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A comparative study of bacterial and mammalian cytochrome c
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4 M. M. RAPPORT, K. MEYER AND A. LINKER, J. Am. Chem. Soc., 73 (I951) 2416. 5 K. MEYER, A. LINKER, ]~. A. DAVIDSON AND B. \¥EISSMANN, Jr. Biol. Chem., 20.5 (1953) 61I. 6 E. A. DAVIDSON AND K. MEYER, J. Biol. Chem., 211 (1954) 6o57 R. \V. JEANLOZ AND E. FORCHIELLI, J. Biol. Chem., 186 (195o) 495. 8 p. j . STOFFYN AND R. W. JEANLOZ, Arch. Biochem. Biophys., 52 (1954) 3739 }3. S. ]~LUMBERG, G. OSTER AND K. MEYER, J. Clin. Invest., 34 (1955) 1454. 10 Z. DISCI~E, J. Biol Chem., i 6 7 (1947) I89. 11 M. V. TRACEY, Biochem. J., 43 (1948) 185. 12 K. MEYER AND E. CHAFFEE, J. Biol. Chem., 138 (1941) 491. 13 K, MEYER AND M. M. RAPPORT, Advances in Enzymol,, 13 (1952) 199. 14 13. WEISSMANN, M. M. RAPPORT, A. LINKER AND K. MEYER, J . Biol. Chem., 205 (1953) 205. is A. COOCEIRO AND R. C. FREIRE, Anais acad. brasil, cienc., 23 (1951) 443. 16 M. M. RAPPORT, K. MEYER AND A. LINKER, J. Biol. Chem., 186 (195o) 615. J7 ~V. E. TREVELYAN AND J. S. HARRISON, Biochem. J., 30 (I952) 298. 19 j . X. KHYM AND D. G. I)OHERTY, J. Am. Chem. Soc., 74 (1952) 3199 . 19 C. H. DOHLMAN AND E. A. }3ALAZS, Arch. Biochem. Biophys., 52 (1955) 445. 20 H. GROSSFELD, K. MEYER AND G. GODMAN, Proc. Soc. Exptl. Biol. Med., 88 (1955) 31. 21 K, MEYER AND E. CHAm~EE, J. Biol. Chem., 133 (194 o) 83. 2a G. BLIX, Acta Soc. Med. Upsaliensis, 56 (195 I) 47. 23 R. AMPRINO, Acta Anat., 24 (1955) 121. 24 S. F. D. ORR, Biochim. Biophys. Acta, 14 (1954) 173. 25 }3. \¥EISSMANN, jr. Biol. Chem., 216 (1955) 783 . 2~ R. MARBETH AND A. WINTERSTEIN, Experientia, 8 (1952) 41. 27 H. SMITH AND R. C. GALLOP, Biochem. J., 53 (1953) 666. 2s Vv. P. DEISS AND A. S. LEON, J. Biol. Chem., 215 (1955) 685 . 29 S. SCHILLER, M. B. MATHEWS, H. JEFFERSON, J. LtlOOWlEG AND A. DORFMAN, J. Biol. Chem., 211 (1954) 717 .
Received December 3rd, 1955
A COMPARATIVE STUDY OF BACTERIAL AND MAMMALIAN C Y T O C H R O M E c* by M A R T I N D. K A M E N * * AND Y O S H I R O T A K E D A * * *
INTRODUCTION It has been shownI that a variety of facultative anaerobic bacteria contains cytochromes of the "c" type which can be obtained in good yield as soluble proteins of high purity. These cytochromes differ radically in electrochemical and biochemical properties from their counterparts in mammalian tissue, although they show close similarities in spectrochemica] properties. In this paper, there are presented and discussed studies which indicate changes in structure apparently associated with differences in function reflecting the physiology of the source material. The cytochrome c, derived from the aerobic denitrifier, Pseudomonas aeruginosa, shown previously by *Most of the work describedin this report was donewhilethe authors were in residenceat the Universityof California, Berkeley, Californiaduringthe summerof 1955, and was made possible by a special grant-in-aidto one of us (M.D.K.) from the C. F. Kettering Foundation,Yellow Springs, Ohio. We record here our special indebtednessto Professor W. M. STANLEYfor opening the Virus Laboratory to us and to Dr. H. K. SCHACHMANand Miss PEARLAPPELfor cooperation in makingthe sedimentationstudies. ProfessorCI-IAOHo LI most generouslyplaced at our disposal t h e facilities of t h e H o r m o n e R e s e a r c h L a b o r a t o r y w h e r e we r e c e i v e d invaluable i n s t r u c t i o n on the p a p e r c h r o m a t o g r a p h y of p r o t e i n h y d r o l y z a t e s fr om Dr. D. CHUNG a n d Mr. A. PARCELLS. A t W a s h i n g t o n U n i v e r s i t y , t e c h n i c a l a s s i s t a n c e in t h e e l e c t r o p h o r e s i s e x p e r i m e n t s w a s p r o v i d e d
References p. 523.
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VERHOEVEN AND TAKEDA2 to be obtainable in high yields and degree of purity, is compared with respect to amino acid composition, spectrochemical, electrochemical and enzymic properties with a typical classical cytochrome c preparation from mammalian heart tissue. METHODS AND MATERIALS
Isolation and purification o/Pseudomonas cytochrome Five h u n d r e d ml of a cell s u s p e n s i o n (IOO g w e t weight) of Ps. aeruginosa were h o m o g e n i z e d in a W a r i n g B l e n d o r in t h e presence of a t r a c e of D o w - C o r n i n g A n t i - F o a m . T h e s u s p e n s i o n r e s u l t i n g w a s h e a t e d to 5 °o C a n d I/IO v o l u m e of I M citric acid added. T h e m i x t u r e w a s allowed to s t a n d at 5 °0 C for five m i n u t e s , a f t e r w h i c h it w a s cooled in a n ice b a t h . A f t e r c e n t r i f u g a t i o n (6ooo r.p.m., 20 m i n u t e s - I n t e r n a t i o n a l R e f r i g e r a t e d Centrifuge) t h e s u p e r n a t a n t fluid was n e u t r a l i z e d to p H 7.0 w i t h 6 N N a O H . A m m o n i u m s u l f a t e was a d d e d slowly to a final c o n c e n t r a t i o n of 4 ° g per i o o ml. T h e p r e c i p i t a t e was r e m o v e d b y centrifugation, as above, a n d t h e s u p e r n a t a n t fluid t r e a t e d w i t h a m m o n i u m sulfate, b r i n g i n g t h e salt c o n c e n t r a t i o n to 65 g / i o o ml. A f t e r s t a n d i n g for several h o u r s in t h e refrigerator, t h e p r e c i p i t a t e c o n t a i n i n g t h e c y t o c h r o m e , was collected b y c e n t r i f u g a t i o n (Sorval h i g h - s p e e d centrifuge, t o p speed for io m i n u t e s ) , t a k e n u p in a s m a l l v o l u m e of distilled w a t e r a n d dialyzed t h o r o u g h l y a g a i n s t distilled water. A t this stage, t h e c y t o c h r o m e a p p e a r e d to be a b o u t 25 % p u r e on t h e basis of spectroscopic a b s o r p t i o n at 27o m/* c o m p a r e d w i t h t h a t of t h e reduced f o r m a t 552 m # . A c c u r a t e e s t i m a t i o n s b a s e d on spectroscopic d a t a could n o t be m a d e b e c a u s e t h e c h a r a c t e r i s t i c e x t i n c t i o n coefficients for t h e bacterial c y t o c h r o m e were n o t identical w i t h t h o s e of m a m m a l i a n c y t o c h r o m e in t h e U. V. region. F u r t h e r purification w a s a c c o m p l i s h e d b y successive a d s o r p t i o n on a l u m i n a Cy gel, followed b y elution with o.I M p h o s p h a t e buffer, p H 6. 5. Electrophoretic a n d centrifugal a n a l y s i s of t h e p r e p a r a t i o n s h o w e d t h e p r o t e i n to be h o m o g e n e o u s a n d all t h e h e m e c o m p o n e n t to be c o n t a i n e d in t h e one p r o t e i n p e a k found. I m p u r i t i e s e s t i m a t e d a t no m o r e t h a n 5 % of t h e t o t a l protein were r e m o v e d b y electrophoresis in t h e u s u a l Tiselius a p p a r a t u s , a n d t h e p u r e h e m e p r o t e i n was recovered f r o m t h e t e s t cell.
Chemical analyses and test materials C y a n i d e h e m o c h r o m o g e n w a s p r e p a r e d as described p r e v i o u s l y 3. Iron a n a l y s i s was p e r f o r m e d b y t h e m e t h o d of DRABKIN4. T h e p r o t e i n w a s h y d r o l y z e d a n d t h e a m i n o acid c o m p o s i t i o n d e t e r m i n e d b y q u a n t i t a t i v e p a p e r c h r o m a t o g r a p h y of t h e d i n i t r o p h e n y l derivatives, as described b y LEVY5. T h e bacterial c y t o c h r o m e u s e d w a s o b t a i n e d b y t h e p r o c e d u r e s o u t l i n e d a b o v e a n d could be e s t i m a t e d to be of a degree of p u r i t y ~ 99 %. T h e m a m m a l i a n c y t o c h r o m e was o b t a i n e d as a p r e p a r a t i o n 1 w h i c h a s s a y e d 0.43 % Fe i n d i c a t i n g a degree of p u r i t y well a b o v e 95 %. T h i s e s t i m a t e was borne o u t b y spectroscopic e x a m i n a t i o n .
Spectroscopic measurements S p e c t r a were d e t e r m i n e d w i t h a B e c k m a n n D U S p e c t r o p h o t o m e t e r b o t h before a n d after t r e a t m e n t w i t h t h e u s u a l oxidizing a n d r e d u c i n g agents.
Electrophoretic measurements E l e c t r o p h o r e t i c mobilities were m e a s u r e d w i t h t h e Klett-Tiselius a p p a r a t u s 1.
Potential measurements F e r r o - f e r r i c y a n i d e r e d o x buffers were used, according to t h e m e t h o d of DAVENPORT AND HILL 6.
Chemical and spectroscopic properties The bacterial pigment was found to concentrate in the same manner as its mammalian analogue when fractionated with ammonium sulfate. The purest sample which assayed b y Miss C. LOWRY of t h e D e p a r t m e n t of B i o c h e m i s t r y , a n d s o m e of t h e iron a n a l y s e s were p e r f o r m e d b y Dr. J. w . NEWTON of t h e Mallinckrodt I n s t i t u t e . F i n a l l y we wish to n o t e a gift of h i g h l y purified m a m m a l i a n c y t o c h r o m e c p r e p a r e d b y Dr. J. B. NEILANDS of t h e D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of California. "* Mallinckrodt I n s t i t u t e of Radiology, W a s h i n g t o n U n i v e r s i t y School of Medicine, St. Louis, Mo. (U.S.A.). *** N o w a t D e p a r t m e n t of N u t r i t i o n , O s a k a U n i v e r s i t y School of Medicine, O s a k a (Japan).
Re/erences p. .523.
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o.45 % Fe showed no appreciable protein contaminants, as determined by examination of the electrophoretic pattern. It was not auto-oxidizable in the physiological range and did not combine with CO in the reduced form. It was readily and reversibly oxidized or reduced by the usual reagents. It reacted with cyanide in alkali to form a cyanide hemochromogen which appeared spectroscopically identical with that obtained from mammalian cytochrome c (Table I). The bacterial pigment showed spectra in the reduced and oxidized states characteristic of mammalian cytochrome c, the two pigments being almost indistinguishable except for slight but reproducible and definite shifts in the maxima of the alpha bands. The bacterial pigment exhibited an alpha absorption maximum at 552 m~, compared to 550 m/~ for the mammalian pigment (Table I). There were appreciable departures from agreement in the shape of the ultra-violet protein absorption curve, as reflected in the ratios of the absorption at 27 ° m~ compared to those at the characteristic alpha and Soret peaks (Table I). TABLE I SPECTROSCOPIC PROPERTIES OF PSEUDOMONAS-CYTOCHROME-552 Bacterial pigment
Absorption m a x i m a (m~) Ferrocytochrome Ferricytochrome Cyanide f e r r o h e m o c h r o m o g e n
Mammalian cytochrome c
552, 52o, 416 53 ° (broad), 4o8-41o 555, 525, 421
55 ° , 520, 415 53 ° , 407 555, 525, 421
Ratios of ferrocytochrome a b s o r p t i o n m a x i m a : 27o/552:0.97 416/552:5.3 552/525 : 1.71
27o/55o: 1.o5 415/55o: 5.1 55o/52o: 1.66
Electrochemical and electrophoretic properties The difference in protein character, hinted at in the spectra, was established when the electrophoretic mobility was measured. The bacterial pigment migrated anodically with a value of 2.12.10 -5 cm2/volt-sec, compared to the cathodic velocity of 8.2. I0 -~ cm2/volt-sec determined for the mammalian cytochrome c at pH 7 (ammonium acetate buffer, 0.I N). The oxidation potentials of the bacterial pigment at a number of pH values, compared to those for the mammalian pigment, are shown in Table II. It is seen that the Pseudomonas aeruginosa cytochrome shares with the c cytochromes of the photosynthetic and other facultative anaerobes the property of an oxidation potential considerably more positive (more oxidizing) than that for mammalian cytochrome c. TABLE II O X I D A T I O N P O T E N T I A L S OF P S E U D O M O N A S - C Y T O C H R O M E - 5 5 2
pH ~ 5.0
Bacterial p i g m e n t Mammalian cytochrome c
0.336 --
6.0
o.3J, o.26 b
7.0
8,0
o.3oo 0.265
0.283 o.265
8.2
0.28o 0.279 . . . . .
Acetate buffer at p H 5 ; p h o s p h a t e buffer, p H 6 and 7 ; " t r i s " buffer, p H 8 and over. R e / e r e n c e s p. 523.
9.0
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Molecular weight From the heme iron content of o.45 %, a minimum molecular weight of 12,400 would be expected. Determinations made using the Spinco analytical ultracentrifuge and employing both sedimentation velocity and sedimentation equilibrium procedures were in agreement with this value, both giving molecular weights near 13,000 (H. K. SCHACHMAN,private communication). The weight calculated from nitrogen recovery and summation of amino acid residues was also in agreement with this value (see next section). Amino acid composition Although THEORELL AND AKESSON prepared mammalian cytochrome c in pure form nearly 15 years ago and gave a preliminary amino acid analysis for the protein at that time:, it appears that only one report has since been published on its amino acid composition. BARBIERI AND ZAMBONIs have analyzed hydrolyzates of cytochrome c, purified according to the procedure of PALEUS AND NEILANDS°, by column chromatography as well as by microbiological assay and have given the following values for the integral amino acid residues: glutamic, 6.4; asparagine, 26.0; cystine, 1.o2; serine, 3.44; glycine, lO.3O; alanine, 3.03; threonine, 2.16; proline, 3.3; valine, 5.83; histidine, 0.87; methionine, 4.63; leucine (and isoleucine) 9.28; phenylalanine, 1.7; tyrosine, 2.16; lysine, 13.4; arginine, 2.07 ; tryptophane, 0.8. The protein assayed o.456% Fe and 12.6% amino nitrogen. Unfortunately, m a n y discrepancies appeared when the results of the column chromatography were compared with those obtained by microbiological assay. Insufficient detail is available to evaluate these fluctuations. It was evident from the electrophoretic behavior of the bacterial protein that it differed in gross chemical composition from the mammalian protein. It was possible to make a semi-quantitative analysis of both preparations using the paper chromatographic procedure of LEVY~ In Table III, results are shown for all amino acids present with the exception of tryptophane, which was largely decomposed under the conditions of acid hydrolysis used. Both groups of workers quoted above appear to agree that approximately one mole of tryptophane is present per mole of protein. Assuming this value, the recovery of total nitrogen in the amino acids was 98-99%. The per cent amino nitrogen was 12.4%. TABLE III AMINO ACID COMPOSITION OF BACTERIAL AND MAMMALIAN PIGMENTS
Integral number o] residues Amino acid Pseud. cyto.-552
Glutamic + aspartic Cystine Serine Glycine + alanine Threonine Proline Valine Histidine Methionine Leucine + isoleucine Phenylalanine Tyrosine Lysine Arginine
Re[erences p. 523.
33 i 3 (32) 3 5 ii 2 (i) 14 4 2 9 2
Mammalian-cyto.-c
(27) 3 I 14 Ii 2 4 4 (i) ii 4 (2) 18 2
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The results shown in Table I I I must be regarded as preliminary because the method used is capable of much better precision than obtained in the short time available for these investigations. In a few cases, reproducible values were not easy to obtain. These are shown in brackets. In all such cases, with the exception of methionine, the m a x i m u m value found is given. In all the other amino acids, the m a x i m u m deviation in 8-1o separate determinations did not exceed 25 % and in most cases was much smaller. From our experience, it seems evident that cytochrome c can be analyzed with a precision better than a few per cent for all its constituent amino acids, using paper chromatography. The results obtained, despite the limitations mentioned, bear out the expectation of gross differences in amino acid composition between the bacterial and mammalian preparations. If it were not for the lumping of aspartic and glutamic acids, the lack of data on amide nitrogen, uncertainty as to whether serine should be considered in the phosphorylated form, and other difficulties, these data could be used to calculate specific volume, as described by McMEEI~IN et al 1°. However, limits on the values possible can be assigned. The range calculated for the bacterial and mammalian preparations are o.73o-o.75o and o.715-o.727, respectively. The molecular weight calculated, based on the results shown in Table I I I and assuming one residue weight of tryptophane per mole protein, is 12,16o for the bacterial and 12,44 ° for the mammalian protein. DISCUSSION EHRENBERG AND THEORELL 11 have presented arguments for a cytochrome c structure in which the ferroheme group is surrounded by four peptide spirals, the iron being coordinated in the fifth and sixth positions out of the porphin plane to histidine residues attached to separate peptide spirals. The old assumption of binding of the side chains of the porphin nucleus through two cysteine residues by saturated thio-ether linkages is retained in this structure. While the amino acid composition of the m a m m a lian cytochrome c is certainly consistent with the requirement of at least two histidines and two cysteines, the corresponding values for the bacterial pigment are just barely adequate. It would appear that extension of the methods of PAI.EUS, TUPPY and othersle, la, including sequence analysis of Fe-containing peptides 14, to specimens of the bacterial "c" cytochromes is desirable to test the general validity of structures based on mammalian cytochrome c alone. The fact that the enzymic behavior of the bacterial pigments differs radically from that of the mammalian analogue also emphasizes the need for structural analysis of cytochrome c proteins derived from a variety of sources. It has been shown ~5 that the bacterial "c" cytochromes obtained from various species of photosynthetic bacteria are invariably inactive in the mammalian cytochrome oxidase system, as is the characteristic cytochrome / of plant chloroplasts 6. Similar inactivity with the mammalian enzyme is encountered among some of the "c" cytochromes of the facultatively anaerobic denitrifiers 1. The behavior toward the mammalian flavoprotein cytochrome c reductase is less specific. In those cases which have been examined it has been found that bacterial cytochrome reductase activity is greater with the bacterial pigment than with cytochrome c~5. In addition to these interesting phenomena involving pigments which m a y be considered as true "c" cytochromes, there are also the observations on cytoehromes of certain strict anaerobes which appear to be hybrid in character
Re]erences p. ,523.
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in t h a t t h e y u n d e r g o s p e c t r a l c h a n g e s a n d h e m o c h r o m o g e n r e a c t i o n s of c y t o c h r o m e "c" but have electrochemical properties more usually encountered in cytochrome b o r m y o g l o b i n TM. VERHOEVEN AND TADEKA h a v e f o u n d 2 t h a t P s e u d o m o n a s a e r u g i n o s a - c y t o c h r o m e 552 c a n f u n c t i o n as a n i n t e r m e d i a t e i n o x y g e n r e d u c t i o n b y t h e i n t a c t b a c t e r i u m . ( H o w e v e r , a e r o b i c a l l y - g r o w n cells c o n t a i n v e r y l i t t l e of t h i s c y t o c h r o m e so t h a t i t s p h y s i o l o g i c a l role is u n c e r t a i n . ) I n c r u d e e x t r a c t s p r e p a r e d f r o m b a c t e r i a l s u s p e n s i o n s , reduced mammalian cytochrome c can link with nitrate, nitrite and other nitrogen o x i d e s , as well as w i t h o x y g e n , b u t o n l y t h e b a c t e r i a l c y t o c h r o m e is a c t i v e i n a p a r t i a l l y purified soluble nitrite reductase system obtained from the crude bacterial extracts. O n t h e o t h e r h a n d , P s e u d o m o n a s a e r u g i n o s a - c y t o c h r o m e - 5 5 2 is n o t a c t i v e e i t h e r i n t h e m a m m a l i a n c y t o c h r o m e o x i d a s e s y s t e m (S. VELICK AND C. 1zi'. STRITTMATTER, p r i v a t e c o m n m n i c a t i o n ; also R. SANADI, p r i v a t e c o m m u n i c a t i o n ) or i n t h e m a m m a l i a n s u c c i n o - d e h y d r o g e n a s e s y s t e m (R. SANADI, p r i v a t e c o m m u n i c a t i o n ) . T h u s i t a p p e a r s t h a t t h e b a c t e r i a l p i g m e n t , i n f u n c t i o n i n g as d i c t a t e d b y t h e p h y s i o l o g y of t h e b a c t e r i u m , is s o m e h o w m o d i f i e d so t h a t - - w h i l e r e t a i n i n g t h e m a i n f e a t u r e s of c y t o c h r o m e c s t r u c t u r e - - i t c a n n o l o n g e r a c t as a s u b s t r a t e for m a m m a l i a n c y t o c h r o m e systems concerned with oxygen reduction. This particular bacterial pigment provides a g o o d e x a m p l e of t h e n e e d for f u r t h e r i n v e s t i g a t i o n s i n t o t h e r e l a t i o n b e t w e e n s t r u c t u r e a n d f u n c t i o n i n c y t o c h r o m e c, u s i n g t h e v a r i e t y of b a c t e r i a l c y t o c h r o m e s n o w a t h a n d . SUMMARY i. Cytochrome c from the aerobic denitrifying bacterium, Pseudomonas aeruginosa, has been prepared in purity greater than 99 %. Its spectrochemical and electrochemical properties have been studied and compared with those of a pure sample of mammalian cytochrome c. 2. The bacterial pigment shows spectrochemical behavior characteristic of mammalian cytochrome c in the visible, except for a shift of the maximum in the alpha band toward the red. Absorption in the U.V. appears less for the bacterial pigment. 3. The bacterial protein is more oxidizing than the mammalian protein, over the whole physiological range, is not active in any of the mammalian systems involved in oxidation of DPNH, succinate, etc., but can serve as a substrate for reduction of oxides of nitrogen when incubated with bacterial extracts. 4. The amino acid composition of the bacterial pigment differs grossly from t h a t of the mammalian compound, as is to be expected from electrophoretic studies which show t h a t the bacterial protein is negatively charged at neutral pH in contrast with the mammalian protein, which is positively charged. 5- The significance of these studies for further researches into the relation between function and structure in cytochrome c is indicated. REFERENCES i M. D. KAMEN AND L. P. VERNON, Biochim. Biophys. Acta, 17 (~955) IO. $ W. VERHOEVEN AND Y. TADEKA, Symposium on Nitrogen Metabolism, Johns Hopkins Univ. Press, Baltimore (I956), p. 159. 3 L. P. VERNON AND M. D. KAMEN, J. Biol. Chem., 211 (I954) 643. 4 D. L. DRABKIN, J. Biol. Chem., 14o (I94 I) 287. A. L. LEVY, Nature, 174 (1954) 126. 6 H. E. DAVENPORT AND R. HILL, Proc. Roy. Soc. (London), B 139 (1952) 327 . H. THEORELL AND A. AKESSON, J. Am. Chem. Soc., 63 (1941) 18o 4. s A. DE BARBIERI AND A. ZAMBON1, Boll. soc. ital. biol. sper., 27 (I951) 3439 S. PALEUS AND J . B. NEILANDS, Acta Chem. Scan&, 3 (195 °) lO24. 10 T. L. McMEEKIN, M. L. GROVES AND N. J. HIPP, J. Am. Chem. Soc., 71 (1949) 3298. 11 H . EHRENBERG AND H . THEORELL, Nature, 176 (1955) 158. 12 H . T u P p Y AND S. PALEUS, Acta Chem. Scan&, 9 (1955) 353. 13 S. PALEUS, A, EHRENBURG AND H . TUPPY, ibid., 9 (1955) 365 . 14 C. L. Tsou, Biochem. J., 49 (1951) 362. 1~ M. D. KAMEN AND L. P. VERNON, dr. Biol. Chem., 211 (1954) 663. la j . vv'. NEWTON AND M. D. KAMEN, Arch. Biochem. Biophys., 58 (1955) 246. R e c e i v e d D e c e m b e r I 4 t h , 1955 34
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4 M. M. RAPPORT, K. MEYER AND A. LINKER, J. Am. Chem. Soc., 73 (I951) 2416. 5 K. MEYER, A. LINKER, ]~. A. DAVIDSON AND B. \¥EISSMANN, Jr. Biol. Chem., 20.5 (1953) 61I. 6 E. A. DAVIDSON AND K. MEYER, J. Biol. Chem., 211 (1954) 6o57 R. \V. JEANLOZ AND E. FORCHIELLI, J. Biol. Chem., 186 (195o) 495. 8 p. j . STOFFYN AND R. W. JEANLOZ, Arch. Biochem. Biophys., 52 (1954) 3739 }3. S. ]~LUMBERG, G. OSTER AND K. MEYER, J. Clin. Invest., 34 (1955) 1454. 10 Z. DISCI~E, J. Biol Chem., i 6 7 (1947) I89. 11 M. V. TRACEY, Biochem. J., 43 (1948) 185. 12 K. MEYER AND E. CHAFFEE, J. Biol. Chem., 138 (1941) 491. 13 K, MEYER AND M. M. RAPPORT, Advances in Enzymol,, 13 (1952) 199. 14 13. WEISSMANN, M. M. RAPPORT, A. LINKER AND K. MEYER, J . Biol. Chem., 205 (1953) 205. is A. COOCEIRO AND R. C. FREIRE, Anais acad. brasil, cienc., 23 (1951) 443. 16 M. M. RAPPORT, K. MEYER AND A. LINKER, J. Biol. Chem., 186 (195o) 615. J7 ~V. E. TREVELYAN AND J. S. HARRISON, Biochem. J., 30 (I952) 298. 19 j . X. KHYM AND D. G. I)OHERTY, J. Am. Chem. Soc., 74 (1952) 3199 . 19 C. H. DOHLMAN AND E. A. }3ALAZS, Arch. Biochem. Biophys., 52 (1955) 445. 20 H. GROSSFELD, K. MEYER AND G. GODMAN, Proc. Soc. Exptl. Biol. Med., 88 (1955) 31. 21 K, MEYER AND E. CHAm~EE, J. Biol. Chem., 133 (194 o) 83. 2a G. BLIX, Acta Soc. Med. Upsaliensis, 56 (195 I) 47. 23 R. AMPRINO, Acta Anat., 24 (1955) 121. 24 S. F. D. ORR, Biochim. Biophys. Acta, 14 (1954) 173. 25 }3. \¥EISSMANN, jr. Biol. Chem., 216 (1955) 783 . 2~ R. MARBETH AND A. WINTERSTEIN, Experientia, 8 (1952) 41. 27 H. SMITH AND R. C. GALLOP, Biochem. J., 53 (1953) 666. 2s Vv. P. DEISS AND A. S. LEON, J. Biol. Chem., 215 (1955) 685 . 29 S. SCHILLER, M. B. MATHEWS, H. JEFFERSON, J. LtlOOWlEG AND A. DORFMAN, J. Biol. Chem., 211 (1954) 717 .
Received December 3rd, 1955
A COMPARATIVE STUDY OF BACTERIAL AND MAMMALIAN C Y T O C H R O M E c* by M A R T I N D. K A M E N * * AND Y O S H I R O T A K E D A * * *
INTRODUCTION It has been shownI that a variety of facultative anaerobic bacteria contains cytochromes of the "c" type which can be obtained in good yield as soluble proteins of high purity. These cytochromes differ radically in electrochemical and biochemical properties from their counterparts in mammalian tissue, although they show close similarities in spectrochemica] properties. In this paper, there are presented and discussed studies which indicate changes in structure apparently associated with differences in function reflecting the physiology of the source material. The cytochrome c, derived from the aerobic denitrifier, Pseudomonas aeruginosa, shown previously by *Most of the work describedin this report was donewhilethe authors were in residenceat the Universityof California, Berkeley, Californiaduringthe summerof 1955, and was made possible by a special grant-in-aidto one of us (M.D.K.) from the C. F. Kettering Foundation,Yellow Springs, Ohio. We record here our special indebtednessto Professor W. M. STANLEYfor opening the Virus Laboratory to us and to Dr. H. K. SCHACHMANand Miss PEARLAPPELfor cooperation in makingthe sedimentationstudies. ProfessorCI-IAOHo LI most generouslyplaced at our disposal t h e facilities of t h e H o r m o n e R e s e a r c h L a b o r a t o r y w h e r e we r e c e i v e d invaluable i n s t r u c t i o n on the p a p e r c h r o m a t o g r a p h y of p r o t e i n h y d r o l y z a t e s fr om Dr. D. CHUNG a n d Mr. A. PARCELLS. A t W a s h i n g t o n U n i v e r s i t y , t e c h n i c a l a s s i s t a n c e in t h e e l e c t r o p h o r e s i s e x p e r i m e n t s w a s p r o v i d e d
References p. 523.
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VERHOEVEN AND TAKEDA2 to be obtainable in high yields and degree of purity, is compared with respect to amino acid composition, spectrochemical, electrochemical and enzymic properties with a typical classical cytochrome c preparation from mammalian heart tissue. METHODS AND MATERIALS
Isolation and purification o/Pseudomonas cytochrome Five h u n d r e d ml of a cell s u s p e n s i o n (IOO g w e t weight) of Ps. aeruginosa were h o m o g e n i z e d in a W a r i n g B l e n d o r in t h e presence of a t r a c e of D o w - C o r n i n g A n t i - F o a m . T h e s u s p e n s i o n r e s u l t i n g w a s h e a t e d to 5 °o C a n d I/IO v o l u m e of I M citric acid added. T h e m i x t u r e w a s allowed to s t a n d at 5 °0 C for five m i n u t e s , a f t e r w h i c h it w a s cooled in a n ice b a t h . A f t e r c e n t r i f u g a t i o n (6ooo r.p.m., 20 m i n u t e s - I n t e r n a t i o n a l R e f r i g e r a t e d Centrifuge) t h e s u p e r n a t a n t fluid was n e u t r a l i z e d to p H 7.0 w i t h 6 N N a O H . A m m o n i u m s u l f a t e was a d d e d slowly to a final c o n c e n t r a t i o n of 4 ° g per i o o ml. T h e p r e c i p i t a t e was r e m o v e d b y centrifugation, as above, a n d t h e s u p e r n a t a n t fluid t r e a t e d w i t h a m m o n i u m sulfate, b r i n g i n g t h e salt c o n c e n t r a t i o n to 65 g / i o o ml. A f t e r s t a n d i n g for several h o u r s in t h e refrigerator, t h e p r e c i p i t a t e c o n t a i n i n g t h e c y t o c h r o m e , was collected b y c e n t r i f u g a t i o n (Sorval h i g h - s p e e d centrifuge, t o p speed for io m i n u t e s ) , t a k e n u p in a s m a l l v o l u m e of distilled w a t e r a n d dialyzed t h o r o u g h l y a g a i n s t distilled water. A t this stage, t h e c y t o c h r o m e a p p e a r e d to be a b o u t 25 % p u r e on t h e basis of spectroscopic a b s o r p t i o n at 27o m/* c o m p a r e d w i t h t h a t of t h e reduced f o r m a t 552 m # . A c c u r a t e e s t i m a t i o n s b a s e d on spectroscopic d a t a could n o t be m a d e b e c a u s e t h e c h a r a c t e r i s t i c e x t i n c t i o n coefficients for t h e bacterial c y t o c h r o m e were n o t identical w i t h t h o s e of m a m m a l i a n c y t o c h r o m e in t h e U. V. region. F u r t h e r purification w a s a c c o m p l i s h e d b y successive a d s o r p t i o n on a l u m i n a Cy gel, followed b y elution with o.I M p h o s p h a t e buffer, p H 6. 5. Electrophoretic a n d centrifugal a n a l y s i s of t h e p r e p a r a t i o n s h o w e d t h e p r o t e i n to be h o m o g e n e o u s a n d all t h e h e m e c o m p o n e n t to be c o n t a i n e d in t h e one p r o t e i n p e a k found. I m p u r i t i e s e s t i m a t e d a t no m o r e t h a n 5 % of t h e t o t a l protein were r e m o v e d b y electrophoresis in t h e u s u a l Tiselius a p p a r a t u s , a n d t h e p u r e h e m e p r o t e i n was recovered f r o m t h e t e s t cell.
Chemical analyses and test materials C y a n i d e h e m o c h r o m o g e n w a s p r e p a r e d as described p r e v i o u s l y 3. Iron a n a l y s i s was p e r f o r m e d b y t h e m e t h o d of DRABKIN4. T h e p r o t e i n w a s h y d r o l y z e d a n d t h e a m i n o acid c o m p o s i t i o n d e t e r m i n e d b y q u a n t i t a t i v e p a p e r c h r o m a t o g r a p h y of t h e d i n i t r o p h e n y l derivatives, as described b y LEVY5. T h e bacterial c y t o c h r o m e u s e d w a s o b t a i n e d b y t h e p r o c e d u r e s o u t l i n e d a b o v e a n d could be e s t i m a t e d to be of a degree of p u r i t y ~ 99 %. T h e m a m m a l i a n c y t o c h r o m e was o b t a i n e d as a p r e p a r a t i o n 1 w h i c h a s s a y e d 0.43 % Fe i n d i c a t i n g a degree of p u r i t y well a b o v e 95 %. T h i s e s t i m a t e was borne o u t b y spectroscopic e x a m i n a t i o n .
Spectroscopic measurements S p e c t r a were d e t e r m i n e d w i t h a B e c k m a n n D U S p e c t r o p h o t o m e t e r b o t h before a n d after t r e a t m e n t w i t h t h e u s u a l oxidizing a n d r e d u c i n g agents.
Electrophoretic measurements E l e c t r o p h o r e t i c mobilities were m e a s u r e d w i t h t h e Klett-Tiselius a p p a r a t u s 1.
Potential measurements F e r r o - f e r r i c y a n i d e r e d o x buffers were used, according to t h e m e t h o d of DAVENPORT AND HILL 6.
Chemical and spectroscopic properties The bacterial pigment was found to concentrate in the same manner as its mammalian analogue when fractionated with ammonium sulfate. The purest sample which assayed b y Miss C. LOWRY of t h e D e p a r t m e n t of B i o c h e m i s t r y , a n d s o m e of t h e iron a n a l y s e s were p e r f o r m e d b y Dr. J. w . NEWTON of t h e Mallinckrodt I n s t i t u t e . F i n a l l y we wish to n o t e a gift of h i g h l y purified m a m m a l i a n c y t o c h r o m e c p r e p a r e d b y Dr. J. B. NEILANDS of t h e D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of California. "* Mallinckrodt I n s t i t u t e of Radiology, W a s h i n g t o n U n i v e r s i t y School of Medicine, St. Louis, Mo. (U.S.A.). *** N o w a t D e p a r t m e n t of N u t r i t i o n , O s a k a U n i v e r s i t y School of Medicine, O s a k a (Japan).
Re/erences p. .523.
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o.45 % Fe showed no appreciable protein contaminants, as determined by examination of the electrophoretic pattern. It was not auto-oxidizable in the physiological range and did not combine with CO in the reduced form. It was readily and reversibly oxidized or reduced by the usual reagents. It reacted with cyanide in alkali to form a cyanide hemochromogen which appeared spectroscopically identical with that obtained from mammalian cytochrome c (Table I). The bacterial pigment showed spectra in the reduced and oxidized states characteristic of mammalian cytochrome c, the two pigments being almost indistinguishable except for slight but reproducible and definite shifts in the maxima of the alpha bands. The bacterial pigment exhibited an alpha absorption maximum at 552 m~, compared to 550 m/~ for the mammalian pigment (Table I). There were appreciable departures from agreement in the shape of the ultra-violet protein absorption curve, as reflected in the ratios of the absorption at 27 ° m~ compared to those at the characteristic alpha and Soret peaks (Table I). TABLE I SPECTROSCOPIC PROPERTIES OF PSEUDOMONAS-CYTOCHROME-552 Bacterial pigment
Absorption m a x i m a (m~) Ferrocytochrome Ferricytochrome Cyanide f e r r o h e m o c h r o m o g e n
Mammalian cytochrome c
552, 52o, 416 53 ° (broad), 4o8-41o 555, 525, 421
55 ° , 520, 415 53 ° , 407 555, 525, 421
Ratios of ferrocytochrome a b s o r p t i o n m a x i m a : 27o/552:0.97 416/552:5.3 552/525 : 1.71
27o/55o: 1.o5 415/55o: 5.1 55o/52o: 1.66
Electrochemical and electrophoretic properties The difference in protein character, hinted at in the spectra, was established when the electrophoretic mobility was measured. The bacterial pigment migrated anodically with a value of 2.12.10 -5 cm2/volt-sec, compared to the cathodic velocity of 8.2. I0 -~ cm2/volt-sec determined for the mammalian cytochrome c at pH 7 (ammonium acetate buffer, 0.I N). The oxidation potentials of the bacterial pigment at a number of pH values, compared to those for the mammalian pigment, are shown in Table II. It is seen that the Pseudomonas aeruginosa cytochrome shares with the c cytochromes of the photosynthetic and other facultative anaerobes the property of an oxidation potential considerably more positive (more oxidizing) than that for mammalian cytochrome c. TABLE II O X I D A T I O N P O T E N T I A L S OF P S E U D O M O N A S - C Y T O C H R O M E - 5 5 2
pH ~ 5.0
Bacterial p i g m e n t Mammalian cytochrome c
0.336 --
6.0
o.3J, o.26 b
7.0
8,0
o.3oo 0.265
0.283 o.265
8.2
0.28o 0.279 . . . . .
Acetate buffer at p H 5 ; p h o s p h a t e buffer, p H 6 and 7 ; " t r i s " buffer, p H 8 and over. R e / e r e n c e s p. 523.
9.0
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Molecular weight From the heme iron content of o.45 %, a minimum molecular weight of 12,400 would be expected. Determinations made using the Spinco analytical ultracentrifuge and employing both sedimentation velocity and sedimentation equilibrium procedures were in agreement with this value, both giving molecular weights near 13,000 (H. K. SCHACHMAN,private communication). The weight calculated from nitrogen recovery and summation of amino acid residues was also in agreement with this value (see next section). Amino acid composition Although THEORELL AND AKESSON prepared mammalian cytochrome c in pure form nearly 15 years ago and gave a preliminary amino acid analysis for the protein at that time:, it appears that only one report has since been published on its amino acid composition. BARBIERI AND ZAMBONIs have analyzed hydrolyzates of cytochrome c, purified according to the procedure of PALEUS AND NEILANDS°, by column chromatography as well as by microbiological assay and have given the following values for the integral amino acid residues: glutamic, 6.4; asparagine, 26.0; cystine, 1.o2; serine, 3.44; glycine, lO.3O; alanine, 3.03; threonine, 2.16; proline, 3.3; valine, 5.83; histidine, 0.87; methionine, 4.63; leucine (and isoleucine) 9.28; phenylalanine, 1.7; tyrosine, 2.16; lysine, 13.4; arginine, 2.07 ; tryptophane, 0.8. The protein assayed o.456% Fe and 12.6% amino nitrogen. Unfortunately, m a n y discrepancies appeared when the results of the column chromatography were compared with those obtained by microbiological assay. Insufficient detail is available to evaluate these fluctuations. It was evident from the electrophoretic behavior of the bacterial protein that it differed in gross chemical composition from the mammalian protein. It was possible to make a semi-quantitative analysis of both preparations using the paper chromatographic procedure of LEVY~ In Table III, results are shown for all amino acids present with the exception of tryptophane, which was largely decomposed under the conditions of acid hydrolysis used. Both groups of workers quoted above appear to agree that approximately one mole of tryptophane is present per mole of protein. Assuming this value, the recovery of total nitrogen in the amino acids was 98-99%. The per cent amino nitrogen was 12.4%. TABLE III AMINO ACID COMPOSITION OF BACTERIAL AND MAMMALIAN PIGMENTS
Integral number o] residues Amino acid Pseud. cyto.-552
Glutamic + aspartic Cystine Serine Glycine + alanine Threonine Proline Valine Histidine Methionine Leucine + isoleucine Phenylalanine Tyrosine Lysine Arginine
Re[erences p. 523.
33 i 3 (32) 3 5 ii 2 (i) 14 4 2 9 2
Mammalian-cyto.-c
(27) 3 I 14 Ii 2 4 4 (i) ii 4 (2) 18 2
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The results shown in Table I I I must be regarded as preliminary because the method used is capable of much better precision than obtained in the short time available for these investigations. In a few cases, reproducible values were not easy to obtain. These are shown in brackets. In all such cases, with the exception of methionine, the m a x i m u m value found is given. In all the other amino acids, the m a x i m u m deviation in 8-1o separate determinations did not exceed 25 % and in most cases was much smaller. From our experience, it seems evident that cytochrome c can be analyzed with a precision better than a few per cent for all its constituent amino acids, using paper chromatography. The results obtained, despite the limitations mentioned, bear out the expectation of gross differences in amino acid composition between the bacterial and mammalian preparations. If it were not for the lumping of aspartic and glutamic acids, the lack of data on amide nitrogen, uncertainty as to whether serine should be considered in the phosphorylated form, and other difficulties, these data could be used to calculate specific volume, as described by McMEEI~IN et al 1°. However, limits on the values possible can be assigned. The range calculated for the bacterial and mammalian preparations are o.73o-o.75o and o.715-o.727, respectively. The molecular weight calculated, based on the results shown in Table I I I and assuming one residue weight of tryptophane per mole protein, is 12,16o for the bacterial and 12,44 ° for the mammalian protein. DISCUSSION EHRENBERG AND THEORELL 11 have presented arguments for a cytochrome c structure in which the ferroheme group is surrounded by four peptide spirals, the iron being coordinated in the fifth and sixth positions out of the porphin plane to histidine residues attached to separate peptide spirals. The old assumption of binding of the side chains of the porphin nucleus through two cysteine residues by saturated thio-ether linkages is retained in this structure. While the amino acid composition of the m a m m a lian cytochrome c is certainly consistent with the requirement of at least two histidines and two cysteines, the corresponding values for the bacterial pigment are just barely adequate. It would appear that extension of the methods of PAI.EUS, TUPPY and othersle, la, including sequence analysis of Fe-containing peptides 14, to specimens of the bacterial "c" cytochromes is desirable to test the general validity of structures based on mammalian cytochrome c alone. The fact that the enzymic behavior of the bacterial pigments differs radically from that of the mammalian analogue also emphasizes the need for structural analysis of cytochrome c proteins derived from a variety of sources. It has been shown ~5 that the bacterial "c" cytochromes obtained from various species of photosynthetic bacteria are invariably inactive in the mammalian cytochrome oxidase system, as is the characteristic cytochrome / of plant chloroplasts 6. Similar inactivity with the mammalian enzyme is encountered among some of the "c" cytochromes of the facultatively anaerobic denitrifiers 1. The behavior toward the mammalian flavoprotein cytochrome c reductase is less specific. In those cases which have been examined it has been found that bacterial cytochrome reductase activity is greater with the bacterial pigment than with cytochrome c~5. In addition to these interesting phenomena involving pigments which m a y be considered as true "c" cytochromes, there are also the observations on cytoehromes of certain strict anaerobes which appear to be hybrid in character
Re]erences p. ,523.
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in t h a t t h e y u n d e r g o s p e c t r a l c h a n g e s a n d h e m o c h r o m o g e n r e a c t i o n s of c y t o c h r o m e "c" but have electrochemical properties more usually encountered in cytochrome b o r m y o g l o b i n TM. VERHOEVEN AND TADEKA h a v e f o u n d 2 t h a t P s e u d o m o n a s a e r u g i n o s a - c y t o c h r o m e 552 c a n f u n c t i o n as a n i n t e r m e d i a t e i n o x y g e n r e d u c t i o n b y t h e i n t a c t b a c t e r i u m . ( H o w e v e r , a e r o b i c a l l y - g r o w n cells c o n t a i n v e r y l i t t l e of t h i s c y t o c h r o m e so t h a t i t s p h y s i o l o g i c a l role is u n c e r t a i n . ) I n c r u d e e x t r a c t s p r e p a r e d f r o m b a c t e r i a l s u s p e n s i o n s , reduced mammalian cytochrome c can link with nitrate, nitrite and other nitrogen o x i d e s , as well as w i t h o x y g e n , b u t o n l y t h e b a c t e r i a l c y t o c h r o m e is a c t i v e i n a p a r t i a l l y purified soluble nitrite reductase system obtained from the crude bacterial extracts. O n t h e o t h e r h a n d , P s e u d o m o n a s a e r u g i n o s a - c y t o c h r o m e - 5 5 2 is n o t a c t i v e e i t h e r i n t h e m a m m a l i a n c y t o c h r o m e o x i d a s e s y s t e m (S. VELICK AND C. 1zi'. STRITTMATTER, p r i v a t e c o m n m n i c a t i o n ; also R. SANADI, p r i v a t e c o m m u n i c a t i o n ) or i n t h e m a m m a l i a n s u c c i n o - d e h y d r o g e n a s e s y s t e m (R. SANADI, p r i v a t e c o m m u n i c a t i o n ) . T h u s i t a p p e a r s t h a t t h e b a c t e r i a l p i g m e n t , i n f u n c t i o n i n g as d i c t a t e d b y t h e p h y s i o l o g y of t h e b a c t e r i u m , is s o m e h o w m o d i f i e d so t h a t - - w h i l e r e t a i n i n g t h e m a i n f e a t u r e s of c y t o c h r o m e c s t r u c t u r e - - i t c a n n o l o n g e r a c t as a s u b s t r a t e for m a m m a l i a n c y t o c h r o m e systems concerned with oxygen reduction. This particular bacterial pigment provides a g o o d e x a m p l e of t h e n e e d for f u r t h e r i n v e s t i g a t i o n s i n t o t h e r e l a t i o n b e t w e e n s t r u c t u r e a n d f u n c t i o n i n c y t o c h r o m e c, u s i n g t h e v a r i e t y of b a c t e r i a l c y t o c h r o m e s n o w a t h a n d . SUMMARY i. Cytochrome c from the aerobic denitrifying bacterium, Pseudomonas aeruginosa, has been prepared in purity greater than 99 %. Its spectrochemical and electrochemical properties have been studied and compared with those of a pure sample of mammalian cytochrome c. 2. The bacterial pigment shows spectrochemical behavior characteristic of mammalian cytochrome c in the visible, except for a shift of the maximum in the alpha band toward the red. Absorption in the U.V. appears less for the bacterial pigment. 3. The bacterial protein is more oxidizing than the mammalian protein, over the whole physiological range, is not active in any of the mammalian systems involved in oxidation of DPNH, succinate, etc., but can serve as a substrate for reduction of oxides of nitrogen when incubated with bacterial extracts. 4. The amino acid composition of the bacterial pigment differs grossly from t h a t of the mammalian compound, as is to be expected from electrophoretic studies which show t h a t the bacterial protein is negatively charged at neutral pH in contrast with the mammalian protein, which is positively charged. 5- The significance of these studies for further researches into the relation between function and structure in cytochrome c is indicated. REFERENCES i M. D. KAMEN AND L. P. VERNON, Biochim. Biophys. Acta, 17 (~955) IO. $ W. VERHOEVEN AND Y. TADEKA, Symposium on Nitrogen Metabolism, Johns Hopkins Univ. Press, Baltimore (I956), p. 159. 3 L. P. VERNON AND M. D. KAMEN, J. Biol. Chem., 211 (I954) 643. 4 D. L. DRABKIN, J. Biol. Chem., 14o (I94 I) 287. A. L. LEVY, Nature, 174 (1954) 126. 6 H. E. DAVENPORT AND R. HILL, Proc. Roy. Soc. (London), B 139 (1952) 327 . H. THEORELL AND A. AKESSON, J. Am. Chem. Soc., 63 (1941) 18o 4. s A. DE BARBIERI AND A. ZAMBON1, Boll. soc. ital. biol. sper., 27 (I951) 3439 S. PALEUS AND J . B. NEILANDS, Acta Chem. Scan&, 3 (195 °) lO24. 10 T. L. McMEEKIN, M. L. GROVES AND N. J. HIPP, J. Am. Chem. Soc., 71 (1949) 3298. 11 H . EHRENBERG AND H . THEORELL, Nature, 176 (1955) 158. 12 H . T u P p Y AND S. PALEUS, Acta Chem. Scan&, 9 (1955) 353. 13 S. PALEUS, A, EHRENBURG AND H . TUPPY, ibid., 9 (1955) 365 . 14 C. L. Tsou, Biochem. J., 49 (1951) 362. 1~ M. D. KAMEN AND L. P. VERNON, dr. Biol. Chem., 211 (1954) 663. la j . vv'. NEWTON AND M. D. KAMEN, Arch. Biochem. Biophys., 58 (1955) 246. R e c e i v e d D e c e m b e r I 4 t h , 1955 34