saturated ammonium sulphate solution or by 2.5 yo trichloroaceticacid.Since it contains considerable amounts of carbohydrate (sheep :carbohydrate by orcinol method 10-13y0,16 glucose by carbazole method22.0-24~6%,~~ hexosamine 8% ; l5 pig : mannose 46%, hexosamine4.4%16), from which the activity has hitherto proved inseparable andmoreover is destroyed by amy1ase,l7 the hormone is considered to be aglycoprotein. Lower values than those quoted above have been given 5for the carbohydrate content of the pure sheep hormone as follows : hexose(orcinol method) 1.3% ; hexosamine 006%.The activity is relatively resistant (as compared with ICSH) to digestionby commercipl trypsin 12*18 so long as no more than 48% of protein isdigested, which may mean that as with ACTH the activity is a propertyof a portion only of the protein molecule and suggests the possibility ofsplitting off an active carbohydrate-peptide.Indeed, Li 19 has recentlyreported the preparation of a biologically active and totally dialysablewill be awaited with interest.11 McShan and Meyer, Proc. SOC. Exp. Biol., 1946, 61, 57.12 McShan and Meyer, J . Biol. Chem., 1940, 135, 473.13 Li, Simpson, and Evans, Science, 1949,109, 445.14 Koenig and King, Arch. Biochem., 1950, 26, 219.1 5 Evans, H. L. Fraenkel-Conrat, Simpson, and Li, Science, 1939, 89, 249.1 6 Gurin, Proc. SOC. Exp. Biol., 1942,49, 48.1 7 McShan and Meyer, ibid., 1939,40, 701 ; J . B i d . Chem., 1938,126, 361.18 Chow, Greep, and van Dyke, J. Endowinol., 1939,1, 440.19 J . Amer. Chem. Soc., 1950, 72, 2815FOLLEY: HORMONES OF THE UTERIOR-PITUITARY GLAND.363material by peptic digestion of FSH protein. Moreover the hormone hasalso been detected in trichloroacetic acid extracts of fresh sheep pituitary,but this material was not ultra-filtrable.20 The activity of an impuresheep-gland preparation remained after 30 minutes at 75" (pH 7-8),12but a pure preparation was more heat-labile.6 The hormone was destroyedby acetylation with keten after 30 minutes but not 5 minutea2l and bytreatment with alkaline cysteine.l2* 22 It seems possible therefore that theactivity is bound up with the presence of amino-groups not immediatelyaccessible t o keten and of disulphide linkages. The hormone contains 4*4y0of tyrosine and 0.6% of tryptophan; cystine is present but not ~ysteine.~Interstitial-cell-stimulating Hormone (ICSH).-The chemistry of ICSHhas been reviewed several time~.l-~Puri$cation.ICSH has been isolated as a biologically and chemicallypure protein in two laboratories; a third group 23 made highly purifiedpreparations. Li, Simpson, and Evans 24s 2s extracted the hormone fromacetone-dried sheep gland with 40 yo alcohol, the gonadotrophins beingprecipitated by raising the alcohol concentration and separated by ammoniumsulphate fractionation. Tested in hypophysectomised rats the hormonewas free from FSH (total dose 3 mg.), thyrotrophin (2 mg.), ACTH (10 mg.),GH (10 mg.), and prolactin (10 mg.), and showed ovarian interstitial cellrepair in hypophysectomised rats in doses of 0.0054.01 mg.Solubility,ultra-centrifugal, and electrophoretic studies showed it to be a homogeneousprotein. Shedlovsky, Rothen, Greep, van Dyke, and Chow 26* 27 obtainedthe hormone from fresh pig gland by extraction with cold saline and thenammonium sulphate fractionation. The protein was homogeneous byelectrophoretic, ultra-centrifugal, and solubility criteria. A significantincrease in the weight of the anterior prostate lobe of the hypophysectomisedmale rat was caused by a minimum of 6.7 pg. of hormone.Chemical Properties. Consideration of the following physico-chemicalproperties shows ICSH from sheep and pig pituitaries to be different proteins :molecular weight (sheep,25 by osmotic pressure : 40,000 ; by ultra-centrifuge : 90,000) ; sedimentation constant (S,,) (sheep : 25 3.6 xcm./sec./dyne ; pig : 27 6.8 x cm./sec./dyne) ; electrophoretic mobility(sheep,26 in buffer at pH 7-40, ionic strength 0.1 : 6.0 x 10-5 cm.2/sec./v.;pig,26 in buffer a t pH 7.86, ionic strength 0.05 : 0.66 x 10-5 cm.2/sec./v.) ;isoelectric point (sheep : 25 pH 4-6 ; pig : 26 pH 7.45).The hormones alsodiffer in carbohydrate content (sheep : 24 mannose 4-45%, hexosamine5-85 % ; pig : 28 mannose 2.8 %, hexosamine 2.2 %) and tryptophan content(sheep : 29 1.0% ; pig : 25 3.8y0). Immunological differences have also2o Geschwind, Hess, Condliffe, Evans, and Simpson, Science, 1950,112, 436.21 Li, Simpson, and Evans, J . BioE. Chem., 1939,131, 259.a2 H. L. Fraenkel-Conrat, Simpson, and Evans, ibid., 1939,130, 243 ; Science, 1940,a4 Endocrinology, 1940, 27, 803.O 8 Gurin, Proc.SOC. Exp. Biol., 1942, 59, 48.em Li, Simpson, and Evans, Science, 1940,92,365.91, 363. 23 Fevold, Ann. N . Y . Acad. Sci., 1943,48,321.J . Amer. Chem. SOC., 1942, 54, 367.Endocrinology, 1942, 80, 650. Science, 1940, 92, 178364 BIOCHEMISTRY.been reported; sheep hormone does not react with specific antibodies topig hormone raised in the rabbit.30 Li and Evans3 report differencesin biological potency of the two proteins in female hypophysectomisedrats (ovarian interstitial tissue repair test) and intact rabbits (ovulationtest), but not in hypophysectomised male rats (ventral prostate repairtest).Sheep ICSH is more readily inactivated by keten 24 (5 minutes) than isFSH, and also by treatment with alkaline cysteine; 22 the activity seemstherefore to depend on amino-groups and disulphide linkages.ICSHdiffers from FSH in that its activity is rapidly destroyed by trypticdigestion l2, l8 but is relatively resistant to arny1a~e.l~Its biological specificity can be summarised thus : it repairs or maintainsthe ovarian interstitial cells in hypophysectomised female rats and willcause ripe follicles to ovulate and luteinise ; in hypophysectomised male ratsit will stimulate the interstitial tissue of the testes and through the resultingincrease in androgen production it will indirectly ‘bring about growth ofthe accessory reproductive organs.Lactogenic Hormone (Prolactin).-The biochemistry of prolactin has beenreviewed many times in the last decade.l* 2* 31 31-34 It was the first A.P.hormone to be obtained pure and has been extensively studied chemically.The biological potency (pigeon crop-gland test) of the pure hormone is30-35 i.u./mg.It seems that Lyons35 must have obtained almost purepreparations as long ago as 1937, since preparations later made by essentiallyhis procedure were found to exhibit this potency 36 and to be homogeneousas judged by solubility and electrophoretic studies.36* 37 The most potentand favoured sources are ox and sheep pituitaries; the activity of pigglands is much less.38 The hormone is less soluble in aqueous solventsthan FSH, ICSH, and thyrotrophin, and extraction with alkaline ethanolgives rather a complex mixture of proteins. Extraction by acid acetone 35or by chloroform39 avoids much of this contamination, but ACTH is stilla major hormonal contaminant though its separation from prolactin presentsno difficulty.Prolactin exhibiting molecular homogeneity as judgedby electrophoretic and solubility criteria was prepared by Li, Lyons, andEvans 361 27 by a slight modification of Lyons’s original method.3s Thisinvolved acid acetone extraction of fresh whole sheep pituitaries and repeatedprecipitation a t the isoelectric point (pH 5.5) in the cold, ACTH beingPurijcation.30 Chow, Endocrinology, 1942, 30, 657.32 White, Ann.N.Y. Acad. Sci., 1943, 43, 341.33 Idem, Vitamins and Hormones, 1949, 7, 253.34 Folley, in ‘‘ Marshall’s Physiology of Reproduction,” 3rd edn. (A. S.Parkes, ed.),35 Proc. SOC. Exp. Biol., 1937, 35, 645; Cold Spring Harbor. Symp., 1937, 5 ,38 Li, Lyons, and Evens, J . Cfen. Physiol., 1941, 24, 303.38 Chance, Rowlands, and Young, J . Endocrinol., 1939, 1, 239.39 Schwenk, Fleischer, and Tolksdorf, J . Biol. Chem., 1943, 147, 535.3l Voss, Ergebn. Physiol., 1941, 44, 96.London, chap. 20 (in the press).198.37 Ibid., 1940, 23, 433FOLLEY : HORMONES OF THE ANTERIOR-PITUITARY GLAND. 365removed by precipitation at pH 6.5. Li, Simpson, and EvansM havedescribed an alternative method of working up the crude acid acetone extract,in which ACTH is removed by utilising the fact that it is soluble in 0.36~-sodium chloride at pH 3.0, while prolactin is not. White, Bonsnes, andLong41 have also prepared the pure hormone from a crude acid acetoneextract.Their preparations were homogeneous by the usual physico-chemicalcriteria. These authors,41 following up an earlier preliminary report>2have reported further on two alternative methods of crystallising prolactinin small yield. Success with this method has been reported from anotherlaboratory but others have faiIed.32*= A novel method of obtaining highlyactive prolactin (30 i.u./mg.) in good yield from fresh sheep pituitarieshas been described by Schwenk, Fleischer, and Tolk~dorf.~~ This involvesextraction with chloroform, and removal of the prolactin from the chloroformgel containing prolactin, ACTH, and inactive proteins by dissolution in acidmethanol. Koenigand King have described a new method of extracting prolactin from sheeppituitaries, useful for large-scale work.14Prolactin, the protein nature of which was suspected longago on account of its solubility relations, exhibits the same physico-chemicalproperties whether prepared from ox or sheep glands except in two, probablyrelated, respects : (a) in NaC1-HCl solutions ox prolactin is less solublethan sheep hormone, though more soluble in citrate buffer a t pH 6.36; 3 6 w 4 3( b ) ox hormone contains more tyrosinc (5.7%) than does sheep hormone(4.5 yo) .439 44 They can therefore be considered as different proteins eventhough it has not been possible to distinguish them immunologically.45The molecular weight has been given as 26,500 (osmotic 22,000(diffusion and vis~osity),~~ 32,000-35,000 (by ultra-centrifuge, on the basisof a spherical molecule),41 and 33,300 (from analytical values for five amino-acids).47 Other physico-chemical data have been given as follows : iso-electric point ; pH 5.73 (electrophoresis, moving boundary method),48* 49pH 5.65 (cataphoresis of coated quartz particles) ; 41 sedimentation constantIS'^^) : 2.65-2-80 x 10-13 cm./sec./dyne ; 41 diffusion constant (D20) : 7-57 xlo-' cm.2/sec.(free diff~sion),~~ 9.0 x 10-7 cm.2/sec. (glass-membranedifbsion) ; 46 partial specific volume : 0.721 ; 46 disymmetry constant (fit,) : 1.29-1.37 ; 46 specific rotation : -40-5" at 25°.46The elementary composition of prolactin is typical of a protein and itcontains no carbohydrate, thiol groups, or phosphor~s.~~ A completeamino-acid analysis of' sheep prolactin has recently been reported by Li 47accounting for 99.9% of the nitrogen and for all the sulphur.60 TheThe degree of purity of this preparation is not known.Properties.40 J .Biol. Chem., 1942, 146, 627.4 2 White, Catchpole, and Long, Science, 1937, 86, 82.O3 Li, Lyons, and Evans, J . Biol. Chem., 1941,140, 43.4 5 Bischoff and Lyons, Endocrinology, 1939, 25, 17.46 Li, J . Biol. Chem., 1942,146, 633.Li, Lyons, and Evans, Science, 1939, 90, 622.4s Idem, J . Amer. Chem. SOC., 1940,62, 2925.50 Li, J . Biol. Chem., 1943,148, 289.41 Ibid., 1942, 143, 447.44 Ibid., 1940, 136, 709.4 7 Ibid., 1949,178, 459366 BIOCIHEMISTRY.biological activity is destroyed or diminished by treatment with keten,61nitrous acid, 62 and phenyl isocyanate,63 indicating the essentiality of freeamino-groups, by iodination of the tyrosine residues, 64 by esterification ofthe free carboxyl groups with methyl alcohol,66 and by treatment withthiol compound^.^^ No evidence that the activity is bound up with aprosthetic group has been obtained; it rather seems from the foregoingthat it depends on the integrity of the whole molecule.Prolactin is a heat-lebile protein though it will survive a certain amountof heating without loss of activity under some conditions of Proteasesdestroy the activity before all material precipitable by trichloroacetic acidis cleaved ; the activity is also destroyed by acid hydr~lysis.~~ The viscosityof prolactin is reversibly increased in presence of urea, indicating de-naturati~n,~' but osmotic pressures indicate no molecular dissociation 43and the biological activity was unchanged after removal of the urea.Treat-ment with another denaturing agent, trichloroacetic acid, also left thebiological activity unchanged.43 Detergents also increase the viscosityof prolactin ; bioassay of the protein-detergent complex indicated someloss of biological potency. 57The best known biological action of prolactin in mammals is the initiationof lactation (lactogenesis), its action on the mammary epithelium apparentlybeing direct,58 though it seems probable that other A.P. hormones are alsoIt also influences the function of the corpus luteum (seeref. 33). No direct metabolic effects in mammals are known; 6o theobservation that prolactin can reduce the fat content of peripheral tissues 61has not yet been confirmed.60Adrenocorticotrophin (ACTH).--lsolation of a " Pure " ProteinExhibiting ACTH Activity.By virtue mainly of dramatic developmentsin the therapy of rheumatoid arthritis and related disorders, ACTH haslately come to the forefront as currently the most important of the A.P.hormones ; its chemistry has several times been reviewed.'* 2* 3* 62s 63 Therichest practicable source appears to be pig pituitaries 64 though it has alsobeen isolated from the pituitaries of sheep and ox. Two groups of workersalmost simultaneously reported the isolation of an ACTH-active proteinsatisfying the currently available criteria of biological and chemical purity.5 1 Li and Kalman, J .Amer. Chem. SOC., 1946, 68, 285.52 Li, Lyons, Simpson, and Evans, Science, 1939, 90, 373.53 Bottomley and Folley, Nature, 1939, 145, 304.54 Li, Lyons, and Evans, J . Biol. Chem., 1941,139,43.5 5 Li and Fraenkel-Conrat, ibid., 1947, 107, 495.6 6 Fraenkel-Conrat, Simpson, and Evans, ibid., 1942, 142, 107.5 7 Li, ibid., 1944, 155, 45.59 Folley and Young, Lancet, 1941, I, 380.60 Li, Ingle, Prestrud, and Negamis, Endocrinology, 1949, 44, 454.61 Reisa, ibid., 1947, 40, 294.62 White in " The Chemistry and Physiology of Hormones," h e r . Assoc. Adv.Lyons, Proc. SOC. Exp. Biol., 1942, 51, 308.Sci., 1944, p. 1.Li and Evans, Vitamin8 and Hw?none8,1947, 5, 198.44 Astwood and Tyelowitz, Fed.Proc., 1942, 1 (Part 2), 4FOLLEY : HORMONES OF THE ANTERIOR-PITUITARY GLAND. 367Li, Evans, and Simpson 65 isolated it from fresh sheep glands by acidacetone extraction followed by fractionation with ammonium sulphate andsodium chloride. The protein behaved as a single substance by ultra-centrifugal, electrophoretic, and solubility criteria and was free from otherA.P. hormones within the limits of sensitivity of the tests used. Later,G. Sayers, White, and Long a6 isolated a protein satisfying similar criteria andexhibiting ACTH activity from pig pituitaries by isoelectric precipitation.The electrophoretic data given in this paper were later corrected.67 Amodification of the latter technique in which sodium chloride fractionationis used instead of isoelectric precipitation at the final step has been described ;the protein so obtained was homogeneous by electrophoretic and solubilitycriteria and as active biologically as that obtained by the method of G. Sayerset al.Physico-chemical data indicate that theACTH-active proteins as isolated from pig and sheep pituitaries are identical.The data are as follows : isoelectric point by moving-boundary electro-phoresis, pH 4.654.70 (sheep 65), pH 4074.8 (pig 66) ; S,, : 2.08 x 10-13cm./sec./dyne (sheep 9, 24M-2-11 x 10-13 cm./sec./dyne (pig 66) ; D,, :10.4 x cm?/sec.(sheep 69) ; disymmetry constant (filf,) : 1.1 (sheep 3* 69),giving an axial ratio of 3 : 1 for an ellipsoid of rotation; molecular weight :20,000 (sheep 69) from sedimentation and diffusion data, 20,000 (pig 66)from sedimentation data but without diffusion data.*The protein contains no carbohydrate, phosphorus, or ~ y s t e i n e .~ ~ Itcontains tryptophan 1 -0 tyrosine 4.5%: methionine 1.93 %, 70 andcystine 7019%,~O the last two acids accounting for all the sulphur.70 Itis very soluble but is precipitated by 2.5% trichloroacetic acid,66 207,sulphosalicylic acid, and 5 % lead acetate.66 The biological activityassociated with it is abolished or diminished by treatment with keten (someamino- and phenolic hydroxyl groups acetylated), 71 nitrous form-Properties of the Protein.6 5 Science, 1942, 96, 450; J. Biol. Chem., 1943, 149, 413.G6 Proc. SOC. Exp. Biol., 1943, 52, 199; J . Biol. Chem., 1943, 149, 425.g8 Fishman, ibid., 1947, 167, 425.69 Burtner, J.Amer. Chern. SOC., 1943, 65, 1238.70 Li, Fed. Proc., 1946,5 (Part 2 ) , 144.71 Li, Simpson, and Evans, Arch. Biochem., 1946, 9, 259.* [Added in proof.] Ghosh, Richards, Merkin, Burns, Brown, G. Sayers, andSmith [Fed. Proc., 1950, 9 (Part I), 1761 have found pig ACTH-protein to be morelabile at high pH than at moderate pH (acid to the isoelectric point). This prompteda re-investigation of its physico-chemical properties by Smith, Brown, Ghosh, andG. Sayers (J. Biol. Chern., 1950, 187, 631). The isoelectric point was pH 4.6, S,, :2.16 x 10-3 cm./sec./dyne, and D,,: 10.8 x sq. cm./sec., giving a molecularweight of 19,400. In electrophoresis experiments on the alkaline side of the isoelectricpoint the main (biologically active) component amounted to 90-94%; in acetatebuffer at pH 4, however, a number of components were present and it was suggestedthat the protein interacts specifically with acetic ion in such a way that primaryparticles are reversibly aggregated.Evidence of rapid dissociation of the hormonewithout loss of biological activity in strongly acid solutions was obtained.Wilhelmi and G. Sayers, ibid., 1948, 176, 175368 BIOCHEMISTRY.aldehyde,7f acetic anhydride,72 or iodine,71 and by esterification of thefree carboxyl groups with methyl alcoh01.~~ An interesting property ofACTH is its remarkable heat stability, a property utilised by Collip 73 formaking partly purified ACTH. Sheep hormone was stable to boiling atpH 7-5 or even for 60 minutes in 0.1M-hydrochloric acid but was inactivatedafter 30 minutes a t 100" in O*lM-sodium hydroxide.65Enzymic and acid hydrolysis of the ACTH-active protein has given results of great interest, showing in confirmationof earlier indications (see below) that the biological activity is associatedwith a molecule much smaller than a protein of molecular weight 20,000.This would accord with its remarkable thermo-stability.Sheep protein-hormone could be digested to a considerable extent by commercial trypsin(18% cleavage, but not 26%) and pepsin (37% cleavage) without loss of'activity.65 Later, Li 72 reported that if the protein were 50% hydrolysed bycrystalline pepsin the nitrogenous fraction soluble in trichloroacetic acid ordialysable was biologically active. Hydrolysis with hydrochloric acid a t37.4", causing 30 yo protein cleavage, also yielded an active non-proteinnitrogen fraction.The average chain length of the biologically activepeptide fraction which evidently was split off by these procedures wasstated to be 7-9 amino-acid residues,72* 74-76 and the average molecularweight determined in the ultra-centrifuge < 1200.75* 76 Fractionation ofthe active peptide mixture by paper chromatography gave a t least six distinctninhydrin-positive spots, one of which was biologically active to theextent of about 13 times the original protein.75* 76 Li 77 has since statedthat his ACTH peptides can be activated 2 or 3 times by boiling them withhydrochloric acid.Fractions showing very high biological activity-up to 120 times thatof an electrophoretically inhomogeneous Armour preparation no.La- 1 -a,itself slightly more active than a " pure '' protein-hormone preparation 78-have been isolated by Lesh, Fisher, Bunding, Kocsis, Waleszek, White,and Hays 79 by subjecting trichloroacetic acid-soluble fractions from pepticdigests of partly purified pig ACTH preparations to counter-current dis-tribution. Ultra-centrifugal studies indicated LL higher molecular weight(2500--10,000) than the average given for the much less biologically activefraction obtained by Li and P e d e r ~ e n . ~ ~ Lesh et ~ 1 . ~ ~ suggest that thelatter type of preparation might consist of low-molecular material containinga small proportion of a very active fraction of higher molecular weight.Raben, Payne, and Astwood 80 have also reported the preparation of ACTHup to 100 times as active as La-l-a.Dialysable material isolated from aConf. Metab. Aspects Convalescence, Josiah Macy Jnr. Foundn., 1948, p. 114." Low-molecular ACTH."79 Li. Abstr. Commun. 1st Int. Congr. Biochem., 1949, p. 386; Trans. 17th Meeting73 J . Amer. Med. Assoc., 1940, 115, 2073.74 Li, Fed. Proc., 1949,8, 219.76 Li and Pedersen, Arkiv Kemi, 1950,1,533.78 M. A. Sayers, G. Sayers, and Woodbury, Endocrinology, 1948, 42, 379.70 Science, 1950, 112, 43.80 Abstr. Papers, Amer. Chem. SOC. 118th Meeting, 1950, l l c .7 6 Li, J . Endocrinol., 1950, 6, xl.7 7 J . Amer. Chem. SOC., 1950,72,2815FOLLEY : HORMONES OF THE ANTERIOR-PITUITARY GLAND.369peptic digest of ACTH protein has been found to be active clinically aswell as by the adrenal ascorbic acid depletion test.81The idea that ACTH may be of relatively small molecular size is by nomeans new; Anselmino, Hoffmann, and Herold 82 long ago claimed thatit was ultra-filtrable through collodion membranes, a contention confirmedsome years later by Tyslowitz,83 using a more specific bioassay method fortesting his ultra-filtrates. More recently, Crooke, C. J. 0. R. Morris, andtheir colleagues g4--86 have prepared ACTH-active ultra-filtrates withoutpreliminary hydrolysis from crude ox ACTH preparations using membranesimpermeable to proteins of mol. wt. 17,500 and 13,700 but passing salmine(mol. wt. 8000). The activity of the total ultra-filtrable solids at theoptimum pH for ultra-filtration (approx.pH 2) was about 1-18 times thatof preparation La-l-a which has now been adopted as the provisionalinternational standard. Later, P. Morris and C. J. 0. R. Morris 87 reportedthe isolation, from the polypeptide mixture. so obtained, of what appears tobe a homogeneous peptide about 8.5 times as active as La-l-a. Furthercharacterisation of this peptide will be awaited with interest. Geschwind,Hess, Condliffe, and Williams 88 have also obtained low-molecularbiologically active material from ACTH-protein by dialysis and also bytreatment with trichloroacetic acid. In the latter case trichloroaceticacid-soluble fractions showing more than ten times the activity of theprotein (calculated per mg.of nitrogen) have been obtained, the activityof the insoluble material being reduced. Moreover, they were able toseparate active from inactive moieties by subjecting the protein to paperchromatography, the active spots exhibiting fluorescence. Similar resultshave since been obtained with trichloroacetic acid extracts of fresh sheeppituitaries .2OThe problem of designing relatively simple chemical procedures capableof giving maximum yields of ACTH in a form suitable for clinical use haslately received attention. Raben, Payne, and Astwood 8o described aprocedure involving extraction of dried pig pituitary powder with glacialacetic acid which gave preparations more active than La-l-a.* Reiss and81 Brink, Meisinger, and Folkers, J. Amer. Chem. SOC., 1950, 72, 1040.82 Klin. Woch., 1934, 13, 209; Arch. Bynaek., 1934, 157, 86.83 Science, 1943, 98, 225.84 Crooke, Henly, and C. J. 0. R. Morris, Abstr. Commun. XVIIth Int. Physiol.Congr., 1947, p. 139.85 Henly, P. Morris, and C. J. 0. R. Morris, Abstr. Commun. 1st Int. Congr. Biochem.,1949, p. 384.Cortis-Jones, Crooke, Henly, P. Morris, and C. J. 0. R. Morris, Biochem. J.,1950, 46, 173. Lancet, 1950, T, 117.Payne, Raben, and Astwood ( J . BioE. Chem., 1950, 187, 719),have given details of methods of preparing highly active ACTH fractions from acetone-dried pig pituitaries. Extraction with glacial acetic acid at 70" is followed by frac-tionation with acetone and ether. The activity of the material precipitated with etherwas increased by adsorption on powdered cellulose, followed by elution with hydro-chloric acid, and solvent partition in presence of picric, benzenesulphonic, or o-mer-captobeneoic acid, gave preparations assaying up to 80 times the potency of theprovisional international standard.Science, 1950, 111, 625.* [Added in proof.370 BIOCHEMISTRY.Halkerston 89 extracted fresh pig glands with acid acetone and, duringfurther purification to remove posterior lobe principles, obtained threeapparently chemically different active fractions. It is not yet knownwhether these represent molecularly distinct hormones or different “ carrier ”proteins associated with the same active moiety.The fact that fractions of enhanced biological activity can be separatedfrom the protein or from A.P. extracts by procedures such as dialysis,ultra-filtration 839 86 (ultra-filtration of the whole activity of the proteinhas been claimed 86), trichloroacetic acid fractionation, and paper chromat o-graphy 88 which would probably not hydrolyse peptide linkages may meanthat the hormone is a relatively low-molecular polypeptide adsorbed onan inactive protein molecule, though the possibility that the ACTH-proteinis a “ mother molecule ” consisting of a dissociation-association systemincorporating an active moiety, cannot yet be excluded. Peptide linkagesare essential for the biological activity since it is destroyed by t r y p ~ i n , ~ ~carbo~ypeptidase,~~ papain,86 and acid hydroly~is.~~The subject at the time of writing is in a state of flux and it is to behoped that tbe results of the extensive experiments now actively proceedingin various countries will soon clarify it. Speculation about the form inwhich ACTH is elaborated by the A.P. or exists in the circulation is clearlypremature at the present stage when we are still awaiting the isolation ofthe pure hormone as it appears in A.P. extracts. If the active moiety isof the size suggested by Lesh et U Z . , ’ ~ then the earlier hopes of preparing itsynthetically will have receded somewhat.Growth Hormone (GH).-A number of reviews of the chemistry of GHhave appeared in the last ten years.l, 2* 3* 62* 63Isolation. GH has been isolated as a molecularly homogeneous protein(by criteria of diffusion, electrophoretic mobility, and solubility), showingno significant contamination with other known A.P. hormones, by Li, Evans,and SimpsonM using salt fractionation of an aqueous calcium hydroxideextract of acetone-dried ox A.P. Later, Wilhelmi, Fishman, and Russell 91described a less laborious method, based on low-temperature ethanolfractionation, by which high yields (stated tc be ca. 3 g./kg. of fresh gland)of crystalline GH were obtained from fresh ox A.P. Li, Evans, andSimpson 92 subsequently crystallised their amorphous protein, bioassayindicating that the process had effected no concentration or fractionationof biological activity.Properties. Physico- chemical data for amorphous GH have beenobtained as follows : l-isoelectric point : pH 6.85; S,, (determined incrn.2lsec. ; 94 disymmetry constant (f/f,) (hydration being neglected andalkaline solution) : 3.1 x 10-13 cm./sec./dyne; 93 D,, : 7.15 x 10-789 J . Pharrn. Pharmacol., 1950, 2, 236.90 Li and Evans, Science, 1944, 99, 183; Li, Evans, and Simpson, J . B i d . Chem.,92 Science, 1948,108, 624.01 Li, J. Phya. Colloid Chem., 1947, 51, 218.1945,159, 353. *l Ibid., 1948, 176, 735.g3 Li and Moskowitz, J . Biol. Chem., 1949, 178, 203FOLLEY : HORMONES OF THB: ANTERIOR-PITUITARY GLAND. 371a prolate ellipsoid assumed) : 1.31, indicating an elongated molecule ;intrinsic viscosity : 7.64 ; 94 molecular weight : 44,250 (osmotic pressure 90),43,600 (analytical data for five amino-acids and total sulphur 94), 46;800(from histidine content 95), 47,300 (analytical data for 14 amino-acids 96),39,300 (diffusion and viscosity determinations "), 44,000 (ultra-centrifuge 93).Data obtained for the crystalline hormone 97 are : D2, : 7.36 xcm.2/sec. ; Szp (determined in glycine buffer) : 3.60 x cm./sec./dyne;molecular weight (ultra-centrifuge) : 49,200.The hormone contains no phosph~rus,~ carbohydrate: or c y ~ t e i n e . ~ ~The total sulphur is accounted for by its content of cystine and methionine.'O, 94Analytical data for 15 amino-acids, which together with the amide-nitrogenaccount for 88.2% of the total nitrogen, corresponding to about 80% of thedry weight, have been determined.O5, 96 From considerations based on thenitrogen partition it is suggested that the GH molecule consists of 369amino-acid residues (26 of whiph are unknown) arranged in two sub-unitswith polar groups located in the outer faces and non-polar groups towardsthe inner planes.96 It is a thermolabile protein more stable in alkalinethan in acid solution.g0 Viscosity,94 osmotic pressure,g4 and sedimentationvelocity 97 determinations indicate that some aggregation of the proteinoccurs in acid solution (pH 4.0) though diffusion measurements at pH 4-0provided no evidence of molecular inhomogeneity.gO Denaturation inacetate buffer 94 or by urea does not destroy the biological activity, butthe activity is destroyed or diminished by treatment with keten,3 nitrousacid: or acetic anhydride?* by i o d i n a t i ~ n , ~ ~ and by proteases.w Theantigenicity of a crystalline growth hormone preparation has been foundto be poor.99Besides re-establishing body growth and influencing chondrogenesisand osteogenesis in hypophysectomised rats (responses used for bioassay 3),GH is diabetogenic in the intact cat,lo0 glycostatic in the hypophysectomisedrat,O1 and galactopoietic in the lactating c0w.1~1Thyrotrophh-Thyrotrophin has not yet been isolated in the pure state,though considerably purified and highly active preparations have beenmade. Reviews of the chemistry of thyrotrophin 2, 3, 62 emphasise itsprobable protein nature but there is some indication that its molecularweight is rather low.The richestcommon sources appear to be pig lo2 and ox lo3 pituitaries ; sheep pituitariesare less potent.lo3 Several methods of preparation of highly potent thyro-It is very soluble and easily extracted from pituitary tissue.95 Franklin, Li, and Dunn, J . Biol. Chem., 1947, 169, 515.B6 Li and Evans, Recent Progr. Hormone Research, 1948, 3, 3.B7 Smith, Brown, Fishman, and Wilhelmi, J . Biol. Chem., 1949, 177, 305.B* Li, Simpson, and Evans, ibid., 1948, 178, 843.BB EIberg end Li, Endocrinology, 1950, 47, 143.loo Cotes, Reid, and Young, Nature, 1949,164, 209.lol Cotes, Crichton, Folley, and Young, ibid., p. 992,loa Rowlands, J. Physiol., 1936, 88, 298.lo8 Jorgensen and Wade, Endocrinology, 1941, 28, 406BIOCHEMISTRY.trophic hormone preparations have appeared during the last decade. J.Fraenkel-Conrat, H. Fraenkel-Conrat, Simpson, and Evans 104 used saltfractionation methods to obtain a purified thyrotrophin preparation froman acetic acid-sodium chloride extract of acetone-dried ox pituitary. Thispreparation was contaminated principally by ICSH and by only smallamounts of FSH, prolactin, ACTH, and GH. Salt fractionation methodswere also used by Fevold, Lee, Hisaw, and Cohn lo5 to obtain a thyro-trophic fraction from an alkaline extract of whole sheep pituitary.Jorgensen and Wade 103 effected considerable purification and concentrationof the activi$y by methods involving adsorption on " Permutit " and pre-cipitation by uranium acetate. More recently Ciereszko lo6 has obtainedhighly purified thyrotrophin preparations from whole fresh ox pituitaryglands by fractionation with acetone, lead acetate, and trichloroaceticacid, the method being essentially a simplification of that previously describedby Bonsnes and White. lo' The preparations so obtained were substantiallyfree from prolactin, GH, and gonadotrophic hormones, and preliminaryelectrophoretic and ultra-centrifugal studies indicated the presence of onlyone protein component.The hormone is not precipitated by trichloroacetic acid,lo6 sulpho-salicyclic acid,lOG or lead acetate,106 and does not sediment easily in theultra-centrifuge,lOB all of which suggest that its molecular weight may berather low. It is very soluble,lM readily adsorbed on a variety of adsor-b e n t ~ , ~ ~ ~ but is precipitated by flavianic,lo3 picric,lo6 or phosphotungsticacid,lo6 uranium acetate,lo6 or mercuric chloride.lo6 Highly purifiedpreparations have been found to contain carbohydrate 104* lo6 but nophosphorus.lo6 The activity is destroyed by proteolysis l8 and by treatmentwith cysteine lo4 or keten.104S. J . F.E. P. ABRAHAM.E. BOYLAND.J. DUCKWORTH.S. J. FOLLEY.D. HERBERT.W. E. VAN HEYNINGEN.G. G. NEWTON.104 J . Biol. Chem., 1940, 135, 199.lo6 J . Biol. Chem., 1945, 160, 585.108 Severinghaus, Levin, and Chiles, ibid., 1938, 23, 285.lo5 Endocrinology, 1940,26,999.lo' Endocrinology, 1940, 26, 990.37