COMBINATION OF CARBON MONOXIDE WITH HEMOGLOBIN
March 5, 195s
actual experiment employed 5.284 mmoles of (CH3)zNP(CH3)l and 7.912 mmoles of HC1. T h e combination occurred in a 50-1111. vertical reaction tube attached t o the highvacuum system, with slow warming from -100' t o room temperature. The volatile product now had physical properties indicative of purity; for example, the molecular weight of the 80% saturated vapor was measured as 97.9; calcd., 96.5. Numerous earlier attempts t o make (CHa)2PCl by the thermal dissociation of (CH3)3PC1z-a method reported by Plets for other RzPCl compounds7-had led only t o a nonvolatile brown solid, HCl, PCls and all of the chloromethanes. Once, however, we obtained a yield of (CH3)zPCl representing a little less than 2 7 , of the phosphorus. It was identified by its mol. wt. (obsd., 97.2; calcd., 96.5), its vapor tension of 34 mm. a t 0' and a chloride analysis (found, 37.17,; calcd., 36.7). Physical Properties of Dirnethylch1orophosphine.-The vapor tensions of pure solid (CH3)2PCl, shown in Table 11, determined the equation logmpmm. = 12.1408 - 2887/T.
TABLE I1 VAPORTENSIONS OF SOLID ( CH3)2PC1 t
("C.)
omm. (obsd.) fimm. (calcd.)
-39.8 0.57 0.59
--'34.0 1.19 1.17
-27.4 2.44 2.47
-18.6 -12.25 11.92 6.31 6.31 11.91
-5.5 22.62 22.63
It was more difficult to obtain consistent measurements for the liquid, which slowly and irreversibly formed a slightly soluble non-volatile solid on standing. After each measurement the apparatus could be cleared by flaming in vacuo t o form volatile materials whose constitution cannot yet be inferred. The vapor tension values shown in Table I11 determine the equation logmpmm.= 7.844 - 1722/T. This would indicate the normal b.p. as 71", but since the Trouton constant (22.9 cal./deg. mole) is a little above normal, the true b.p. probably is nearer to 73".
TABLE I11 VAPORTENSIOSSOF LIQUID(CH&PCI 1 ("(2.)
Pmm. (obsd.) omm. (calcd.)
0.00 34.6 34.7
2.4 39.5 39.4
9 . 4 1 5 . 3 19.83 5 6 . 6 74.9 9 1 . 5 56.2 74.9 9 2 . 0
21.7 99.5 100.9
33.1 168.7 166.5
From the two vapor tension equations the heat of fusion is calculated as 5.33 kcal./mole and the m.p. as -2.0'. The directly observed melting range was - 1.4 t o - 1.0". Aminolysis of Dimethylch1orophosphine.-Samples of (CH3)zPC1 (0.437 mmole) and (CH&XH (0.947 mmole) were measured as gases and allowed to warm together from (7) V. M. Plets, Dissertation, Kazan, 1938, cited by G. hi. Kosolapoff, "Organophosphorus Compounds," John Wiley and Sons, New York, N. Y., 1950, p. 47.
[CONTRIBUTION NO. 1467 FROM
Hemoglobin Studies.
THE S T E R L I N G
1109
-78" to room temperature during 8 hr. T h e excess amine was distilled through a trap a t -78' and measured its 0.071 mmole, meaning that 0.876 mmole of the amine had combined (calcd., 0.876). T h e volatile product trapped out a t -78" had a vapor tension of 12.1 mm. a t O", corresponding t o pure (CH3)zNP(CH8)2; weight 42.2 mg., or 0.402 rnmole (92.0% of the expected 0.437). T h e non-volatile product was analyzed for C1-: found, 0.437 mmole, strictly in accord with the equation (CHs)zPCl 2( CHa)zNH + (CH3)zSP( CH3)z (CHI izSHzCl This result is a further confirmation of the formula (CH3)ZPC1. I t appears that 8% of the expected (CH~)ZN.P(CHP)Z remained with the (CH3)~NH~Cl-inwhat form cannot be judged. Ammonolysis of Dimethylaminodimethylphosphine.--An initial micro-scale experiment showed that ammonia attacks (CH3)2NP(CH3)z fairly rapidly a t room temperattre, displacing more than 2(CH,)zNH per NH3 used up, and forming polyphosphino-amines. For more accurately q uantitative results, 2.3725 g. (22.57 mmoles) of ( C H ~ ) Z N P ( C H ~ ) Z and 53.2 mg. (3.13 mmoles) o! NHs were heated for 22 hr. in a 100-ml. sealed tube a t 46 Then the volatile components were separated by high-vacuum fractional ccndensaThe retion, with U-traps a t 0, -23, -112 and -196". covery of h7H3 was 0.50 mmole (used, 2.63) and of (CHI)ZNP(CH3)2, 16.60 mmoles (used, 5.97 mmoles). The dimethylamine weighed 268.4 mg. (5.96 mmoles; mol. u t . 45.3 vs. calcd. 45.1; vapor tension 556 mm. a t 0" us. known value, 562). The yield of almost pure [(CHd hPI2SH was 273.5 mg. (1.995 mmoles); and after very thorougt refractionation, the pure product melted in the range 39-40 and showed a vapor tension of 4.1 mm. a t 30" (literature values, m.p. 39.5" and 4.13 mm. a t 30°).4 Thus the overall reaction balance would require that the slightly volatile oily liquid residue contain 1.98 mmoles of (CHB)&' groups and 0.635 N, reasonably interpreted as 0.65 mrnole of [ ( C H ~ ) Z P ] ~ NT.h e stoichiometry of the whole experinieiit is well represented by 9( CHa)zNP(CH3)z 4NHa ---f 3[(CHa)zPlzNH [(CHz.)zPIaN 9(CHa)zNH The recovery of some ammonia after exposure to a great excess of the aminophosphine suggested that the reaction is sensibly reversible. Another approach to this complex equilibrium system was tried in an experiment wherein 3.44 mmoles of (CHa)*NP(e&),and 0.479 mmole of [ (CH3)2P]2SHreacted partially during 20 hr. a t 105-130" to form 0.564 mmole of (CH3)zNH and 0.216 mmole of [(CHa)zP],N. The presence of 1.1 mg. (0.068 mmole) of NHa again indicated the reversibili .y of the original ammonolysis. Los ANGELES7, CALIFORNIA
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CHEMISTRY LABORATORY, YALE UNIVERSITY]
I. The Combination of Carbon Monoxide with Hemoglobin and Related Model Compounds
BY JUI H. W A N G , AKITSUGUNAKAHARA~ AND EVERLY B. FLEISCHER RECEIVED SEPTEMBER 16, 1957 The nature of bitiding of carbon monoxide to hemoglobin, heme and Fe(I1)-dicysteinate was studied by means of infrared and magnetic measurements. T h e formation constant of carbonmonoxyheme was determined and compared tc, that of carboxyhemoglobin. A possible cause for the unusual resistance of hemoglobin derivatives toward oxidation by molecular oxygen was proposed.
Although i t has long been well-known that hemoglobin and myoglobin combine reversibly with molecular oxygen, whereas aqueous solutions of free heme do not, it appears that no satisfactory explanation has yet been proposed t o account (1) On leave from the Department of Chemistry, Osaka University, Nakanoshima, Osaka. Japan.
for this important difference in their chemical property. The mere formation of a coordination bond between a donor group of globin and the ferrous iron in heme is clearly inadequate to prevent the irreversible oxidation of the latter by molecular oxygen, for the ferrohemochromogens forrned by combining heme with ammonia, pyridine, imida-
J. H. WANG,A. NAKAHARA AND E. B. FLEISCHER
1110
zole, etc., are readily oxidized to the ferrihemochromogens when aqueous solutions of the former are shaken with air. In the absence of oxygen, heme and ferrohemochromogens do combine reversibly with carbon monoxide. But these carbonmonoxyheme compounds are oxidized rapidly when their aqueous solutions are exposed to air. By analogy, it has been conjectured that heme also combines with molecular oxygen, but the resulting “oxyheme” is so unstable in aqueous solutions, that i t immediately goes over t o the ferric state.* It is of interest t o know whether the greater stability of carboxyhemoglobin and oxyhemoglobin is due t o the difference in the primary valence force which binds the diatomic molecule to the respiratory pigment or due to secondary interactions which may exist in the protein molecule. For example, the binding of carbon monoxide t.o hemoglobin has been suggested by Pauling3 to be represented by structure I.
Vol. 80
Preparation of Crystalline Dicarbonmonoxy-Fe(I1)-dicysteinate.-The disodium salt of dicarbonmonoxy-Fe(I1)-dicysteinate, N~?[F~(SCHZCH~’HZCOO)Z(CO)~].~H~O, was prepared according t o the method of Schubert? in the compleie absence of air. Preuaration of Crvstalline Carbonmonoxvheme-Pvridine Complex.-This was prepared in the absence of ai; in a n apparatus similar to t h a t described b y Schubert.’ One gram of hemin, Fisher (recrystallized) was dissolved in 40 cc. of pyridine, reduced by a slight excess of sodium dithionite. Carbon monoxide was then passed through the solution, and the product was precipitated by diluting the mixture with saturated aqueous solution of sodium chloride. The crystalline ppt. was filtered, washed and dried in the absence of air. Measurement of Infrared Spectra.-The above three compounds were made into KBr-disks for infrared measurements with a Perkin-Elmer Model 21 IR-spectrometer. -1fter the infrared measurements the carboxyhemoglobin ;uid oxyhemoglobin disks were dissolved in water. The water sr~lutionsshowed t.he same visible absorption bands as the original proteins. On the other hand, the carbontnonoxyhenie-pyridine cornplex was so unstable t h a t its infrared spectrum changed rapidly when the KBr disk was exposed t o air.
The infrared spectra of crystalline carboxyhemoglobin and carbonmonoxyheme-pyridine cornplex between 1500 and 3000 cm.-‘ are shown in Fig. 1. It may be noticed in Fig. 1 that both carboxylieinoglobin and carbonmonoxyheme-pyridine coniplex showed a sharp absorption peak at 1970 CIII.-~. Since infrared spectra of oxyhemoglobin and henioI I1 chromogen, respectively, did not show this nbBut because of the existence of bridge carbonyl sorption peak, this peak can be safely assigned to groups in iron enneacarbonyl? and iron tetracar- the stretching vibration of the bound CO molecule. bony1,j one may wonder if the binding of carbon The observed frequency, 1970 ctn.-l, is consistent monoxide to hemoglobin could be represented by with Pauling’s structure I, but definitely rules out structure I1 where X represents an unknown donor structure I1 which predicts a corresponding fregroup of the globin molecule. Although the weight quency in range 1700-1800 cm.-l. Furthermore, of opinion favors structure I, it is desirable to dis- the fact that both carboxyhemoglobin and carbonprove structure I1 hy means of a single unambigu- monoxyheme-pyridine complex possess a sharp ahous experiment. Measurement of the infrared ab- sorption peak a t 1970 cm.-’ shows that there is no sorption spectrum could serve this purpose. It difference in the primary valence force which binds may also yield information regarding the nature of the CO molecule t o the Fe(I1) in these two coinbinding of carbon monoxide to heme in carbon- pounds. iiionoxyhenie as coinpared t o that in carboxyThe infrared spectrum of dicarbonmonoxy-Fehemoglobin. (11)-dicysteinate also showed a sharp absorption peak a t 1970 ern.-'. In addition, there are two Infrared Studies other pealis at 2025 and 2030 cm.-’, respectively. The infrared absorption spectra of crystalline These frequencies cannot yet be assigned definitely. carboxyhemoglobin, carbonmonoxyheine-pyridine The infrared spectra of the complexes formed by complex and dicarbonmonoxy-Fe(I1)-dicysteinate the diethyl ester of carbonmonoxyheme with pyriwere measured. The experimental methods are dine and imidazole, respectively, in ether solution outlined below. were examined. Each of these also contained Preparation of Crystalline Oxyhemoglobin and Carboxy- a sharp absorption peak a t 1970 cm-l. 0
‘0’
hemoglobin.-Three times recrystallized oxyhemoglobin mas prepared from fresh horse blood by the method described by Keilin aud Hartree .6 The carboxyhetiloglobit] was made by reducing a coucentrated solution of the three times recrystallized oxyhemoglobin with a very slight excess of sodiuni dithionite, S a 2 S 2 0 4 and , then equilibrating the solution with carbon iiionoxide. The final crystalline samples of carboxyhemoglobin and ox~-hemoglobin were obtained by recrystallization from thcoaqueous solution in a rotary vacuum evaporator a t about 2 . _.
( 2 ) See R. Lemberg and J. W.Legge, “Hematin Compounds and Bile Pigments,” Interscience Publishers, Inc., New York. h-.Y . , 1949, p . 184. (3) L. Pauling, “Haemoglobin,” (edited by F. J . W .Koughton a n d 1. C. Kendrew), Interscience Publiihers. Inc., S e w York, P;.Y.. 1919,
Im. (4) IO) ( 5 ) R . K . Sheline, i b i d . , 73, 1615 (1951). (B) D . Keilin and E. F. Hartree, Pi.oc. Roy. S O L .( L c i i d u i z ) . B117, 1
(11335).
Magnetic Studies The magnetic properties of hemoglobin, oxyhemoglobin and carboxyhemoglobin were carefully studied by Pauling and Coryell.8 In this work, the magnetic susceptibilities of carbonmonoxyheme, Fe(I1)-dicysteinate complex and dicarbonmonoxyFe(I1)-dicysteinate complex, respectively, were measured. Apparatus for Magnetic Susceptibility Measurements.1 Gouy apparatus with a compensation vessel was used. The compensation vessel, 30 cm. long and 0.70 mm. o.d., was suspended from one arm of an air-damped Sartorius microbalance. The maximum \-arialice of the microbalance (7’) 31. Schubert, T H I S J O U R X A L , 55, 4563 ( 1 9 3 3 ) . (8) L . Pauling and C. D . Coryell, Pvoc. A’utl. A c a d . Sci. L‘. 159, 210 (1936).
S.,22,
March 5 , 1958
COMBINATION OF CARBON MONOXIDE WITH HEMOGLOBIN
1111
Equilibrium Data It may be speculated that perhaps because of the existence of some secondary interactions in carboxyhemoglobin, the CO molecule is more firmly hleld to hemoglobin than t o heme, and consequently carboxyhemoglobin is more resistant t o oxidation by air than carbonmonoxyheme. I n order t o investigate this possibility, the equilibrium constant for the reaction between carbon monoxide and heme was determined spectroscopically and compared t o that for hemoglobin.
Car boxyhemoglobln
Because of the pronounced difference in absorption spectra of heme and carbonmonoxyheme solutions, respectively, in the range 500-600 mfi, the equilibrium constant for the reaction between carbon monoxide and heme can be readily determined by spectroscopic and manometric measurements. If the reaction is CO heme heme-CO tlieii the equilibrium constaut is
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Pyrt d f n e - heme - C O I
aiitl
I
I
3000
2500
,
,
,
,
I
2000
the tlegrec of saturation is
L
1500
Wave number (cm.-I). I'ig. 1.--Infrared spectra of carboxyhemoglobin and carbonmonoxyheme-pyridine complex. was 3 micrograms. The magnetic field, about 12,000 gauss, was produced by a Varian Model V-4004 Four Inch Electromagnet with Two Inch tapered poles. Magnetic Susceptibility Measurements.-The compensation vessel was calibrated with air and oxygen-free water. Both ends of the vessel were sealed with a rubber cap. In general, the solution was injected into the upper compartment, which was prefilled with an inert gas, by means of a hypodermic syringe. The lower compartment was filled with the corresponding solvent medium. The average probable error of the measured paramagnetic susceptibilities by the present procedure is about 2.5%. In order t o check the present procedure for systematic errors, the total molar susceptibility of potassium ferricyanide in aqueous solution was determined. The measured value xmol = 2195 c.g.s. units is only 0.7y0 higher than the accepted value of 2180 c.g.s. units.
The total molar susceptibility of Fe(I1)-dicysteinate in aqueous solution was found to be 9256 X lop6 c.g.s. unit a t about 20". The molar diamagnetic susceptibility was estimated from Pascal's c.g.s. unit. additivity rule t o be -151.4 X This gave a molar paramagnetic susceptibility of 9407 X c.g.s. unit and a permanent magnetic moment of 4.74 B.M. This corresponds t o four unpaired electrons per Fe(I1). Pauling and Coryell8found that the magnetic moment of both hemoglobin and heme showed the existence of four unpair
