THE PROSTHETIC GROUP OF SULFHEMOGLOBIN

BY FELIX IIAUROWITZ*

(From the Institute of Biological and Medical Chemistry, University of Istanbul, Istanbul, Turkey)

(Received for publication, September 23, 1940)

Treatment of hemoglobin with H&S and 02 gives rise to the for- mation of a green pigment, sulfhemoglobin. In contradistinction to the transformation of hemoglobin into HbOz, HbCO, Meth-Hb, and other hemoglobin derivatives the transformation into sulf- hemoglobin is not reversible. The nature of this reaction is as yet unknown (1). An attempt.was made therefore to isolate the prosthetic group of sulfhemoglobin and to clear up its constitution.

Difficulties arose first of all in preparing pure sulfhemoglobin. The typical absorption band in the red region of the spectrum after having reached a certain intensity showed a gradual de- crease of its extinction during prolonged treatment with II&S and 02 (Fig. 1). Probably the nascent sulfhemoglobin is autocatalyti- tally destroyed later on by traces of HzOz, the formation of which from HzS and 02 has been proved (2, 3).

Contrary to hemoglobin and oxyhemoglobin, sulfhemoglobin is not split into a hemin and globin by the action of dilute acids. Keither boiling acetic acid and NaCl (Schalfejef) nor oxalic acid and acetone (4) cause the formation of hemin. We treated sulf- hemoglobin therefore with pepsin and HCZ, a method used first by von Zeynek (5) for the splitting of Hb. By repeated digestion with pepsin the bulk of the protein compound was split off. But contrary to the results with oxyhemoglobin 5 to IO per cent of the globin adhered to the pigment. Instead of pure hemin (6) a brown hemin-protein compound was obtained. It will be designated in

* The experiments described herein were performed up to April, 1939, in the Medical-Chemical Institute of the German University in Prague and were continued later in the Institute for Biological and Medical Chem- istry of the University of Istanbul.

771

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772 Prosthetic Group of Sulfhemoglobin

the following as sulfheminproteose, in analogy to heminproteoses produced by the action of trypsin on hemoglobin (6). A similar sulfheminproteose was obtained by digestion of sulfhemoglobin with papain.

FIG. 1. Absorption curves. of hemoglobin-sulfhemoglobin mixtures. Curve I = 10 hours treatment, Curje II = 20 hours treatment with SH, and 02.

lo g

6:

4.4

4.2

4.0

1.8

3.b FIG. 2. Absorption curves of hemin (Curves 1, 3, 5) and of sulfhemin-

proteose (Curves 2,4,6) in 1 per cent NaOH (Curves 1 and 2), after addition of NaCN (Curves 3 and 4) and after reduction with Na&Oa (Curves 5 and 6).

Sulfhemoglobin contains 0.32 to 0.35 per cent of Fe, as does pure hemoglobin. This iron is not split off by treatment with pepsin- HCl. Its linkage to the pigment is just as strong as in hemin. The absorption spectrum of the sulfheminproteose is a typical hemin spectrum and resembles closely the spectrum of protohemin, both with regard to the position of the absorption bands and to

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F. Haurowitz 773

their intensity (Fig. 2). Just like hemin, the sulfheminproteose is converted into a bright red cyanide derivative by XaCN and into a typical ferro compound (hemochromogen) by Ka2S204. The sulfheminproteose contains 2 to 3 per cent of iron and 5 to 10 per cent of sulfur, corresponding approximately to 7 S atoms for each Fe atom. The bulk of this sulfur is adsorbed colloidal sulfur, which can be removed to some extent by dissolving the proteose in NaOH and reprecipitating with acetic acid, and to a greater extent by extraction with hot benzene.

From diffusion experiments with alkaline solutions of a sulf- hemiproteose containing 3 per cent of Fe a molecular weight of at least 19,000 results for sulfheminproteose. This corresponds to about 10 hemin molecules per molecule of sulfheminproteose. Apparently the sulfheminproteose just as hemin (7) is associated in its alkaline solutions.

Sulfheminproteose is insoluble in a saturated solution of HBr in glacial acetic acid and does not lose its iron, even after pro- longed treatment with this reagent. The cleavage of sulfhemin- proteose was achieved by the action of concentrated HCZ at 100’. Under these conditions the iron is split off, the protein component is hydrolyzed, and the bulk of the pigment is obtained as an insoluble precipitate which can be dissolved in dilute NaOH or in concentrated sulfuric acid. The absorption maxima of these solutions are as follows:

629 mp, 576 mp, 545 mp, 509 mp in N XaOH 603 ‘I (593 “ ), 556 “ in concentrated HZSOc

These are typical porphyrin absorption bands. But contrary to the behavior of most prophyrins the precipitated porphyrin is insoluble in dilute HCl and in organic solvents. Its elementary analysis corresponds to the formula C34H36K~0&S2, differing from protoporphyrin, C34H34N404, by an excess of 2 S and 4 0 atoms (perhaps 2 H atoms). Oxidative destruction of the porphyrin with HXOa (8) produces neither methylsulfonic acid nor other alcohol-soluble S compounds. 1c’o S-containing ether-soluble substances were obtained after oxidation of the porphyrin with chromic acid (9) nor after reductive cleavage with tin and HCl. We conclude from these experiments that no sulfur-containing pyrroles have been formed in these reactions. The sulfur is not

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774 Prosthetic Group of Sulfhemoglobin

removed from sulfheminproteose after heating with resorcinol to 190” (Schumm (lo)), but the absorption spectrum is shifted after this treatment 5 rnp towards the blue region. This corresponds perfectly with the analogous reaction of protohemin, whose ab- sorption spectrum is shifted in the same direction after fusion with resorcinol, owing to a loss of the vinyl groups (Schumm). Ap- parently the S atoms of the insoluble porphyrin are not linked to its vinyl side chains.

Sulfoporphyrins with SOaH groups have been described by Treibs (11). But no such groups could be detected in our por- phyrin. Titration of the porphyrin with NaOH indicated the presence of only two acid groups, apparently the COOH groups of protoporphyrin. Methylation gave rise to two methoxy groups, most probably corresponding to a methylation of the two car- boxyls.

We conclude from the failure to obtail sulfur-containing pyrrole derivatives, and from the result of the pyro reaction with re- sorcinol, that the S atoms are linked to the methene C atoms of the porphyrin, probably in the following way.

CHs CH=CH2 CHs CH=CHz CHs CH=CH

-4 SOsH - so2

/

-\ /=CH- N

--NN/=C- -\N/=C-

It must be emphasized that the above reaction of the vinyl groups has nothing to do with the appearance of the typical absorption bands of sulfhemoglobin, since a similar spectrum is obtained by the action of H2S and oxygen on mesohemoglobin, in which the vinyl groups are replaced by ethyl groups.

The green color of sulfhemoglobin has led some authors to the opinion that sulfhemoglobin is related to the green bilirubinoid pigments (12, 13) with an open porphin ring. But contrary to the different green pigments described by Warburg (14), Edl- bather (15), Lemberg (16), and Barkan (17) and their coworkers sulfhemoglobin and sulfheminproteose do not lose their iron after treatment with dilute HCI. Since sulfhemoglobin is different from the green pigments mentioned above, the question arises

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F. Haurowitz 775

whether the typical absorption spectrum of blood observed after treatment with phenacetin or with aniline derivatives is correlated to sulfhemoglobin or to one of the green pigments with labile iron.

With regard to the iron linkage only one of the green pigments resembles sulfhemoglobin; viz., the green pigment produced by the action of KCN and Hz02 on Hb (Davis (18), Barkan and &hales (12)). Its iron is not split off by dilute HCl. It differs from sulfhemoglobin by its absorption band in the red region, which does not appear before addition of a reducing agent (12). In contradistinction to our statement Barkan and Schales (19) have found an increase of labile iron after treatment of Hb with a great excess of H&S and Oz. Their results are confirmed below, but no significant increase of labile iron was found in preparing sulfhemoglobin from Hb according to our own method and during digestion with pepsin-HCl. We conclude therefore that the formation of labile iron has nothing to do with the formation of sulfhemoglobin and that it must be attributed to the secondary destruction of sulfhemoglobin by HzOz.

Sulfhemoglobin can be prepared from Hb under very mild conditions; i.e., in cold neutral solution. The typical absorption spectrum of sulfhemoglobin appears almost instantaneously. Hence profound chemical reactions of the porphyrin nucleus :an be excluded as a first cause of the altered spectrum. Michel (20) has proved recently in solutions of HbOz partially converted to sulfhemoglobin that the latter contains only 1 atom of extra sulfur per iron atom. Most likely the typical absorption spectrum of sulfhemoglobin develops as soon as one of the methene groups is substituted by a sulfur-containing group. The alteration of the spectrum may be due to a coordinating bond between iron and the S-containing group.

EXPERIMENTAL

Preparation of Suljhemoglobin-Crystalline HbOz was prepared from horse blood corpuscles with the aid of toluene (21) or alcohol (22). A 20 per cent solution of crystalline Hb was perfused alter- nately with H&S and 02 or was slightly shaken in a mixture of both gases (H&3: 02 = 2: 1). The progress of the reaction was con- trolled spectroscopically (1). After 10 hours the spectrum showed no further appreciable alteration. Now the precipitated sulfur

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776 Prosthetic Group of Sulfhemoglobin

and denatured protein were filtered off and the absorption curve of the clear dark green solution Sulfhemoglobin I was deter- mined (Fig. 1, Curve I). For that purpose 1 ml. of the solution was diluted with 99 ml. of 0.1 per cent sodium carbonate, reduced by a trace of NazSz04, and measured in a Hiifner spectrophotom- eter. A part of Sulfhemoglobin I was treated again for 10 hours with the gas mixture (Sulfhemoglobin II) and the absorption measurement repeated (Fig. 1, Curve II). The protein concen- tration had fallen within these 10 hours from 17.5 to 15.5 per cent. The curves of Fig. 1 indicate the extinction coefficients of a 0.1 per cent protein solution.

Iron Determination-Iron was determined by the method of Kennedy (23). 1 ml. of 19.3 per cent SHb = 0.65 mg. of Fe = 0.35 per cent Fe. In 5 ml. of the same solution 0.022 mg. of labile iron was found by Barkan’s (24) method, or less than 1 per cent of the total iron. Other preparations of sulfhemoglobin contained 0.32 to 0.35 per cent of Fe and 1 to 5 per cent of the total iron in the labile form.

Action of Acids on Sulfhemoglobin-The dark green solution of Sulfhemoglobin I was heated in the usual way with glacial acetic acid and NaCl. No hemin crystals were formed. The solution of sulfhemoglobin (20 per cent) gave a dark green precipitate after addition of acetone, but no pigment was extracted from this pre- cipitate by a 4 per cent solution of oxalic acid in acetone. A solu- tion of HbOZ prepared from the same blood after addition of acetone gave a brown precipitate, the pigment of which was extracted almost quantitatively by the treatment with oxalic acid solution.

Treatment of XHb with Pepsin-HCZ-480 ml. of a 10 per cent solution of Sulfhemoglobin I were adjusted to pH 1 to 2 by addi- tion of HCl. 0.5 gm. of pepsin was added and the mixture kept at 38” for 2 days. The pigment was then precipitated, almost quantitatively, by neutralizing the free HCl with sodium acetate, with Congo paper as indicator. The precipitate was filtered off, washed with 0.1 per cent acetic acid, and treated again with pepsin and HCl. This was repeated two or three times until a negative biuret test was obtained in the clear filtrate. Now the sulf- heminproteose was dissolved in warm 1 per cent NaOH, precipi- tated again with acetic acid, washed with water, dried at reduced

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F. Haurowitz 777

pressure, and extracted with boiling benzene in a Soxhlet appa- ratus. 6.036 gm. of a dark brown powder were obtained. It contained 2.2 per cent Fe, that is a total of 133 mg. of Fe, whereas the collected clear and slightly brownish filtrates of the repeated precipitations contained altogether 11.9 mg. of iron.

Action of Papain on SHb-500 ml. of 10 per cent sulfhemoglobin solution were kept at 38” with 50 ml. of 0.1 M phosphate buffer solution, pH 6.3, 1 ml. of toluene, and 0.25 gm. of papain. As for the rest, the treatment resembled the procedure with pepsin. After repeated action of papain 4.6 gm. of sulfheminproteose were isolated, containing 3.7 per cent of Fe and 8.8 per cent of S. Determinations of sulfur were performed according to Waelsch and Klepetar (25).

Properties of Sulfheminproteose-For spectrophotometric meas- urement a 0.0002 N solution of sulfheminproteose in 1 per cent NaOH was prepared from sulfheminproteose containing 2.4 per cent of Fe (100 ml. of this solution contained 1.12 mg. of iron). An equimolecular solution of pure protohemin in NaOH was pre- pared. Both solutions were measured in a Hiifner spectropho- tometer (a) directly, (b) after addition of an equal volume of 10 per cent NaCN, and (c) after further addition of 30 mg. of NazSz04 to 10 ml. of solution (b). Because of the slow reaction of sulf- heminproteose with cyanide and hyposulfite in the cold, all solu- tions were kept 20 minutes in a boiling water bath. The result of the experiment is shown in Fig. 2.

200 mg. of sulfheminproteose were kept 15 minutes at 190” with 20 gm. of resorcinol. Then the resorcinol was dissolved in water and the pigment precipitated by addition of acetic acid and solid NaCl and reprecipitated from alkaline solution with acetic acid. The sulfur content decreased after fusion with resorcinol from 7.0 to 2.55 per cent, whereas the Fe content remained un- changed at 2.9 per cent. The maximum of the first hemochromo- gen band, after treatment with NazSz04 in 0.01 N NaOH and addi-

tion of 5 per cent pyridine, was determined spectroscopically as follows :

Sulfheminproteose. .552-553 mp; after fusion with resor- cinol..............................542mp

Protohemin. . . . . . ,559 mp; after fusion with resorcinol. .547 “

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778 Prosthetic Group of Sulfhemoglobin

In eight different preparations of sulfheminproteose 2 to 4 per cent of iron and 7 to 10 per cent of sulfur were found. Most probably the percentage of iron depends on the integrity of the sulfhemoglobin, from which the sulfheminproteose has been prepared, and on its destruction by HzOz.

1.5 gm. of sulfheminproteose were dissolved in 150 ml. of 1 per cent NaOH and placed in the diffusion apparatus, No. 36b MG4 of Schott (Jena). The diffusion of the pigment in 1 per cent NaOH was measured calorimetrically. In the course of 7 days 3.85 per cent of the total pigment had passed the sintered glass membrane, corresponding to 0.55 per cent in 24 hours. The same experiment with 10 per cent sucrose solution brought about the diffusion of 4.1 per cent of the total sugar in 24 hours. Its con- centration was determined by refractometry.

Preparation of a Porphyrin from Xulfhemoglobin-2.0 gm. of sulfheminproteose in a flask with a reflux condenser were digested with 100 ml. of 25 per cent HCl in the boiling water bath. After 8 hours the clear, slightly olive-colored solution was decanted from the insoluble pigment and replaced by fresh HCl. This was repeated once or twice until no ferric ions were detectable in the liquid. During the hydrolysis a considerable sublimate of crys- talline sulfur accumulated in the reflux condenser. The insoluble brown pigment was centrifuged off and dissolved in warm diluted NaOH. It was precipitated then by acetic acid and washed with water, alcohol, ether, and hot benzene. Finally 0.609 gm. of dry pigment was obtained. Analysis of two different preparations gave the following results.

C Porphyrin I.. . . . . .60.0 5Y27 232 CO9 1;3

0 (calculated) 17.3

‘I II . . . . . . . .60.4 5.18 7.54 7.65 0 16.8 C~~H~BNJO&. Cal-

culated...........59.1 5.20 8.09 9.24 0 18.5

Nitrogen was determined by the Dumas method. Kjeldahl nitrogen determinations gave low values, which could be prevented by addition of potassium persulfate. The collected hydrolysis liquids, after centrifugation of the pigment, contained appre- ciable amounts of hydrogen sulfide, but neither sulfate nor sulfite.

A solution of 0.797 gm. of the porphyrin in 13.8 ml. of 0.25 N

NaOH required 7.5 ml. of 0.1 N HCl for neutralization with

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F. Haurowitz 779

phenolphthalein as indicator. This corresponds to 2.35 equiva- lents of NaOH for each 4 N atoms (= one porphin ring). 0.1 gm. of the porphyrin was suspended in dry methanol, saturated with HCl gas, and evaporated at reduced pressure. In 31.1 mg. of the dry residue (15 mm., 100”) O-methyl was determined in the micro apparatus of Pregl. Titration of the methyl iodide accord- ing to Kirpal and Biihn (26) required 8.28 ml. of 0.01 N silver nitrate, corresponding to 1.9 methyl groups for each 4 N atoms. The methylated porphyrin was insoluble in pure methanol, but soluble after addition of a drop of concentrated HCI. Apparently the methylated product still contains basic groups, most probably the basic pyrrole nitrogen atoms of the original porphin ring.

Comparison of Xulfhemoglobin with Other Green lib Derivatives- The green pigment of Edlbacher and von Segesser (15) was pre- pared according to these authors from horse blood corpuscles by treatment with ascorbic acid and oxygen. The green amyl alco- holic solution of the pigment lost its iron after shaking with N HCI. The amyl alcohol was evaporated at room temperature at reduced pressure over solid KOH and the green residue was dissolved in ether and drawn, for chromatography, through a column of pure talcum. Colorless substances (lipids) were extracted from the column by chloroform, whereas the green pig- ment was developed by ethyl alcohol. It was practically free of iron (1.0 mg. = 0.001 mg. of Fe).

Twice recrystallized hemoglobin from horse blood was trans- formed into a green pigment by treatment with KCN and Hz02 according to Barkan and Schales (12). The solution was neu- tralized by addition of oxalic acid and precipitated by addition of twice its volume of acetone. The precipitate was washed with acetone and then treated with a solution of 4 per cent oxalic acid in acetone. No pigment passed into the acid solution from the precipitate.

Mesosuljhemoglobin-Mesoporphyrin was transformed into mesohemin by the usual treatment with ferric chloride and sodium acetate in glacial acetic acid. Globin was prepared from ox blood according to Laporta (27). Slightly alkaline solutions of meso- hemin and globin were coupled according to the procedure of Hill and Holden (28) and the solutions of mesohemoglobin then per- fused with a mixture of HZS and 02. The spectrum of the alkaline

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780 Prosthetic Group of Sulfhemoglobin

mesomethemoglobin with its two bands in the green region was replaced by the spectrum of mesosulfhemoglobin, with a charac- teristic sharp band in the red region at 617 rnp. The analogous band of sulfhemoglobin is situated at 626 rnp. But it is well known that all spectra of meso derivatives show a typical shift to the short wave region in comparison with proto derivatives.

SUMMARY

1. Sulfhemoglobin is formed by the action of HzS and 02 on hemoglobin. Prolonged action of the gas mixture destroys a part of the newly formed sulfhemoglobin, apparently by inter- mediary formation of HzOz.

2. Contrary to hemoglobin and its derivatives, sulfhemoglobin is not split into hemin and globin by dilute acids. Digestion with pepsin furnishes sulfheminproteose, i.e. a hemin, to which a part of the protein is firmly bound. In contradistinction to other green pigments (verdohemochromogens) sulfhemoglobin and sulfheminproteose lose no iron after treatment with dilute HCl.

3. Sulfhemoglobin contains 2 to 4 per cent sulfur, sulfhemin- proteose 7 to 10 per cent sulfur. The bulk of this sulfur is ad- sorbed colloidal sulfur.

4. Hydrolysis of sulfheminproteose with boiling concentrated HCl furnishes an iron-free porphyrin, which differs from proto- porphyrin by an excess of 2 S and 4 0 atoms. Most probably it contains two SOz bridges between the porphin nucleus and its side chains.

BIBLIOGRAPHY

1. Haurowitz, F., Z. physiol. Chem., 161, 130 (1926). 2. &hales, O., Ber. them. Ges., 71, 447 (1938). 3. Drabkin, D. L., and Austin, J. H., J. Biol. Chem., 112, 51 (193536). 4. Hamsik, A., Z. physiol. Chem., 176, 173 (1928). 5. von Zeynek, R., Z. physiol. Chem., 30, 126 (1900); 49, 472 (1906). 6. Haurowitz, F., Z. physiol. Chem., 188, 161 (1930). 7. Zeile, K., and Meyer, H., Naturwissenschuften, 27, 596 (1939). 8. MBmer, K. H., Z. physiol. Chem., 93, 181 (1914). 9. Fischer, H., and Lindner, F., Z. physiol. Chem., 168, 156 (1927).

10. Schumm, O., Z. physiol. Chem., 178, 1 (1928). 11. Treibs, A., Ann. Chem., 606, 196 (1933). 12. Barkan, G., and &hales, O., Z. physiol. Chem., 263, 83 (1938). 13. Lemberg, R., Au&al. Chem. Inst. J. and Proc., 6, 170 (1939).

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14. Warburg, O., and Negelein, E., Ber. them. Ges., 63, 1816 (1930). 15. Edlbacher, S., and von Segesser, A., Naturwissenschaften, 26, 461,

557 (1937). 16. Lemberg, R., Legge, J. W., and Lockwood, W. H., Biochem. J., 29,

1322 (1935); 33, 754 (1939). 17. Barkan, G., and Schales, O., 2. physiol. Chem., 246, 96 (1937). 18. Davis, J. G., Proc. Sot. Agric. Bacterial., Rep. No. 386 (1937). 19. Barkan, G., and &hales, O., 2. physiol. Chem., 264, 241 (1938). 20. Michel, H. O., J. Biol. Chem., 126, 323 (1938). 21. Heidelberger, M., J. BioZ. Chem., 63, 34 (1922). 22. Zileer, V., Biochem. Z., 179, 348 (1926). 23. Kennedy, R. P., .I. BioZ. Chem., 74, 385 (1927). 24. Barkan, G., KZin. Woch., 16, 300 (1937). 25. Waelsch, H., and Klepetar, G., 2. physiol. Chem., 211, 47 (1932). 26. Kirpal, A., and Btihn, T., Monatsh. Chem., 36, 853 (1915). 27. Laporta, M., Boll. Sot. Ital. biol. sper., 6, No. 7 (1931). 28. Hill, R., and Holden, F., Biochem. J., 20, 1336 (1926).

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Felix HaurowitzSULFHEMOGLOBIN

THE PROSTHETIC GROUP OF

1941, 137:771-781.J. Biol. Chem. 

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