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THE MECHANISM OF PORPHYRIN FORMATION

THE R6LE OF THE TRICARBOXYLIC ACID CYCLE*

BY DAVID SHEMIN AND JONATHAN WITTENBERG

(From the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, New York)

(Received for publication, December 9, 1950)

In the present investigation, which is a continuation of our study on the biosynthetic mechanism of porphyrin formation, we have incubated samples of duck blood (1) with CY4-methyl-labeled and with C14-carboxyl-labeled acetate and have determined, by complete chemical degradation (2) (Fig. l), the carbon atoms in the protoporphyrin derived from the carbon atoms of acetate. The experiments with deuterioacetate (3) suggested that some of the carbon atoms of the side chains of protoporphyrin are derived from the methyl group of acetic acid, since these carbon atoms and the methene bridge carbon atoms are the only ones bonded to hydrogen. More re- cently it has been demonstrated that the methyl groups (carbon atom 6) of protoporphyrin and the p-carbon atoms of the pyrrole rings to which the methyl groups are attached (carbon atom 4) are derived from the methyl group of acetic acid, whereas the carboxyl groups of the porphyrin are derived from the carboxyl group of acetate (4). However, these 10 carbon atoms did not account for all the Cl4 activity found in hemin biosynthetically derived from C4-carboxyl- and C14-methyl-labeled acetate.

Previous studies on the biosynthesis of protoporphyrin have demonstrated the Ale of glycine (5). The nitrogen atoms of glycine are utilized for both types of pyrrole rings (Fig. 1, rings A and B, C and 0) (6, 7) ; 8 a-carbon atoms of glycine are utilized for each porphyrin molecule (8,9), but its carboxyl group is not utilized (8, 10). It was found that 4 (Y- carbon atoms of glycine (2) occupy comparable positions in both types of pyrrole rings (see Fig. 1, carbon atoms numbered 2) and that 4 more are utilized for the methene bridge carbon atoms (2, 9). Glycine therefore accounts for 8 of the 34 carbon atoms of the porphyrin and in this publication the remaining 26 of the carbon atoms are shown to be derived from acetate via a 4-carbon atom unsymmetric compound.

In this paper the numbering system for porphyrins is the same as that previously employed (2). Uroporphyrin serves as the parent compound.

* The work was supported by grants from the Rockefeller Foundation, from the National Institutes of Health, United States Public Health Service, and from the American Cancer Society on the recommendation of the Committee on Growth of the National Research Council.

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316 MECHANISM OF PORPHYRIN FORMATION

The carbon atoms of protoporphyrin would therefore be numbered as shown in Fig. 1. With this system it is comparatively easy to designate a carbon atom; e.g., carbon atom A4 refers to the ,&carbon atom of pyrrole ring A of protoporphyrin to which the methyl group is attached.

EXPERIMENTAL

Measurement of Radioactivity-All samples were assayed for their radioactivity as barium carbonate, and were counted as in the previous investigation (2). The results are reported as counts per minute of an “in- finitely thick” sample in a standard dish and as total activity, which is equivalent to counts per minute times the number of carbon atoms in the compound. Total activity is proportional to the activity per mole of the compound (2). In the experiments with methyl-labeled acetate and carboxyl-labeled acetate counts of less than 10 and 3 per minute above back- ground, respectively, were not considered significant.

Methyl-Labeled Sodium Acetate (C14H3COONa)-2 mc. of isotopic sodium acetate (Tracerlab, Lot 12) were diluted with non-isotopic sodium acetate to give an activity of 6.34 X lo6 c.p.m. as barium carbonate and contained approximately 0.16 mc. per mM.

Carboxyl-Labeled Sodium Acetate (CIJ$Y400Na)-Two samples were prepared by the reaction of CY402 with methyl magnesium iodide. Sample H61 had an activity of 11.6 X lo5 c.p.m. and contained approximately 0.23 mc. per mM, and Sample H62 had an activity of 4.85 X lo5 c.p.m. and contained approximately 0.1 mc. per mM.

Although both of these samples of carboxyl-labeled acetate were used in the experiments for heme synthesis, more of Sample H61 than of Sample H62 was employed. The average activity of the carboxyl-labeled acetate used can be calculated to be 10.6 X lo5 c.p.m. containing about 0.21 me. per mM.

Incubation of Duck Blood and Preparation of Labeled Hemin-Nineteen ducks were exsanguinated, providing a total of 2025 ml. of blood. In no case were samples of blood from different ducks mixed before incubation.

Labeled hemin was obtained by incubating whole duck blood with isotopically labeled sodium acetate and isotopically labeled glycine as previously described (1). For each ml. of blood 1 mg. of anhydrous isotopic sodium acetate and 1 mg. of glycine labeled with 32 atom per cent excess N15 were added as an isotonic, solution. These amounts are sufficient to give maximal incorporation of isotopes into the hemin.

In order to insure that the rates of synthesis of hemin were the same in the experiment with methyl-labeled acetic acid as in that with carboxyl- labeled acetic acid, the blood of each duck was divided into two parts. One-half was incubated with methyl-labeled acetic acid plus glycine, and the other half was incubated with carboxyl-labeled acetic acid plus glycine.


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HEMIN +

ME

PROTOPORPHYRIN Ix

HEMATINIC ACID

FH2 COOH

MESOPORPHYRIN MESOPORPHYRIN

CH2 CH3

GH3 CH3GH2

THYLETHYLMALEIMIDE I)

Z-(3)-METHYL-3-(2) ETHYLTARTARIMIDE

METHYLETHYLMALEIMIDE

CH3-CO-COOH __)

6-4-5

+

CH,-COOH W

6-4

+

CO2

CH3NH2

6

+

CO2 4

CH3- CH2-COOH --)

9-8-3

CH3-CH2- CO- COOH + +

9-8 -3-2 CO2 2

c CH3NH2 + CO2 9 9

(II) CH3-COOH -)

c

-I-

9-8 CO2 8

w CO2 6

CH3-CH2NH2 +

9-8

+

COP 3

FIG. 1. Protoporphyrin degradation. The letters and numbers designate positions of the carbon atoms.

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In the experiment with carboxyl-labeled acetate a total of 800 ml. of duck blood was incubated with the acetate Sample H61 and a total of 212 ml. with acetate Sample H62. 2.012 and 0.354 gm. of hemin were isolated from the blood samples which had been incubated with Samples H61 and H62, respectively, and then pooled. The ratio of the Cl4 activities of the two isolated hemin samples was the same as the ratio of activities of the two acetate samples, demonstrating that the utilization of acetate was the same for both samples of hemin. The average activity 10.6 X lo6 c.p.m. of the carboxyl-labeled acetate used for hemin synthesis can be calculated from the following equation, which is based on the amounts of hemin isolated.

(11.6 X 105 X 2.012) + (4.85 x lo5 X 0.354) = 1. 6 x lo6 c p m 2.012 + 0.354

. . .

The rates of synthesis of hemin in the experiments with methyl-labeled acetate and the two samples of carboxyl-labeled acetate were practically the same. This is demonstrated by the finding of equal N16 concentrations in the hemin samples. The hemin prepared from methyl-labeled acetate, from carboxyl-labeled acetate Sample H61, and from carboxyl- labeled acetate Sample H62 contained 0.145, 0.147, and 0.152 atom per cent excess N15, respectively.

The labeled hemin samples were diluted with non-isotopic hemin in order to provide more material for the degradation experiments. The 2.676 gm. of hemin obtained from the methyl-labeled acetic acid were diluted with 3.285 gm. of non-isotopic hemin. The 2.366 gm. of hemin obtained from the carboxyl-labeled acetic acid were diluted with 3.789 gm. of non-isotopic hemin. Since the rates of synthesis were the same, one can compare the activities in the carbon atoms of the diluted samples of the hemin from the two experiments. By multiplying the activities of the carbon atoms found in the carboxyl-labeled experiments by 0.696, the values observed in the two experiments are made comparable.

6.34 X lo6 X z X g = 0.696

10.6 X lo6 . .

Degradation of Labeled Hemin

The method of degradation of hemin used in this paper represents an extension of the method previously presented (see Fig. 1) (2).

Preparation of Protoporphyrin-The hemin was converted to protoporphyrin by treatment with powdered iron in boiling formic acid (11). The yields in each case were about 99 per cent.

Preparation of Mesoporphyrin-The protoporphyrin samples were converted to mesoporphyrin by hydrogenation in 100 per cent formic acid


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D. SHEMIN AND J. WI’M’ENBERG 319

solution with colloidal palladium catalyst (12) and isolated as previously described (2). The yield in each case was 96 per cent.

Oxidation of Mesoporphyrin to Methylethylmaleimide and Hematinic Acid-A solution of mesoporphyrin in 20 per cent sulfuric acid was stirred for 18 hours at room temperature with a small excess of chromic acid (13, 14). The products of the oxidation were collected by ether extraction and separated and purified as previously described (2).

The yield of methylethylmaleimide, purified by repeated sublimation, was in both cases 44 per cent of theoretical. The product derived from methyl-labeled acetic acid had a melting point of 65.0-66.4” (Kiister (13) reported 67”; Fischer et aE. (14) 70”) ; that from carboxyl-labeled acetic acid melted at 63.8-65.4”.

GHgOsN. Calculated. C 60.4, H 6.5 From methyl-labeled acetic acid. Found. “ 60.6, “ 6.3

I‘ carboxyl- “ ‘I “ “ ‘I 60.2, “ 6.5

The hematinic acid was isolated as previously described (2) and recrys- tallized from ethyl acetate-petroleum ether. That from methyl-labeled acetic acid (yield 52 per cent) melted at 114.2-115.0” (Kiister (15), 113.5- 114.5”) ; that from carboxyl-labeled acetic acid (yield 38 per cent) at 114.0- 114.6”.

GHIO~N. Calculated. C 52.5, H 4.9 From methyl-labeled acetic acid. Found. “ 52.5, “ 4.8

‘I carboxyl- ‘I “ “ ‘( “ 52.2, “ 5.1

Conversion of Hematinic Acid to CO2 and Methylethylmaleimide-Hema- tinic acid was decarboxylated by heating a suspension in nearly saturated ammoniacal ethanol for 2 hours at 175” (15, 16). The reaction mixture was diluted with water, acidified, and the COZ formed was aerated into barium hydroxide solution. The yields of barium carbonate were 70 to 78 per cent. The methylethylmaleimide was isolated by extraction with ether, steam distillation, and repeated sublimation (2). The yield in the methyl-labeled acetic acid experiment was 52 per cent, m.p. 63-65”; in the carboxyl-labeled acetic acid experiment, 42 per cent, m.p. 65.5-66.5”.

GHsOgN. Calculated. C 60.4, H 6.5 From methyl-labeled acetic acid. Found. “ 60.5, “ 6.5

‘I carboxyl- “ “ “ “ “ 60.5, “ 6.5

Preparation of Pyruvic and or-Ketobutyric Acids-The methylethylmaleimide samples were oxidized with sodium chlorate and osmium tetraoxide in aqueous solution by the method of Milas and Terry (17). Titration of aliquots of the reaction mixture with periodic acid indicated that the oxidation was complete after about 20 hours at 25”. The resulting tartari- mide was cleaved, without isolation, by addition of 1 equivalent of sodium


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metaperiodate (18). After 20 minutes, the solution was extracted with ether for 5 hours in a continuous extractor. The ether was taken to dry- ness and the residue dissolved in 1 to 2 ml. of 0.5 N HCl and heated for 1 hour at 80”. The solution was then mixed with 1 to 2 gm. of anhydrous silica gel and transferred, with the aid of a small volume of butanol-chloroform solution, to a chromatographic column in which the pyruvic and a-ketobutyric acids were separated on silica gel by a modification (2) of the procedure of Isherwood (19). The solvent phases were 0.5 N HzS04 and butanol-chloroform mixture.

The keto acids were isolated from the column effluent as their 2,4- dinitrophenylhydrazones. A non-acidic impurity was removed by dissolv- ing the pyruvic hydrazone in dilute sodium bicarbonate, the a-ketobutyric hydrazone in a large volume of half saturated lithium carbonate, and by filtering and reprecipitating the hydrazones with dilute hydrochloric acid. The hydrazone of pyruvic acid was crystallized from 95 per cent ethanol; that of a-ketobutyric acid from acetic acid, as previously described (2). The yields, based on methylethylmaleimide, ranged from 20 to 40 per cent. All the samples of pyruvic acid hydrazone melted at 217-218” and those of a-ketobutyric acid hydrazone melted at 198-200”; and no de- pression in melting point was observed when the products were mixed with authentic samples of the hydrazones.

2,4-Dinitrophenylhydrazone of pyruvic acid CHON 8 6 6 4. Calculated. C 40.3, H 3.0 Carboxyl-labeled acetate From rings A and B Found. ‘( 40.3, ‘( 3.0

“ I‘ c “ D I‘ “ 40.4, “ 3.1 Methyl-labeled acetate From rings A and B “ <‘ 40.5, “ 3.3

“ ‘I (-J “ D “ I‘ 40.3, “ 3.2

2,4-Dinitrophenylhydrazone of a-ketobutyric acid CdSaOaN,. Calculated. C! 42.6, H 3.6 Carboxyl-labeled acetate From rings A and B Found. “ 42.5, “ 3.6

‘I “ (J “ D ‘L ” 42.5, ‘I 3.6 Methyl-labeled acetate From rings A and B ‘I “ 42.6, ‘I 3.6

“ “ c “ D I‘ “ 42.5, “ 3.5

Decarboxylation of a-Keto Acid Hydrazones-Pyruvic acid dinitrophenylhydrazone was oxidized with an excess of potassium permanganate in 5 per cent sulfuric acid, according to the procedure devised by Krebs (20) for the determination of cu-ketoglutaric acid. The yields of barium carbonate were only 71 to 82 per cent of the expected amounts. This we believe to be due to incomplete oxidation of the benzene ring, and not to an incom-


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D. SHEMIN AND J. WITTENBERG 321

plete release of COZ from the carboxyl group, since the yields of acetic acid were practically quantitative (2). On this assumption it is possible to correct the observed activity values by applying the corresponding yield factors (0.71 to 0.82). The corrected values represent the activities which should have resulted from complete oxidation.

The pyruvic acid dinitrophenylhydrazone samples were suspended in water and dissolved by the addition of 5 per cent sodium hydroxide. They were immediately reprecipitated by the addition of dilute sulfuric acid, and the mixture was diluted to bring the volume to about 100 ml. per mM of compound and the sulfuric acid concentration to 5 per cent. An excess of potassium permanganate dissolved in 5 per cent sulfuric acid was added, and the mixture held at 60” for 14 hours. The carbon dioxide liberated was aerated into barium hydroxide solution.

The excess permanganate was then reduced with hydrogen peroxide, and the solution filtered; the acetic acid formed was steam-distilled and converted to the sodium salt.

a-Ketobutyric acid hydrazone was decarboxylated in the same manner, except that the reaction was run at room temperature to minimize oxidation of the propionic acid by the permanganate. The yields of propionic acid ranged from 101 to 106 per cent.

The yields of barium carbonate were 62 to 73 per cent. Appropriate corrections were similarly applied to the radioactivities of the a-ketobutyric acid carboxyl group.

Decarboxylation of Propionic and Acetic Acids-The propionic acid samples were converted into CO2 and ethylamine by a modification (21) of the Schmidt reaction. Phares (22) has demonstrated that by this reaction’ each carbon atom of acetic and propionic acids can be analyzed separately. The ethylamine was oxidized with alkaline permanganate to acetic acid.

The samples of acetic acid, both from the decarboxylation of propionic acid and of pyruvic acid hydrazone, were similarly decarboxylated by the Schmidt reaction. The resulting methylamine was oxidized with alkaline permanganate. The carbon dioxide formed in each step was collected as barium carbonate.

DISCUSSION

Before the results are discussed in greater detail, it may be worth while to evaluate the experimental data in the light of certain relationships which were found to exist among different parts of the molecule. In the degradation methods used to locate each carbon atom, derived from the

1 The authors wish to express their sincere thanks to Dr. E. F. Phares of the Oak Ridge National Laboratory for furnishing thea with his unpublished methods for these degradations,


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carbon atoms of acetate in the porphyrin, crystalline and analytically pure products were obtained only through the or-keto acid stage. In the degradation of the keto acids, the derivatives were not obtained in crystalline form. As contaminants may have been present, or the degradation not completely specific, the Cl4 values ascribed to the individual carbon atoms are not as reliable as those for the crystalline porphyrins, the maleimides and the hydrasones of the cw-keto acids. It is considered that the activities of the single atoms can be more correctly evaluated from the data found for the analytically pure compounds.

The Cl4 activities of the crystalline degradation products reveal certain relationships among different parts of the porphyrin molecule. In the experiments in which methyl-labeled and carboxyl-labeled acetates were used, all the Cl4 activity of the porIjhyrin molecule resides in the products obtained from the chromic acid oxidation of the porphyrin.

The activity of pyrrole rings A and B is equal to that of pyrrole rings C and D (Tables I and II). In the carboxyl-labeled acetate experiment the methylethylmaleimide samples of both types of rings are used for this comparison, since the carboxyl group of rings C and D are highly radioactive and are not found in rings A and B (Tables I and II). Moreover the activities of the hydrazones of pyruvic and cr-keto butyric acids, repre- senting, respectively, the methyl side and the vinyl side of the pyrrole ring from A and B, are equal and their sum is equal to the total activity of the methylethylmaleimide. The same is true also for the two keto acids of C and D (Table II). Not only do all four hydrazones in each of the experiments have the same activity, but the hydrazones contain the same number of radioactive carbon atoms: 3 in the methyl-labeled acetate experiment and only 1 in the carboxyl-labeled acetate experiment. There- fore it is reasonable to expect that a definite pattern of activity exists among these active carbon atoms and that comparable carbon atoms, i.e. those bearing corresponding numbers in the porphyrins (Fig. l), would have the same activities. As this seems to be the case (see Table I), it appears valid, especially in view of evidence presented below, to average the activities of comparable carbon atoms. We have done this and shall use these average values in this discussion. The average Cl4 activities for each pair of carbon atoms, 6 and 9, 4 and 8, and 5 and 3, are given in Fig. 2. The sum of the total activities for these three pairs of carbon atoms (see Fig. 2) is 2795 c.p.m. in the methyl-labeled acetate experiment, which is in good agreement with the values found for the total activity of each of the four keto acids, 2720, 2780, 2750, and 2740 c.p.m. (Table I).

In the degradation of the hemin, the pyrrole units are isolated as pairs, rings A and B and rings C and D, since pyrrole A is identical with B and pyrrole C is identical with D, However, the conclusions we have drawn


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TABLE I

Distribution of Cl4 in Protoporphyrin from C%Vfethyl-Labeled Acetic Acid and from @Warboxyl-Labeled Acetic Acid

Glycine labeled with 32 atom per cent excess Nr5 was also added to the duck blood incubation mixture. The undiluted hemin samples contained 0.145 and 0.148 atom per cent excess N15 in the experiments with CWmethyl-labeled and W-carboxyl- Iabeled acetic acid respectively. -

E

/

Compound analyzed

(a)

Mesoporphyrin

Methylethylmaleimide

Hematinic acid

Methylethylmaleimide from hema tinic acid

Carboxyl group of hematinic acid Carboxyl group of hematinic acid1

Pyruvic acid 2,4-dinitrophenylhydrazone

a-Ketobutyric acid 2,4-dinitrophenylhydrazone

Pyruvic acid 2,4-dinitrophenylhydrazone

a-Ketobutyric acid 2,4-dinitrophenylhydrazone

Pyruvic acid, carboxyl group

a-Carbon atom of pyruvic acid p-Carbon atom of pyruvic acid

?ositions in proto-

porphyrin

Total

porphy rin

Rings A and B

Rings C and D

Rings C and D

ClO, Dl( ClO, Dll

A6, B6 A4, B4 A5, B5 A9, B9 A8, B8 A3, B3 A2, B2 C6, D6 C4, 04 C5, 05 C9, D9 C8, 08 C3, 03 C2, 02

A5, B5

A4, B4 A6, B6

tc

3 3

'0 '0

Factors to calculate

Ital activit!

Cc)

34

7

8

7

1

9

10

9

10

,711 x ’ ,811 x ’

1 1

-

YA i

<

7 7

dethyl-labeled Carboxyl-labeled acetate acetate

ctivit) ‘ound

(a

:.#.m

699

Total ,$y Activi

(d&x fount

(e) If) __-

c.p.m. c.p.m

3,770 123

-.

830 5,810 47

740 5,920 234

863 6,040 46

80 80 1816

302 2,720 16. .4

278 2,780 14. .9

306 2,750 16. .2

274 2,740 13 .6

155

973 1207

770 27

973 1. 1,210 0

.7

.4

.3 - -

COP ected total elf-

vity,* Cc) x

kii’,,?

(9)

.p.m.

1910

228

1310

225

L260 1080

103

104

102

95

109 0 0


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TABLE I-Concluded

Compound analyzed

(4 (8)

Carboxyl group of cr-ketobutyric acid

a-Carbon atom of a-ketobutyric acid

p-Carbon atom of a-ketobutyric acid

-r-Carbon atom of or-ketobutyric acid

Carboxyl group of pyruvic acid

a-Carbon atom of pyruvic acid p-Carbon atom of pyruvic acid

Carboxyl group of a-ketobutyric acid

or-Carbon atom of cu-ketobutyric acid

p-Carbon atom of a-ketobutyric acid

T-Carbon atom of oc-ketobutyric acid

F ‘ositions in Factors tc proto- calculate

porphyrin total activi

(4 ---

AZ, B2 0.691 X 0.62t x

83, B3 1

A8, B8 I

89, B9 I

C5, D5 0.81$ X 0.81f X

C4, 04 1 C6, D6 1

C2, 02 0.716 x 0.74$ x

C3, 03 1

C8, 08 1

C9, D9 1

Methyl-labeled acetate

T- rota1

ac- :ivity,

($)”

.;t:eJ 0 , i

(4

c.p.m.

7

776

854

1185

(4

c.p.m.

(

77f

85~

1,19(

147 83:

811 81: 1005 l,Ol(

5j i (

767j i 76

869{ 5 86!

1128( 3 1,13(

krboxyl-labeled acetate

(f)

c.p.m.

COP rected total

activity,* Cc) x

t&z

Cd

c.p.m.

0.2 0 136 95

0.: 0

1.c 0

26.i 106 2.: 0 l.f 0

0.1 0 126 88

0.:

1.:

0

0

* Total activity found X 0.696 to make both experiments comparable (see “Ex- perimental”).

t Calculated, difference between total activity of hematinic acid and methylethylmaleimide derived from hematinic acid.

$ Corrections for incomplete oxidation (see “Experimental”). 0 This sample of ol-ketobutyric acid 2,4-dinitrophenylhydrazone was diluted for

subsequent degradation reactions. 0.079 gm. of the isotopic hydrazone was diluted with 0.145 gm. of a non-isotopic sample of the hydrazone. The activities were therefore multiplied by 2.83.

from the data are concerned with each of the carbon atoms of each of the pyrrole rings. This appears to be valid since, as shown below, the biosynthetic mechanism for the dissimilar pairs of pyrrole rings is considered to be the same and it is reasonable to expect that each pyrrole of each pair is made in the same manner.


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TABLE II Cl4 Activity of Fragments of Protoporphyrin Molecule

Porphyrin fragment Methyl-labeled acetate

Porphyrin (mesoporphyrin) Rings A and B

Methylethylmaleimide Pyruvic acid

c.p.??k.

23,770

a-Ketobutyric acid

11,620 5,440 5,560

Pyruvic acid + a-ketobutyric acidt Rings C and D

Hematinic acid Methylethylmaleimide

Pyruvic acid

11,000

a-Ketobutyric acid

11,840 12,080

5,500 5,480

Pyruvic acid + a-ketobutyric acidt 10,980 Rings A + B + C + Dj 23,460

T Total activity*

Carboxyl-labeled acetate

c.p.m.

2910

456 206 208 - 414

2620 450

u)4 190 - 394

3080

* The total activities are those of Table I multiplied by 2, since the fragments are derived from pairs of pyrrole rings.

t Addition of activities found for pyruvic and a-ketobutyric acids. $ Addition of activities of methylethylmaleimide from rings A and B and hema-

tinic acid from rings C and D.

d4H3COOH Experiment CH3Ci400H Experiment

N H H

FIG. 2. Average activities of comparable carbon atoms in all pyrrole units. The activities are given in parentheses. The pyrrole unit represented contains a car- boxy1 group which is found only in rings C and D of protoporphyrin.

From the data the following conclusions on the biosynthetic mechanism of porphyrin formation can be drawn. It can be seen from Table II that the total activity of the porphyrins biosynthetically produced from CY4-


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methyl-labeled and carboxyl-labeled acetate resides in the carbon atoms other than the methene bridge carbon atoms. This is consistent with the previous finding that the a-carbon atom of glycine is the source of the methene bridge carbon atoms (2, 9).

In Tables I and II it can be seen that the total activity of pyrroles A and B is equal to that of pyrroles C and D in hemin made from methyl- labeled acetate. This comparison holds for pyrroles A and B and pyrroles C and D in hemin made from carboxyl-labeled acetate if one excludes, for the moment, those carboxyl groups in C and D which are not found in A and B. Also it can be seen from Table I and Fig. 2 that the carbon atoms of A and B that occupy similar positions in C and D have the same activities. This supplements our previous finding (2) that the carbon atoms A2 and B2 (derived from the a-carbon atom of glycine) have the same activity as the carbon atoms C2 and 02 (also derived from the a!- carbon atom of glycine) and that both types of pyrrole utilize glycine nitrogen equally for their formation (6). All these findings are most readily explained by the hypothesis that in the biosynthesis of protoporphyrin a pyrrole is formed which is the common precursor of both types of pyrrole structure found in protoporphyrin. This was first suggested by Turner (23) but no evidence has heretofore been offered.

It can be seen from Table I that all the carbon atoms of protoporphyrin, with the exception of the 8 which originate from the a-carbon atom of glycine, are derived from acetic acid. The methyl group of the acetic acid contributes more carbon atoms than the carboxyl group. This is in agreement with the previous finding that methyl-labeled acetate gave rise to hemin containing more Cl4 activity than hemin from carboxyl-labeled acetate (4, 9). It can also be seen from Table I and Fig. 2 that carbon atoms 4, 6, 8, and 9 in all four rings are derived from the methyl group of acetic acid, that carbon atoms 3 and 5 in all four rings are mainly derived from the methyl group of acetate and partly from the carboxyl group of acetate, and that carbon atoms ClO, DlO, the carboxyl groups of protoporphyrin, are mainly derived from the carboxyl group of acetate and partly from the methyl group of acetate. Radin, Rittenberg, and Shemin (4) have found that carbon atoms 4 and 6 are derived from the methyl group of acetate and that carbon atoms ClO, DlO are derived from the carboxyl group of acetate. Muir and Neuberger (9) suggest that there is some utilization of the carboxyl group of acetate for one or both carbon atoms of the vinyl side chains. This was based on measurements of the CY activity of hemin derived from C14-carboxyl-labeled acetate and the deuterohemin (hemin minus the two vinyl groups) made from the labeled hemin. This is contrary to our finding. The measurements in our paper are far more direct and do not rely on small differences.


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Examination of the CL4 activities of the different carbon atoms in the porphyrin reveals a pattern and relationship among these different carbon atoms. In the porphyrin made from methyl-labeled acetate, not only do the methyl group carbon atoms (A6, B6, 66, D6) of each pair of pyrroles have similar activities, but their activity is also equal to that of the terminal carbon atoms of the vinyl groups (A9, B9) and to the corresponding carbon atoms of the propionic side chains (C9, 09). The methyl-bearing carbon atoms in all the pyrrole rings (A4, B4, C4, 04) have the same activity as the proximal carbon atoms of the vinyl side chains of rings A and B (A8, BS) and their counterparts in the propionic acid side chains of rings C and D (C8, OS). Also the carbon atoms numbered 5 in the pyrrole rings (A5, B5, C5, 05) have the same activity as all the ring carbon atoms to which the longer side chains are attached (A3, B3, 63, 03) (see Table I and Fig. 2). Similarly, in the experiment with carboxyl- labeled acetate all carbon atoms numbered 5 and 3 have the same activity (see Table I). These data strongly suggest that not only are the two types of pyrrole unit in protoporphyrin made from the same precursors but also that in each pyrrole ring the same compound is utilized for the methyl side of the structure and for the vinyl and propionic acid sides of the structure. This conclusion is supported by the finding, as pointed out earlier; that the pyruvic acid and a-ketobutyric acid fragments of the pyrrole units have the same activities in the experiments with methyl-labeled acetate and that with carboxyl-labeled acetate (Tables I and II).

On examination of the structure of protoporphyrin and in view of the quantitative distribution of Cl4 among the carbon atoms in the experiments, it can be seen that a 3-carbon atom compound would satisfy the data as the precursor of the methyl sides of the pyrrole units (carbon atoms 6, 4, and 5) and the same compound would also be consistent with the data as the precursor of the vinyl sides of pyrrole units A and B (carbon atoms 9, 8, and 3) if we exclude the carbon atom numbered 2, which is derived from the a-carbon atom of glycine. However, it would appear that a 4-carbon atom compound would be necessary as the precursor for the propionic acid sides (carbon atoms 10, 9, 8, and 3) of pyrrole units C and D, again exclusive of the carbon atom numbered 2. If, as suggested above, each side of each pyrrole unit utilizes the same compound, the precursor which condenses with glycine to form the pyrrole unit must be either a 3- or a 4-carbon atom compound. If a 3-carbon atom compound were utilized, subsequent carboxylations must have occurred on positions C9 and D9. On the other hand if a 4-carbon atom compound were utilized, decarboxylations must have occurred on all numbered 6 positions and on positions A9 and B9 subsequent to pyrrole formation. It can be decided which of these alternative mechanisms operates in the synthesis


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of protoporphyrin by correlating some of the data obtained in the experiments with methyl-labeled and carboxyl-labeled acetate. This correlation is valid since the experiments were so carried out that the synthesis of heme from carboxyl-labeled acetate proceeded to the same extent as with methyl-labeled acetate, for the IV concentrations of the hemin samples in the two experiments were practically identical (0.148 and 0.145 atom per cent).

The activities of the carboxyl group of the porphyrin (ClO, DlO) made from carboxyl-labeled acetate can be compared to that of the carbon atoms adjacent to the carboxyl group (C9, 09) of the porphyrin made from methyl-labeled acetate. (Th e activities of the two differently labeled hemin samples are made comparable by application of a factor, as shown in experimental section and in Table I.) It can readily be seen from Table I that the activities of carbon atoms Cl0 and DlO ((1260 + 1080)/Z = 1170 c.p.m.) in protoporphyrin made from carboxyl-labeled acetate are equal, with the limits of error, to those of carbon atoms C9 and D9 (1130 c.p.m.) in the porphyrin made from methyl-labeled acetate. This equality, i.e. the same degree of dilution, makes it appear that the acetic acid enters as a unit and that the utilization of acetic acid for pyrrole formation is via a d-carbon atom compound. If Cl0 and DlO had been introduced by carboxylation, the activity of these carbon atoms would have been much lower. Moreover, it has been shown (4) that carboxyl- labeled acetate gives rise to labeled carbon dioxide in the system used and that radioactive carbon dioxide is not incorporated into hemin (4, 24).

As shown above, in the methyl-labeled acetate experiment the activities of the methyl groups (A6, B6, C6, and D6) and the activities of the terminal carbon atoms of the vinyl groups (49, B9) are equal to those of the corresponding carbon atoms of the propionic acid side chains (C9, D9). Therefore the activities of A6, B6, C6, and 06 and A9 and B9 (average activity, 1140 c.p.m.) are also equal to those of the carboxyl groups of heme (ClO, DlO) made from carboxyl-labeled acetate. It would appear, therefore, from this distribution and the evidence of the utilization of acetic acid as part of a 4-carbon atom unit that in some intermediate stage in the formation of protoporphyrin a pyrrole or porphyrin was formed bearing carboxyl groups attached to the 4 methyl carbon atoms and to the terminal carbon atoms of the vinyl side chains. Further, it would appear that the common precursor pyrrole originally formed contained acetic acid and propionic acid side chains in its /3 positions (see Fig. 3). Although the evidence presented thus far makes it seem highly probable that each of the four pyrrole rings bore two carboxyl groups in some stage of synthesis and that a 4-carbon atom compound was utilized for pyrrole synthesis, more evidence will be furnished later for these conclusions.


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The data obtained from these acetate experiments can readily be explained by assuming the participation of the tricarboxylic acid cycle in porphyrin formation. Acetic acid has been shown to be the direct source of the y-carbon atom and the y-carboxyl group of Lu-ketoglutaric acid (25-28). This compound is adduced in the following argument merely

GOOH

GOOH AHe &He hH2

&Hz------{OX

I C?X ,AHa-COOH

k2N

FIG. 3. Hypothetical scheme for formation of common precursor pyrrole from glycine and a succinyl derivative.

TABLE III Relative Distribution of 04 Activity in Carbon Atoms of c+Ketoglutaric Acid Resulting

from Utilization of P-Labeled Acetate in Tricarboxylic Acid Cycle

The results are expressed in counts per minute.

a-Ketoglutarfc acid

COOH I

‘3%

‘3%

c=o I

COOH

From CY-methyl-labeled acetate (activity of methyl group = 10 c.p.m.)

From C’“-carboxyl-labeled acetate (activity of carbowl group = 10 c.p.m.)

1st

0

10

0

0

0

-- 2nd 3rd

0 0

10 10

5 7.5

5 7.5

0 2.5

No. of cycles in tricsrboxylic acid cycle

.- 03

0

10

10

10

5

- 1st

10

0

0

0

0

10

0

0

0

5

10

0

0

0

5

10

0

0

0

5

to exemplify the possible participation of the tricarboxylic acid cycle as a whole in this synthesis. If one starts with methyl-labeled acetate with a relative activity of 10 in the methyl group after endogenous dilution, the cr-ketoglutaric acid formed on the first turn of the cycle would contain Cl4 activity only in the -y-carbon atom, and the relative activity would be 10 (see Table III). When it has been converted to the symmetrical succinic acid, the activities of the methylene carbon atoms would (dilution by endogenous succinic acid being ignored) be 5 and 5 and those of the oxalacetate eventually formed would contain half of the activity of the y-car-

2nd 3rd --


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bon atom of the a-ketoglutarate. The recycling of this oxalacetate with the labeled acetate would result in cY-ketoglutarate having the relative activities shown in Table III. If cr-ketoglutarate or an unsymmetric compound derived from it were utilized for heme synthesis after a finite number of cycles, most of the carbon atoms of the heme would contain CY4. Moreover, the porphyrin carbon atoms originating from the y position of the a-ketoglutarate would have the highest activity and the 2 adjacent carbon atoms in the heme would theoretically have somewhat lower but equal activities. It can be seen from Table I and Fig. 2 that a pattern exists on both sides of each pyrrole unit, consisting of 3 carbon atoms, 1 with the highest activity and 2 adjacent carbon atoms having somewhat lower activity. The comparable carbon atoms numbered 6 and 9 have the highest activities and the carbon atoms numbered 4 and 5 on the methyl side and 8 and 3 on the opposite side have somewhat lower activities. The relationship of activities of carbon atoms numbered 6 and 4 was in agreement with that previously found (4). However, the comparable carbon atoms numbered 4 and 8 are slightly more active, on the average, than those numbered 5 and 3. This inequality of activities of these carbon atoms (numbered 4 and 8; 5 and 3) would at first suggest that the tricarboxylic acid cycle is not functioning as postulated theoretically above. However, carbon atoms numbered 5 and 3 are also in part derived from the carboxyl group of acetate (Table I and Fig. 2) and when corrected for this dilution, the average activities of these two pairs of adjacent carbon atoms are equal. The contribution of the carboxyl groups of acetate to positions 5 and 3 is 100 counts on the average. The addition of 100 counts to these carbon atoms having an average activity of 788 c.p.m. gives a total of 888 c.p.m., a figure close to the 877 c.p.m. found for the average activity of carbon atoms 4 and 8 in the product from methyl- labeled acetate (see Fig. 2). Therefore it would appear that acetate is utilized through the tricarboxylic acid cycle, and the relative activities found fit those theoretically predicted on the basis of the distribution of activities in a-ketoglutarate.

The distribution of activity in the porphyrin in both experiments elimi- nates the dicarboxylic acids (succinate, fumarate, malate, oxalacetate) and pyruvic acid as direct precursors of the porphyrin. Carboxyl-labeled acetate would label equally the carboxyl group of these dicarboxylic acids, and, if any of these acids were utilized directly, the carboxyl groups of protoporphyrin (ClO, DlO) and the carbon atoms numbered 3 and 5 would have had equal activities. If pyruvate formed in the methyl-labeled acetate experiment was utilized for porphyrin formation, carbon atoms 6, 4, 9, and 8 would be equally labeled, whereas carbon atoms 5 and 3 would be lower than carbon atoms 4 and 8.


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Lemberg and Legge (29) have suggested that 2 moles of a-ketoglutaric acid condense with glycine, with the elimination of the cr-carboxyl group of the keto acid to form a pyrrole bearing acetic acid and propionic acid side chains. Muir and Neuberger (9) have adopted the suggestion of Lemberg and Legge with a modification. They believe that the keto acid condenses with hydroxyaspartic acid, since it has been shown that 8 a-carbon atoms of glycine are utilized in porphyrin formation. How- ever, this view is incompatible with the distribution of the a-carbon of glycine in the porphyrin (2).

Our data also appear to eliminate cw-ketoglutaric acid as an immediate precursor of the porphyrin. The mode of utilization of carboxyl-labeled acetate in the tricarboxylic acid cycle would result in a-ketoglutaric acid labeled only in both carboxyl groups, even after repeated cycles (see Table III) (27, 28). If cr-ketoglutaric acid were directly utilized, with the elimination of the ac-carboxyl group, the protoporphyrin would contain Cl4 only in the carboxyl group. Actually, however, carbon atoms numbered 3 in the porphyrin contain some C14. These atoms correspond to the car- bony1 carbon atom of a-ketoglutaric acid and should contain no Cl4 unless the conversion of a-ketoglutaric acid to succinic acid were reversible. From the best evidence to date it would appear that this reaction is irreversible in higher animals, and until this reaction is shown to be reversible another intermediary compound must be postulated.

The postulated unsymmetric 4-carbon intermediate mentioned above must take into account the finding of some activity in carbon atoms 3 and 5 in the experiment with carboxyl-labeled acetate. The low activity in carbon atoms 3 and 5, in conjunction with the high activity in the car- boxy1 groups of heme produced from carboxyl-labeled acetate, can be explained by presupposing that the compound utilized is derived in greater part from an unsymmetrical compound and in lesser part from a symmetrical compound.

A 4-carbon atom unsymmetric intermediate arising from both cr-ketoglutaric and succinic acids would explain our findings. This compound may be the semialdehyde of succinic acid (30) or, more likely, a succinyl- coenzyme complex. The succinyl-coenzyme complex may be formed in a manner analogous to the formation of acetyl coenzyme A from both pyruvate and acetate (31, 32). cr-Ketoglutaric acid labeled in both carboxyl groups would on decarboxylation yield a succinyl derivative labeled only in the carboxyl group. This compound in turn would be oxidized to succinic acid, a symmetrical compound. If the latter reaction is reversible (XOC-CHZ-CH2-COOH % HOOC-CHzCHz-COOH), the succinyl derivative arising from the symmetrical succinate would contain equal activity in the carboxyl group and in the other terminal carbon


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atom. However, since the succinyl derivative is presumably formed more extensively from cr-ketoglutarate than it is from succinate, the carboxyl group of the pooled intermediate arising from both processes would contain more activity than the other terminal carbon atom. 2 molecules of the succinyl derivative may then condense with glycine to form a pyrrole containing a carboxymethyl group and a carboxyethyl group in its fi positions (see Fig. 3).

Corroborative evidence for the formation of an unsymmetrical 4-carbon compound from a-ketoglutarate and succinate was obtained in the experiment with methyl-labeled acetate. As shown in Table III, methyl-labeled acetate is converted to a-ketoglutarate with Cl4 activity in all its carbon atoms except the y-carboxyl group. However, the carboxyl groups of protoporphyrin made from methyl-labeled acetate have Cl4 activity equivalent to that found in positions 3 and 5 in protoporphyrin made from carboxyl-labeled acetate (Table I). The same mechanism described above would account for this finding. However, an alternative explanation can be advanced, namely the conversion of pyruvate to acetate, but this conversion does not take place appreciably in this biological system (4).

The formation of protoporphyrin may therefore be visualized as follows (Fig. 3): Four of these mono-pyrroles are condensed, with the loss of the ac-carboxyl group of the pyrrole and with the addition of a compound originating from the a-carbon atom of glycine by a mechanism outlined previously (2). The tetrapyrrole first formed would be uroporphyrin III, which by decarboxylation of the carboxymethyl side chain would be converted to coproporphyrin III. The latt,er by decarboxylation and dehydrogenation of the propionic acid side chains of pyrroles A and B would yield protoporphyrin. An alternative pathway may be suggested, in which the first decarboxylation occurs at the monopyrrole stage. De- carboxylation of the carboxymethyl group and condensation of these derived pyrrole compounds would result in the formation of coproporphyrin, thus by-passing uroporphyrin. It has been pointed out (33) that this sequence is compatible with all the facts known. It is significant that the side chains of all the naturally occurring porphyrins can theoretically be derived from carboxymethyl and carboxyethyl groups.

SUMMARY

Whole duck blood was incubated with acetic acid labeled with Cl* in the carboxyl group and with acetic acid labeled with Cl* in the methyl group. The heme resulting from each of these acids was degraded in order to locate the positions of the isotopic carbon atoms that originated from acetic acid. The degradation was so carried out that each carbon atom from each pair of pyrrole rings (A and B; C and 0) could be traced


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individually. It was concluded that both types of pyrrole in protoporphyrin are derived from a common precursor pyrrole. All the carbon atoms of the porphyrin, except the 8 (carbon atoms 2 and the bridge carbon atoms) originally present in the methylene group of glycine, can be derived from acetic acid. The results further show that in protoporphyrin the groupings comprising, respectively, carbon atoms numbered 6, 4, 5 and 9, 8, 3 probably originate from one and the same compound. This compound contained a carboxyl group, from which the carbon atoms numbered 10 originated, which during the synthesis of protoporphyrin was lost from all 4 carbon atoms numbered 6 and 2 of the carbon atoms numbered 9.

The relative distribution of the Cl4 activities among the carbon atoms of the porphyrin can be explained by the utilization of acetate for porphyrin formation through a compound arising from the tricarboxylic acid cycle. This compound is not succinate, fumarate, malate, oxalacetate, pyruvate, or acetoacetic acid, nor, if the a-ketoglutarate-succinate reaction is irreversible, can it be ac-ketoglutarate. The precursor utilized for pyrrole formation is thought to be an unsymmetrical 4-carbon compound which can arise both from ar-ketoglutaric acid and succinic acid. It is suggested that this compound is a succinyl derivative, possibly a succinyl-coenzyme complex, formed from succinate as well as by oxidative decarboxylation of a-ketoglutarate.

A mechanism is proposed wherein 2 moles of the succinyl derivative condense with glycine to yield a-carboxy-&carboxymethyl-@‘-carboxyethyl- pyrrole, and it is suggested that four of these pyrroles condense to form a porphyrin which is converted to protoporphyrin by decarboxylation and dehydrogenation.

BIBLIOGRAPHY

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(1946). Altman, K. I., Casarett, G. W., Masters, R. E., Noonan, T. R., and Salomon, K., J. BioZ. Chem., 176, 319 (1948).

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David Shemin and Jonathan WittenbergTRICARBOXYLIC ACID CYCLE

FORMATION: THE RÔLE OF THE THE MECHANISM OF PORPHYRIN

1951, 192:315-334.J. Biol. Chem.

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