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cinate, from the four carbon chain, was isolated by chromatography on Dowex 1*and on pa.per using aqueous pheno1.I The succinate isolated from the oxidation product of synthetic spermidine,8 labeled in the four carbon chain, contained about 33y0 of the radioactivity u t on the Dowex 1 (6) We are very much indebted to Mrs. Elizabeth Klemperer who column, while less than 0 . 3 h of the radioactivity was reponsible for many of the measurements in this investigation. HARVARD UNIVERSITY JACQUES R. FRESCO was found in the succinate from the oxidation CAMBRIDGE, MASSACHUSETTS PAUL DOTY products of isolated spermidine and synthetic spermidine8 labeled in the three carbon chain. RECEIVED ,MAY 23, 1957 The low level of activity in the succinate shows that the isolated spermidine is not. labeled in the INCORPORATION OF T H E CARBON CHAIN OF four carbon chain and is consistent with labeling METHIONINE INTO SPERMIDINE'J in the three carbon chain. Sir: TABLE I1 The cumulative work of several laboratories has Total radioactivity in spermidine fractions, c.p.m. indicated the metabolic importance of the polyfiH f ATP - ATF amines putrescine, spermidine and spermine in 7.2 2 . 1 x 104 520 diverse biological systems. Tabor, et ~ l . have , ~ 8.2 2 . 5 X IO3 795 shown that putrescine is the source of the fourIncubation, four hours a t 37"; incubation mixture, 100 carbon chain of spermidine and spermine. Recent Mg. 2 c " - ~ ~ - m e t h i o n i n(3.8 e X lo6 c.p.m.) per vessel, 0.01 kf results obtained by the author show that the side putrescine, 0.008 M glutathione, 0.003 M MgClZ, 0.~30001,I1 chain of methionine is an efficient precursor of the pyridoxal phosphate, 0.1 M buffer, 2.5 ml. 1 : 1 extract of 74A-3b, 0.02 M KrATP as indicated, total volume 5 ml.; three carbon chain of spermidine in Neurospora buffers are p H 7.2, potassium phosphate + Tris hydrochlocrassa. ride (1 : l ) , pH 8.2, Tris hydrochloride. Neurospora crassa strains 74A-3b (wild type) and A possible mechanism for this reaction is the 38706 (blocked in the conversion of homocysteine to methionine) were grown a t room temperature transfer of the methionine side chain from S-adenoon Fries minimal medium supplemented with 50 syl methionine to putrescine in a manner similar pg./ml. of ~ ~ - m e t h i o n i n e - 2 C ' ~ The . spermidine to the transfer of the methyl group of thi.s com~ h e almost was isolated by grinding the mold with sand and pound first observed by C a n t ~ n i . T 5% TCA4 and chromatography of the extract on absolute ATP requirement for the incorporation of 0.6 X 20 cm. columns of Dowex 50-X2 using an HCI C14 methionine into spermidine by a cell free exgradient. The spermidine containing fractions tract of 74A-3b as shown in Table I1 is corisistent were combined and dried; spermidine trihydrochlo- with this hypothesis. Further evidence for the ride was crystallized from methanolic hydro- presence of radioactivity in the spermidine from gen chloride and ethyl acetate. The specific activi- the PH 7.2 incubation mixture was obtained by ties of the added methionine and the isolated paper ionophoresis in PH 8.2 borate buffer. Tabor, spermidine are compared in Table I. Amine et aZ.,1° have extended these studies in extracts of concentrations were determined as described by E. coli and have found further evidence that SRosenthal and Tabor.6 "Radiopurity of the iso- adenosylmethionine is an intermediate. ( 6 ) J. K. Palmer, Bull. 589 Conn. Agric. Expt. Station (1955). lated spermidines was checked by paper ionophore(7) H. K.Berry, H. E. Sutton, L. Cain and J. S. Berry, Biochemical sis in pH 8.2 borate buffer and by descending paper Institute Studies IV. No. 5109, Univ. of Texas, Austin, Texas, 1951, chromatography (3 parts 1-propanol :1 part 0.2M p. 22. sodium acetate buffer). I n all cases the radioac(8) Kindly supplied by Dr. E. Jackson of the National Imtitute of tivity moved in one spot and was in the same Arthritis and Metabolic Diseases. (9) G. L. Cantoni, "Phosphorus Metabolism," Vol. 11, Johns Hopposition as added authentic spermidine trihydro- kins Press, Baltimore, Md., 1952, p. 129. chloride. (10) H. Tabor, S. M. Rosenthal and C. W. Tabor, personal communication. TABLE I
relative to DNA. If this structural proposal is the correct one, it may be possible for ribonucleic acid, also, t o assume a double-stranded configuration in solution through pairing of only the adenine nucleotides.6
Strain
74A-3b 38706
Specific Activities Methionine Spermidine (initial) (isolated) c.p.m./pmole c.p.m./smole
6.36 X lo4 1 1 . 4 X lo'
3 . 3 8 X 104 6.34 X 10'
Ratio Spermidine/ Methionine
0.53 0.56
Spermidine was degraded by oxidation with alkaline permanganate. The small yield of suc(1) A preliminary report of this work was presented before the American Society of Biological Chemists: Federation Proc., 16, 189 (1957). (2) This work was supported in part by Research Grant C-3436(A) from the National Cancer Institute, National Institutes of Health, Bethesda 14, Md. (3) H. Tabor, S. M. Rosenthal and C. W. Tabor, Federation P70C.. 15, 367 (1956). (4) Abbreviations used are: TCA, trichloroacetic rcid: ATP, adenosine triphosphate; Tris, tris-hydroxymethylaminomethane. (5) S. M. Rosenthal and C. W. Tabor, J. Pharmacol. and E x p t l . Thrr., 116, 131 (1956).
RADIOISOTOPE SERVICE,VETERANSADMINISTRATICIN HosPITAL, AND DEPARTMENT O F BIOCHEMISTRY, DUKE~JXIVERSITY, SCHOOL OF MEDICINE,DURHAM,NORTH CAROLINA, AND NATIONAL INSTITUTE OF ARTHRITISAND METABOLIC INSTITUTES O F HEALTH, BETHICSDA 14, DISEASES, NATIONAL MARYLAND RONALD C. GREENE RECEIVED MAY9, 1957 T H E HETEROGENEITY OF POLYSACCHARIDES A S REVEALED BY ELECTROPHORESIS O N GLASS-FIBER PAPER
Sir: Glass-fiber paper' has been used in conjunction with a borate buffer p H 9-10 for the electrophoretic separation2of a wide variety of organic substances (1) M. J. O'Leary, R. B. Hobbs, J. K. Missimer and J. J Erving, T a p p i , 87, 446 (1954). (2) D. R. Briggs, E. F. Garner, R. Montgomery and F. Smith, A d . Chcm., 28, 1333 (1956).
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including polysaccharides3 and it has been reported logical examination has recently indicated the that the method can be used for differentiating cer- heterogeneity of gum arabic. lo tain specimens of glycogen from various sources4; The gums from the seeds of guar (Cyamopsis dextrans and other bacterial polyglucosans also tetragonaloba), locust bean (Ceratonia siligua L.), may be di~tinguished.~The advantage of using Kentucky coffee bean (Gymnocla,dus dioica) , ivory the glass-fiber paper rather than cellulose paper is nut (Phyteleplws macrocarpa), tara (Cnesulpiniu that complexes betweefi the polysaccharide and the spinosa), and those from flat (Linum usitatissimum) fiber are avoided5 and the location of polysac- and Plantago bnceolata, all proved to be heterocharides of all types is no longer a p r ~ b l e n i . ~ geneous, .~ two components being detected in each St'hile it is possible to separate certain polysac- case. charides such as fructans, glucans and pentosans The hemicelluloses separated from corn hulls, which have entirely different structures, using a wheat straw, flax straw and ramie grass, and the borate buffer,6 the inhomogeneity of a single so-called hemicelluloses from aspen wood, slash polysaccharide cannot be ascertained in the same pine, IobIolIy pine and Western Hemlock all way, probably because of molecular association. proved to be heterogeneous in nature. On the We have found, however, that by replacing the other hand the crystalline xylans11 prepared from borate buffer with sodium hydroxide sonsider- birch wood and barley straw by partial hydrolysis' able information can be obtained. By this tech- and also a specimen of the galactoaraban of sugar nique it has been shown that many polysaccharides beet pulp proved to be homogeneous. These two hitherto assumed to be essentially homogeneous crystalline xylans had almost identical mobilities can be separated into two or more components. on glass-fiber paper while the galactoaraban moved All glycogen specimens from a wide variety most rapidly, probably because of the more readily of sourcesi thus far encountered have been found ionizable character of the primary alcoholic groups. to be composed of two fractions, the major one Potassium hydroxide has been found to be a better moving a t about ten times the rate of the other. electrolyte than sodium hydroxide for the separaIn all cases the slow-moving component moved at tion of certain hemicelluloses; this observation the same rate but some of the glycogen specimens may be related to the long-established factI2 that can be differentiated by the rate of movement it is better to use potassium hydroxide than sodium of the faster moving component. Thus a specimen hydroxide for methylating hemicelluloses by the of liver glycogen obtained from a pregnant rabbit8 methyl sulfate method. can be distinguished by the fact that the faster The fructans from rye flour (graminin), dahlia moving component moves more slowly than the and perennial rye grass which have relatively corresponding component of normal rabbit liver low molecular weights were found to be honioglycogen. geneous while the fructans from the ti (Cordyline -4mylopectins from a wide variety of starches terminalis) root, and from cocksfoot (Dactylis have not only been found to be heterogeneous but glomevata) contained small amounts of a second also to differ from one another; three components slow moving component. have been detected in corn amylopectin, one of Carraggenin already recognized as being heterowhich corresponds to one of the components of corn geneous13 was found to consist of several compoamylose which is likewise heterogeneous. Re- nents; one of the components corresponded t o the Kpeated precipitation fails to separate all the amylose fraction precipitated with potassium chloride, component(s) from amylopectin and both corn which was found to be homogeneous. Similarly and potato amylose display heterogeneity even agar has been found to be a mixture, a result in after repeated crystallization. The reason for agreement with the recent reportl4 that it consists this is not yet clear, but it is of interest to note that of a neutral (agarose) and an acidic (agaropectin) amylitol, obtained from corn amylose by sodium component. Floridean starch also exhibits heteroborohydride reduction, moves on the glass paper geneity as also does laminarin from both Laminaria largely as a single component and shows a mobility digitata and L. cloustoni. The reduced (SaRHd quite distinct from that of amylose. laminarin from L. cloustoni displays homogeneity. Gums from Acacia ayabicum, -4.pychnantha, -4. Glass-fiber paper electrophoresis has also shown srnegal, A . seyal, Anogeissus latqolin (gum ghatti) that yeast mannan and the glucomannans from and gum tragacanth (Astragnlus sp.) were all found Konjak mannan and Iles mannan are heterogeneous to be heterogeneous whereas Black Wattle gum while the three mucopolysaccharides, heparin, from .1.decirrrens and mesquite gum from Prosopis hyaluronic acid, and chondroitin sulfate which injiili'ora appeared to be homogeneous. Immuno- dividually behave as homogeneous polymers can be separated from each other with relative ease. ( 3 ) D. R. Briggs, E. F. Garner and F. Smith, J ' a l w e , 178, 134 The movement of carbohydrate polymers on the (1956). (4) J. A . Cifonelli, €3. A. Lewis. R . Xontgomery and F.Smith, p . 30. glass paper appears to depend on the ionization of of Abstracts 129th A.C.S. meeting, Dallas, 'Texas, 1956. the alcoholic groups under the influence of the ( 5 ) Cf.E. J. Bourne, 4 . B. Foster and P. lf. Grant, J . Chem. SoC.. alkaline electrolyte. This is supported by the 4311 (1956). ( 6 ) Cf. K. W ,Fuller and D. H. Xorthcote, Biochem. J . , 64, 657 (1986). (7) R I . Abdel-Akher and F. Smith, TmS JOURNAL, 7 3 , 9 9 4 (1951). ( 8 ) S. Peat. P. J. P. Roberts and W. J. Whelan, Biochem. J . , 51, X V I I (1952). (9) 41.Abdel-Akher. J. K . Hamilton and F,Smith, T H I S TOT?RNAI.. 7 3 , 4691 (1951).
(10) Cf.>I Heidelberger, , J. Adams and Z. Disrhe, rbid., 7 8 , 2853 (1956). (11) A. P. Yundt, ibid., 7 1 , 757 (1949). (12) H. A . Hampton, W. h7,Haworth and E. 1.. Hirst, J . Chum. Soc., 1739 (1929). (13) D. €3. Smith and W. H. Cook, A v c h . B z o c l w m Biopiiyr., 46, 2 3 2 (1953). (14) C. Araki a n d K . Arai, Bull., Chein. SOC.J n p n n , 29, ,548 (19.56).
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fact that the nm-reducing neutral oligosaccharides, sucrose, raffinose, stachyose, and the a- and pSchardinger dextrins display considerable mobility, and like the free sugars, glucose and maltose, show no evidence of heterogeneity. Electrophoretic separations were carried out on strips of glass-fiber paper (7 X 40 cm.) a t 120 volts and 110 milliamperes for 10 hours:15 The mobilities of the polysaccharide components are expressed conveniently as the ratio of the distance of migration of the component from the origin to the distance moved by the faster and major corrlponent of calf liver glycogen whose mobility is taken as 100 In a typical experiment the two components of glycogen moved 4.0 and 0,4 cm., thus having mobilities of 100 and 10, respectively. Likewise the three components of the seaweed polysaccharide from Laminaria cloustoni had mobility values of 10, 105 and 130 whereas the apparently homogeneoils polysaccharides, birch wood xylan, sugar beet galactoaraban, Dahlia inulin and levosine (from rye flour) have mobility values of 110, 160, 130 and 140, respectively. The components of a number of the above polysaccharides are now being separated on a larger scale with the object of ascertaining the structural significance of the phenomenon of heterogeneity as revealed by glass paper electrophoresis. It seems evident even a t this early stage of the work that heterogeneity in polysaccharides especially those of fairly high molecular weight may be the rule rather than the exception. It is also conceivable that this phenomenon is analogous to the heterogeneity already recognized in proteins16 and it may have some bearing on the specificity of the chemical components of the various species of plants and animals.
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0.0i 0.u2 0.03 0.04 Concentration (grams/cc. 1. Fig, 1.-Viscosity data for native ferrihemoglobin I(@) and for the same protein exposed to pH 3.5 for about 10 to 15 minutes (0). t represents flow time for the solution of concentration c, and to flow time for solvent. All nieasurements were performed at ionic strength 0.04 a t 2 5 " . 0
data obtained from flow times in an Ubbelohde viscometer. To obtain intrinsic viscosities ( [VI) from these data one must add 0.5 to the limiting value of the ordinate a t zero concentration, so as t o correct for the density difference between solution and solvent3 and for the kinetic energy of the effluent solution. One obtains [77] = 3 . 5 cc.1 gram for the native protein in neutral so:lution, in good agreement with a determination by Cohn and P r e n t i ~ s . At ~ pH 3.5, after the configurational change has occurred, [77] = 13.5 cc./gram. The configurational change thus clearly involves a (15) The glass fiber sheets were obtained through the courtesy of Dr marked expansion of the protein molecule, much R B Hobbs of the Bureau of Standards, Washlngton, D C , to whom like that observed for serum a l b ~ m i n . Hemo~ the authors express their thanks (16) J R Colvin, D. B Smith and W H Cook, Chem R e v , 64, 687 globin dissociates into half-molecules, a t a pH (1954). much higher than that a t which the conjiguraDEPARTMENT OF AGRICULTURAL BIOCHEMISTRY tional change occurs.6 If this dissociation is asUNIVERSITY O F MINNESOTA BERTHAA LEWIS sumed to occur without appreciable change in [VI, ST. PAUL,MINNESOTA F. SMITH then, by Einstein's equation, the radius of 5n 7 , 1957 RECEIVEDFEBRUARY equivalent sphere (for half-mqlecules) is 26.5 A. in the native state and 41.5 A. in the expanded state. T H E ACID DENATURATION OF FERRIHEMOGLOBIN1 The finding here reported is of special in.terest because it suggests a simple explanation for the Sir : A series of important studies by Steinhardt and uptake of protons observed by Steinhardt and Zaiser2 has demonstrated that hemoglobin under- Zaiser.2 For expansion implies penetration of goes a drastic configurational change below pH 4. solvent into the molecular domain, which proThe reaction is slow enough to permit spectroscopic foundly affects the titration curve by reducing all and titrimetric observation of both the native and electrostatic interactions.' Specifically, the factor the altered form near pH 3.5. It is character- w,which occurs in the usual equation for the titrabecomes much smaller and the ized by a change in absorption spectrum, and by tion the binding of about 36 protons per molecule (mol. titration curve itself becomes steeper. Equations wt., 67,000); i.e., near p H 3.5 the "denatured" derived earlier' enable this effect to be calcula.ted if form contains about 36 more bound protons than a spherical model is assumed. For spherical halfmolecules with the radii given above we get, a t 25' the native form. This communication reports the viscosity change (3) C. Tanford, J . Phys. Chem., 59, 798 (1955). (4) E. J. Cohn and A. M. Prentiss, J . Gen. Physiol., 8 , 619 (1927). accompanying the reaction. Figure 1 shows the 1588 (1954); (1) This work was supported by research grant RG-2350 from the National Institutes of Health, Public Health Service. (2) Summarized by J. Steinhardt and E. M. Zaiser, Advances i n Protein Chem., 10, 186 et sep. (1955).
( 5 ) J. T. Yang and J. F. Foster, TAISJOURNAL, 76, C. Tanford, J. G . Buzzell, D. G. Rands and S. A. Swanson, ibid., 77, 6421 (1955). (6) E . 0 . Field and J . R P. O'Brien. Biochem. J.,60, 656 (1955). (7) C. Tanford, J . Phyr Ch~7n..69, 788 (1955).
