TIIE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 249, No. 14, Issue of July 25, pp. 4495-4503, 1974

Printed in U.S.A.

Heme-Spin Label Studies of Hemoglobin

I. PREPARATION AND PROPERTIES OF HEME-SPIN-LABELED I;ERRIHEMOGLOBIN*

(Received for publication, May 15, 1973)

TOSHIO ASAKURA~

From the Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 191?‘4

SUMMARY

In order to investigate the protein conformation in the vicinity of the heme and the spin states of the heme-iron in dissolved hemoglobins, a nitroxide spin label was covalently attached to one of the propionic acid groups of the porphyrin ring. The optical spectra of the heme-spin labeled ferri- hemoglobins were identical to those of native hemoglobins indicating that the spin labeling did not affect significantly the electronic structure of the prosthetic group. The EPR spectra of the nitroxide moiety in the fluoride, cyanide, and azide derivatives of the heme-spin-labeled hemoglobin and of the corresponding acid methemoglobin in solution were not identical, suggesting that the protein conformation in the vicinity of the label is different in each of these hemoglobin derivatives.

The resonance amplitude of the nitroxide in heme-spin- labeled hemoglobin was sensitively influenced by the high spin heme-iron located in the center of the porphyrin ring due to the magnetic dipolar interactions between them. The degree of the dipolar interaction depended on the magnetic moment and electron-spin relaxation time of the heme-iron, as well as the distance between the nitroxide and heme-iron. From the strength of this interaction, the distance between the heme-iron and the nitroxide radical was estimated as 11.8 A. Since the spin label attached to heme is sensitive to changes in the magnetic moment of the iron, the heme-spin- labeled ferrihemoglobin can also be used for studying the thermal equilibrium between high and low spin electronic states of the heme-iron. By comparing the nitroxide reso- nance amplitudes of the hydroxide form with those of the high spin acid met form and the low spin cyanide form of hemoglobin, the ratio of high to low spin components of the hydroxide was calculated as 53:47 at 0”. This ratio was increased at higher temperatures due to the shift of the equilibrium composition in favor of the high spin form.

Optical and EPR spectra of free spin-labeled protohemin were also investigated. EPR spectra of protohemin were sensitive to dimer formation of hematin in aqueous alkaline solution.

Two empirical indices are presented for convenient nu-

*This work was supported by National Institutes of Health Grants GM-12202 and HE-14679.

$ Recipient of a National Institutes of Health Career Develop- ment Award 5-K04-GM 47463.

merical expression of the mobility of a nitroxide label from its EPR line shape.

X-Ray crystallographic studies by Perutz and associates (1, 2) have provided three-dimensional structures of human and horse hemoglobins and have made possible discussions of the relations between hemoglobin structure and function at the molecular level. Structural studies of dissolved hemoglobin are the next urgent requirement for determination of the physio- logically important regulation of hemoglobin function, especially the molecular mechanism of the effect of pH, organic phosphates, and COn on oxygen binding by hemoglobin. The intermediate structure between oxy and deoxy forms of hemoglobin is of po- tential value in understanding the mechanism of heme-heme in- teraction as well as the sequence of oxygen binding to hemoglobin tetramers.

For this purpose, the spiu label technique developed by MC- Connell et al. (3) has been used in studies of hemoglobin in solution by labeling the heme group directly with nitroxide- free radicals. Preliminary experiments with this heme-spin label method showed that conformational changes in the vicinity of the heme and the spin state of the heme-iron are reflected in the nitroxide EPR spectra (4-7).

The present paper describes the preparation and properties of heme-spin-labeled ferrihemoglobin in which all four subunits contain mono-spin-labeled protoheme.

METHODS AND MATERIALS

Hemoglobin and ApohemoglobirL-Crystalline hemoglobin was obtained from human blood by the method of Drabkin (8). The heme concentration was measured as a pyridine hemochromogen spectrum with the millimolar extinction coefficient of 34.4 rnM-l at 556 nm. The apohemoglobin was prepared by a modification of Teale’s acid-butanone method (9, 10). A millimolar extinction coefficient of 65 rnM-l cm-l at 236 nm was used to determine the tetrameric apohemoglobin concentration (11).

The hemoglobin crystals were dissolved in cold-distilled water at a final concentration of approximately 0.2 mM and bubbled with carbon monoxide gas to convert oxyhemoglobin to the carbonomonoxy form. The pH of the solution was then adjusted to 2.5 by the addition of 1 N HCl while stirring gently with a Vor- tex mixer. The solution was immediately mixed with 2 volumes of cold 2-butanone containing a few drops of 1 N HCl and shaken by inverting the test tube 2 to 3 times. The mixture was allowed

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to stand at 0” for a few minutes until the organic phase was clearly separated from the colorless lower globin phase. The butanone phase which contained free hemin was siphoned off. The colored butanone phase remaining on the top of the aqueous globin solu- tion was completely removed by repetitive addition and removal of cold 2-butanone. Each new solution of 2-butanone was care- fully added so that no mixing occurred with the aqueous globin solution. The remaining aqueous phase was treated again with 2 volumes of butanone as described above. The globin solution was dialyzed first against several changes of cold-distilled water and then against 10 mM potassium phosphate buffer, pH 7.0. The slight precipitates formed during the dialysis were removed either by centrifugation or by filtration through Whatman No. 5 filter paper. The globin solution can be stored for a few days at 0”.

Preparation of Mono- and Di-spin-labeled Protohemin-A nitrox- ide compound, 2,2,5,5-tetramethyl-3-aminopyrrolidine-l-oxyl was prepared by decarboxylation of 2,2,5,5-tetramethyl-3-car- boxamide-1-oxyl according to the method of Morrisett (12). The starting material was kindly supplied by Dr. H. R. Drott or was purchased from Eastman Kodak Co. Di-spin-labeled protohemin in which two spin labels are covalently bound to positions 6 and 7 of the porphyrin ring, was prepared from protohemin (Sigma) and 2,2,5,5-tetramethyl-3-aminopyrrolidine-1-oxyl as described elsewhere (4).

Mono-spin-labeled protohemin, spin-labeled at only one of the two propionic acid groups at positions 6 and 7 of the porphyrin ring, was prepared by the partial reaction of protohemin with the nitroxyl-free radicals, followed by purification by column chromatography on silicic acid (Bio-Gel SA). Conditions for the chromatography were the same as that used for the preparation of protohemin monomethyl ester (13).

The pyridine hemochromogen spectra of the di- and mono-spin- labeled protohemins are identical with that of protohemin. A millimolar extinction coefficient of 34.4 rnM-i cm-i at 556 nm was used to determine the concentration of these spin-labeled hemins.

Identification of Spin-Labeled Protohemin-The pyridine hemo- chromogen spectra of the di- and mono-spin-labeled hemins have the same absorption maxima as that of protohemin, indicating that positions 2 and 4 of the porphyrin ring were intact. Optical spectra of mono- and di-spin-labeled protoheme in dimethyl- sulfoxide were also identical with those of protohemin (cf. Fig. 2). Di-spin labeled protohemin was soluble only in organic solvents such as ethyl acetate and chloroform, and completely insoluble in aqueous alkali. Mono-spin-labeled protohemin has a solu- bility intermediate between that of di-spin-labeled protohemin and free hemin. By optical and EPR measurements, the ratio of hemin to nitroxyl-free radical was calculated as approximately 1:2 in di-spin-labeled hemin and 1:1 in mono-spin-labeled hemin.

TABLE I

Thin layer chromatography of spin-labeled protohemin

Hemin Lutidine-water Hexane-chloroform-

(20: 1) methanol (1:1:0.3)

Protohemin.. 0.05 0.1 Mono-spin-labeled protohemin. 0.45 0.33

Di-spin-labeled protohemin. 0.95 0.56

Thin layer chromatography on glass fiber impregnated with silicic acid (ITLC-SA, Gelman Instrument Co.) showed that the di-spin- labeled hemin moved as a single spot with RF values of 1.0 and 0.52 in the solvent systems, lutidine-water (1:2), and hexane- chloroform-methanol (1:1:0.3). Free protohemin had RF values between 0 and 0.1 in these two solvent systems. The mono-spin- labeled protohemin had intermediate RF values under the same conditions. These results are summarized in Table I. The in- frared spectrum of the di-spin-labeled protohemin in chloroform shows carbonyl stretching vibrations of amide groups at 1655 and 1525 cm-i. The mono-spin-labeled protohemin exhibited the above-mentioned amide bands together with the free carboxylic acid group having an absorption at 1705 cm-i.

Further reaction of the mono-spin-labeled protohemin with the nitroxide produced a heme indistinguishable from the di-spin- labeled protohemin.

Recombination of Apohemoglobin with Spin-labeled Heme-Re- combination was carried out by essentially the same technique as that used for the recombination of apocytochrome c peroxidase with protoheme alkyl esters (14). A stoichiometric amount of spin-labeled protohemin (0.5 mM) dissolved in dimethylsulfoxide was added dropwise to 0.01 mM apohemoglobin in 10rn~ potas- sium nhosohate buffer. nH 7.0. at 0”. The solution was stirred for 5 min, and the mix&e was’dialyzed against three changes of cold-distilled water for 6 to 9 hours. The mixture was then dia- lyzed against 5 mM potassium phosphate buffer, pH 6.0, followed by column chromatography on carboxymethylcellulose previously equilibrated with the same buffer. The crude spin-labeled hemo- globin was eluted with 50 mM potassium phosphate buffer, pH 7.0, from the column. Since the material contains some dena- tured low spin component (cf. Fig. 5A, Trace II), the spin-labeled ferrihemoglobin was reduced enzymatically to the ox; form with ferredoxin, ferredoxin reductase, and TPNH-generating system (15). A heat treatment for this oxyhemoglobin was carried out at 40” for 10 min. After centrifugation at 10,000 X g for 10 min, the supernatant was oxidized with excess potassium ferricyanide, which was removed immediately with gel filtration on Sephadex G-25. The ferrihemoglobin eluted shows an absorption spectrum indistinguishable from that of native ferrihemoglobin in potas- sium phosphate buffer, pH 6.0 (cf. Fig. 50, Trace VI).

Dejinition of Mobility Index-Perhaps the most rigorous defini- tion of the mobility of a free radical is by the correlation time for relaxation of its electron spin. However, calculation of the cor- relation times for radicals in the “slow tumbling region” at present, requires a considerable amount of computer time (16). Therefore, two empirical equations were used which allow a convenient expression of the spectra.1 changes by a “mobility index.” Index A is used for homogeneous systems and Index B is used for complex EPR spectra where two or more subspectra are observed. The Index A is calculated from two parameters: the peak to peak line width of the central resonance (P in Fig. IA) and the distance from the central point to the outer line peak in the higher magnetic field (D in Fig. 1A).

Index A = P2D2/550

Table II illustrates the relations of the Mobility Index A to vari- ous EPR spectra of 2,2,5,5-tetramethyl-3-aminopyrrolidine-l- oxyl dissolved in water-glycerol mixed at various temperatures. When the label is tumbling very fast, the index shows a value less than 1, while the index is about 100 when the label is strongly

FIG. 1. Notation of various parameters used for the calculation of mobility index A and B.

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TABLE II

Comparison of mobility Indices A and B with rotational correlation times (T)

The EPR spectra of 2,2,5,5-tetramethyl-3-aminopyrrolidine- 1-oxyl were measured under conditions described in the table with a Varian E-4 EPR spectrometer. The mobility Indices A and B were calculated as shown in the text.

lability Index P hobhty Index E 1

06

27

15 4

34.7

77.0

I

5

26

33

47

I

4

15

40

70

a The rotational correlation time (T, ns) was estimated from the Stokes law (17).

immobilized. Table II also compares Index A with the rotational correlation time ‘T which was estimated from the results obtained by Hsia using the Stokes equation (17). It can be seen that the Mobility Index A is approximately proportional to the correlation time although there is no theoretical justification for this relation.

The Mobility Index B is used to compare mobility changes in complex systems which contain more than two discernable spec- tra. This index is equal to the sum of the products of the relative amplitude (h,/H) and the cube of the distance (di) from the point where the central resonance crosses the base-line (Fig. 1B).

Mobility Index B = (l/H X 10s) c hi.dia

The sign of hi is negative when the curve is below the baseline. The product is calculated at 1-G intervals, and the results are shown in Table II.

Measurements-The optical and EPR measurements were carried out with a Perkin-Elmer Coleman 124 spectrophotometer and a Varian E-4 spectrometer with variable temperature con- trols.

RESULTS

Optical Spectra of Spin-labeled Protohemin-Absorption spectra of di-spin-labeled protohemin in dimethylsulfoxide are shown in Fig. 2. The spin-labeled hemin shows a typical high spin-type spectrum in dimethylsulfoxide in the presence of 2 PM perchloride acid. The sharp Soret band is indicative of monomeric ferri- heme. Dimethylsulfoxide may furnish axial ligands for the heme-iron under these conditions (18). Addition of KCN con- verts the spectrum to that of the low spin di-cyanide complex. On the other hand, upon addition of aqueous alkali, the hemin is converted to a complex which has relatively low Soret band (Fig. 2). This compound is probably a dimeric form of the hemin (18). All of the optical spectra of di-spin-labeled proto-

FIG. 2. Optical absorption spectra of di-spin-labeled proto- heme (Di-SL-Protoheme). The hemin (10 PM) was dissolved in dimethylsulfoxide in the presence of 2 PM perchloric acid (-), 0.1 mM potassium cyanide (-----), and 0.1 mM NaOH (- - -).

1 L h4SL.Pl-l in DMSO

m

,-a ‘I -__- -y- .____

k

1 -J

FIG. 3. EPR spectra of mono-spin-labeled protoheme (MSL. PH) in dimethylsulfoxide at 20’. The hemin (50 PM) was dis- solved in dimethvlsulfoxide in the presence of 10 FM perchloric acid (I); 0.1 mM fiaOH (II), and 0.5 rnM potassium cyanide (IN). The microwave power of 20 mwatts and the modulation amplitude of 0.5 G were used.

hemin are identical with those of protohemin and of mono-spin- labeled protohemin, indicating that the modification of the propionic acid groups at positions 6 and 7 of the porphyrin ring produces no significant effect on the electronic structure of the porphyrin ring, although the modification alters the solubility of the spin-labeled protohemins significantly.

Effect of Iron Spin State and Dimerizaiion on EPR Spectra of Spin-Labeled Protoheme in Dimethylsuljoxide-As shown in Fig. 3, both the line shape and the resonance amplitude of the EPR spectra of the mono-spin-labeled protohemin in dimethylsulfox- ide are strongly affected by the changes in the spin state of the heme-iron. The low spin cyanide complex (Spectrum ZZZ in Fig. 3) shows three sharp lines typical of rapidly tumbling ni- troxyl-free radicals, while the high spin complex (Spectrum Z in Fig. 3) exhibits relatively weak, broad lines. This broadening in the high spin complex is probably due to the magnetic dipole interaction between spin label and heme-iron (5, 19). The high spin heme-iron has a larger magnetic moment as well as a longer electron spin relaxation time, and both of these factors produce a decrease in the resonance amplitude. By contrast, the low spin iron of the cyanide complex has little effect on the spin label be- cause of the small magnetic moment and the very short relaxa- tion time of the iron.

The effect of temperature on the EPR spectrum of the mono- spin-labeled protohemin is shown in Fig. 4. At higher tempera-

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MSL PHI” DMSO 0

/ : e

8~~

20 G

FIG. 4. Effect of temperature on the EPR spectra of low spin cyanide (top) and high spin ferric forms (bollom) of mono-spin- labeled protoheme (ML%-PH). The experimental conditions are the same as in Fig. 3.

tures, the low spin cyanide complex shows sharper lines. This change is simply due to the increased tumbling rate at higher temperatures. On the other hand, the opposite effect is ob- served in the high spin complex, which shows weaker signals at higher temperatures (Fig. 4B).

An interesting observation is that the resonance amplitudes of the alkaline forms are highly concentration dependent. The molar resonance amplitude of the alkaline complex becomes higher at the lower concentrations. This increase of the res- onance amplitude upon dilution may be due to the dissociation of the dimer into the monomer form. Addition of cyanide to the alkaline complex gradually changes the resonance amplitude to that of the cyanide form. Thus, the El’R spectra of the spin labels attached to heme are not only sensitive to the spin state of the heme-iron, but may also reflect the extent of dimerization of the heme.

Recombination of Spin-labeled Wemin with Apohemoglobin- Although dialkyl hemins combine with apohemoproteins, the affinity of the dialkyl hemins for apoprotein is low (14, 19). In addition, di-spin-labeled protohemin sometimes exhibits compli- cated EPR spectra due to the magnetic dipolar interaction be- tween the two spin labels attached at positions 6 and 7 of the porphyrin ring (5). This problem is eliminated by using the mono-spin-labeled hemin. In this case, the only possible mag- netic interaction is between the spin label and the heme-iron in the same heme group, so that from the extent of the magnetic interaction the iron-label distance can be estimated (5).

The titration of apohemoglobin with mono-spin-labeled proto- hemin shows a quantitative binding of 4 moles of heme per mole of apoprotein. The optical spectrum taken immediately after elution of the first column chromatography on carboxymethyl- cellulose exhibits a spectrum characteristic of a mixture of high and low spin compounds (Fig. 5A, Trace I). The two peaks at

wove Length ( nm I

FIG. 5. Absorption spectra of mono-spin-labeled hemoglobin at various purification steps. A-Z, hemoglobin (80 NM) immedi- ately after elution from carboxymethylcellulose column; A-II, I + 0.2 M potassium ferricyanide. The excess ferricyanide was removed by gel filtration with Sephadex G-25. B-III, I1 + 0.1 M potassium fluoride; B-IV, methemoglobin fluoride prepared from native hemoglobin; C-V, ZZZ + ferredoxin reductaee sys- tem (15) ; D-VI, V + potassium ferricyanide; D-VIZ, VI + ferre- doxin reductase system.

534 and 570 nm are in part due to the oxyhemoglobin which can be oxidized to the met form upon addition of potassium ferri- cyanide (Fig. 5A, Trace II). Since the spin-labeled hemin used for the recombination experiment is 100% ferric form, the hemo- globin must have become reduced and oxygenated during the preparation. This reduction of the ferric heme-iron has recently been attributed to the spin labels. The visible spectrum of the ferricyanide-treated hemoglobin (Fig. 5A, Trace ZZ) still contains unidentified low spin-type absorptions at around 530 and 570 nm. Although the over-all spectrum resembles that of ferric hemo- protein which is a thermal mixture of high and low spin com- pounds, this cannot be the explanation because the spectrum is insensitive to changes in temperature. The low spin peaks are therefore attributed to contamination by hemichrome-type denatured protein. Since the ratio of this hemichrome type com- pound to that of the high spin complex is always approximately 1: 1, the formation of the high and low spin compounds may be attributed to the two isomers of 6 or 7 mono-spin-labeled proto- hemin (see below). As shown in Fig. 5B, Trace ZZZ, the low spin component of the ferrihemoglobin does not react with fluoride, while the high spin component forms a typical fluoride complex. If all the heme reacted with fluoride, we would expect Spectrum IV in Fig. 5B. Separation of the denatured component on carboxymethylcellulose column did not occur when the ferric sample was used. As shown elsewhere (15, 20), spin-labeled ferric hemoglobin can be reduced enzymatically to the oxy form without destroying the free radical (Fig. 5C, Truce V). After this reduction, the hemoglobin obviously contains some nonre- ducible impurity which has a broad absorption between 600 and 700 nm. This impurity can be completely removed by a heat treatment at 40” for 10 min, followed by gel filtration on Sepha- dex G-25 (Fig. 50, Trace VIZ). The oxidized product of this oxyhemoglobin with ferricyanide now shows a spectrum identical with that of native high spin methemoglobin, as shown in Fig.

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FIQ. 6. Optical spectra of mono-spin-labeled ferrihemoglobins. The hemoglobins were dissolved in 0.1 M potassium phosphate buffer, pH 6.0.

- MSL PH-HI-CN

- MSLPH-HI’

-----. MSL PH-HI F

(pH60,20’C)

, 20G ,

Fro. 7. EPR spectra of cyanide, fluoride, and acid met forms of mono-spin-labeled hemoglobin. The hemoglobin (0.5 mM) was dissolved in 0.1 M potassium phosphate buffer, pH 6.0. The microwave power, 20 mwatts; the modulation amplitude 0.5 G.

50, Truce VI. The following experiments were performed with this purified ferrihemoglobin.

Absorption Spectra of Spin-labeled Methemoglobin and Its Com- plexes with Cyanide, Azide, and Fluoride-Sugita and Yoneyama (21) reported that the alkylation of the two propionic acid groups at positions 6 and 7 of the porphyrin ring did not affect the optical and oxygen-binding properties of hemoglobin. As shown in this and the following papers (22), the optical proper- ties of the synthetic hemoglobins containing mono-spin-labeled protohemin and its complexes with various ligands are also iden- tical (within experimental errors) to those of native hemo- globin (Fig. 6).

EPR Spectra of Mono-spin-labeled Hemoglobin and Its Com- plexes with Various Ligands-In Fig. 7, the EPR spectra of syn- thetic hemoglobin containing mono-spin-labeled protohemin in 0.1 M potassium phosphate buffer, pH 6.0, are compared to those of the cyanide, aside, and fluoride complexes. Although all these spectra belong to those of so-called moderately immobilized labels, the mobility indexes are slightly different from one an- other indicating that there are differences in the heme environ- ments of the various hemoglobin derivatives (Table III). The cyanide and aeide complexes have relatively large resonance amplitudes, while the fluoride complex has a small amplitude. Differences in the resonance amplitudes are attributed to differ- ences in spin states (i.e. magnetic moment and electron spin re- laxation times) of the heme-iron (5). It should be noted that the resonance amplitude of the acid met form of the spin-labeled hemoglobin is intermediate between the high spin fluoride and

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TABLE III Mobility indices of spin-labeled jerrihemoglobins at 80'

Ferrihemoglobin Mobility index A Mobility index B

Methemoglobin fluoride. 16.1 38.4 Acid methemoglobin. 11.1 19.0 Alkaline methemoglobin. 8.9 17.5 Methemoglobin cyanide.. . 7.1 14.9 Methemoglobin azide. 6.3 10.9

the low spin cyanide complexes. This observation was initially attributed to the presence of both high and low spin components in the spin-labeled hemoglobin as seen optically in Fig. 5A, Truce ZZ. Nevertheless, it was found that the amplitude is still smaller than that of the high spin fluoride complex, even after purification of the hemoglobin to the completely high spin form which has an absorption spectrum shown in Fig. 50, Trace VI. Since the magnetic moment of the acid methemoglobin is similar to that of the high spin fluoride complex (22), this cannot be the cause for the difference in the resonance amplitude. The de- creased resonance amplitude must therefore be attributed to differences either in the electron spin relaxation time of the heme-iron or in the distances between the spin label and the heme-iron in the two high spin hemoglobins. To distinguish between these two possibilities, the solut.ion EPR spectrum of the fluoride and acid methemoglobin were measured at room temperatures (23). From the peak to peak line width of the g = 6 high spin iron signal, the electron spin relaxation times of the heme-iron are estimated at 0.5 and 0.2 ns for the fluoride and acid methemoglobins, respectively. Thus, differ- ences in the resonance amplitudes of the EPR signals of the label between the high spin fluoride and acid methemoglobin molecules can be clearly explained by the difference in their electron spin relaxation times. It should be pointed out that the EPR spectra of the label in the cyanide and aeide complexes reveal two peaks in the lower magnetic field, indicating two different environments for the labels. The splitting may be due to the different heme environments between cy and @ subunits of hemoglobin or some other reason such as the isomeric states of the label relative to the protein as suggested by McConnell et al. (24).

Ionization of Spin-labeled Ferric Hemoglobin in Alkaline Solu- tion-The absorption spectra of the spin-labeled ferric hemo- globin at various pH values are shown in Fig. 8. The pK values for the acid-alkali transition of the spin-labeled ferric hemoglobin is 7.6 which is about one pH unit lower than that of native hemo- globin (25). The room temperature absorption spectrum of the alkaline form clearly shows the presence of two components which are thermal mixtures of high and low spin forms as will be de- scribed below,

Effect of Temperature on EPR Spectra of Spin-labeled Hemo- globin-The temperature-dependent spectral changes of various complexes of the spin-labeled ferrihemoglobins are shown in Fig. 9. In all complexes examined, the labels are less immo- bilized at higher temperatures, and there are concomitant in- creases in the resonance amplitudes. These changes are com- pletely reversible at temperatures between 0 and 30”. The mo- bility indices and the resonance amplitudes at various tempera- tures are illustrated in Fig. 10.

George et al. (26) and Iizuka and Kotani (27) showed that bc th the acid and alkaline forms of ferrihemoglobin are thermal m.x- tures of high and low spin electronic states. This property ap- pears to be unaltered after spin labeling of the propionic acid

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groups at positions 6 and 7 of the porphyrin ring. Spin-labeled ferrihemoglobin in alkaline solution clearly exhibits a mixed spectrum of high and low spin compounds (Fig. 8), and the resonance amplitudes of the hemoglobin hydroxide also shows intermediate values between those of the high spin fluoride and low spin cyanide complexes (Fig. 10). If the alkaline form of the spin-labeled ferrihemoglobin is a thermal mixture of high and low spin compounds, then changing the temperature would

0.6

0.6 Q E

u 8 5 0.4

5 s

0.2

C

SL- Hemoglobin (Fe+++)

2 70

3 73

4 76

5 8.1

6 6.6

7 9.7

560 m 660 ’ 7

X (nm)

3

FIG. 8. Effect of pH on the optical absorption spectra of mono- spin-labeled ferrihemoglobin at 20”. The hemoglobin was dis- solved in 1 mM potassium phosphate buffer, pH 6.2. The pH was adjusted by adding 1 to 2 pl of concentrated NaOH solution.

MSLPH-H: (pIi

- 30.C ---- 20’ -I. 10.

---- 0.

, 20 G ,

I 20 G

MWPH-Hi-F (pH 6 01 - 30-c --I 20. - _.._..__ , 0. ----- 0.

be expected to influence the equilibrium and a change should therefore be observable in the absorption spectrum and in the magnetic moment. This change in the magnetic moment of the heme-iron should secondarily affect the EPR spectrum of the spin label located close to the heme-iron (5). Such a tempera- ture-dependent magnetic effect is obviously seen in temperature-

HiCN

HiOH

Hi*

HiF

. . . HiOH HiCN I . 0 IO 20 30

Temperature ( ‘C)

FIG. 10. Effect of temperature on the central resonance ampli- tude and mobility index A of EPR spectra of mono-spin-labeled ferrihemoglobins. The central resonance amplitudes are nor- malized to hemoglobin concentrations and the settings of the EPR spectrometer. The dotted line was obtained by assuming that the hydroxide complex is temperature independent.

, 20G ,

ML PH-HI-CN (pH 6 0)

- 30.C ----- 20. _- 10. ------ 0.

2OG, ,

MSL PH-HI-N, (pH60)

- 30. ---- 20.

_^.” 10. --s-e* 0.

FIG. 9. Effect of temperature on the EPR spectra of mono-spin-labeled ferrihemoglobin in the presence of various liganda. The ex- perimental conditions were the same as in Fig. 10.

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dependent rhanges in the resonance amplitudes in Fig. 10. The expected increase of the resonance amplitude of the hydroxide form of heme spill-labeled hemoglobin from increased mobility is suppressed at higher temperatures, indicating that the thermal equilibrium is shifted in favor of the high spin heme at higher temperatures.

Thcoret.ical treatments for clcctron paramagnetic resonance line shape of slowly tumbling molecules have been developed us- ing the adiabatic assumption (28, 29) or perturbation assump- tion (30). More recently, Freed et al. (16), and Gorden and Messenger (31) presented a theory of rotational motion of mole- cules with the rotational diffusion equation.

In the present paper, attempts were made to find empirical equations which simply express the spectral changes numerically. These equations were obtained on the basis of the fact that the distance between the central peak and outer peak (.!I and di), and the line width (P) of the central resonance increase as spin labels are more immobilized. It was empirically found that P2D2 in Index A and hi.dia in Index B has certain parallelism with the correlation times as shown in Table II. Although the two equations used in this paper have no theoretical basis, rela- tively small spectral changes are distinguishable numerically by simple calculations. These two indices, however, are not ap- plicable in cases of rapid anisotropic mot.ion of the spin label (cf. Ref. 32).

It has been known that hemin can exist in a dimeric form in aqueous alkaline solut.ions or in alkaline dimethyl sulfoside (20, 33-36). Schugar et al. (37) reported that the low magnetic moment of hematin solution is consistent with the idea of anti- ferromagnetic spin eschange behavior, associated with an Fe-O- Fe bridge. The infrared spectrum of aqueous alkaline hemin solution also suggests an osobridged structure (38). The present result obtained by heme-spin label method clearly shows that the spin labels att,ached to alkaline hematin have no significant magnetic interaction with the heme-iron indicating that the heme-iron is in either a low spin or antiferromagnetic state. The magnetic interaction observed in concentrated solutions may be mainly due to that between spin labels of different molecules.

The mono-spin-labeled protohemin used in the present experi- ment is assumed to be a 1 :l mixture of the two isomers of 6 or 7 mono-spin-labeled protohemin. Although these isomers cannot be separated, the formation of an equal amount of the high and low spin complexes upon binding with apohemoglobin suggests that the protein may distinguish these two isomers.’ The con- clusion must await the x-ray crystallography of the crystalline hemoglobin. The opt,ical spectra of both ferric and ferrous hemoglobins containing 6 or 7 mono-spin-labeled protoheme are identical with those of natural hemoglobin. The modification of the carboxyl end of the propionic acid chains may be too far removed from the porphyrin ring to affect its elect,ronic proper- ties, although these modifications cause marked changes in the solubility of the hemin. A second reason why the modifications at the 6 and 7 carboxyl groups do not affect the optical and oxygen-binding properties of hemoglobin may be the orientation of the heme group in the heme pocket. As reported by Perutz

1 Recently, we have succeeded in the chromatographic separa- tion of the two isomers of 2- or 4-monovinylmonoformyldeutero- hemin. Although the physical properties of these two isomers are similar to each other, it was found that apohemoglobin can dis- tinguish these two isomers and shows different oxygen-binding properties (43).

TAIJLI,: IV

Distances between heme-iron and spin label in spin-labeled jerrihemoglobin

Ferrihemoglobin P A/h-i* Distance

11s A

Acid methemoglobin. _, 0.2 0.53 11.5 Methemoglobin fluoride. 0.5 0.36 11.8

jL ISstimated from the EPIt spectrum of the iron signal (23). * Ratio of the central resonance amplitudes of heme-spin-la-

beled acid met and fluoride methemoglobin to that of cyanide hemoglobin.

(I, 2), the propionic acid groups in hemoglobin make polar con- tact with protein in water so that their contributions to the bind- ing energy should be small. In fact., modifications of the propi- onic groups cause no significant effect on the oxygen-binding properties of hemoglobin (20, 21) while chemical modification at posit.ions 2 and 4 give large effects (21, 39). Completely oppo- site results were obtained in cytochrome c peroxidase (14, 40) and horseradish peroxidase (41, 42) where the modification at positions 6 and 7 resulted in the marked decrease in the perox- idase activity, while t,hose at the 2 and 4 positions showed no effect on the enzymatic activities.

Estimation of Iron Spin Label Dislance-As previously re- port.ed (5), the distance between the spin label moiety and heme- iron in dissolved hemoproteins is estimated using the magnitude of the interaction C and the experimentally determined line width of the heme-iron signal. Such a calculation was made here again using purified hemoglobin which contains no hemi- chrome compound. The theoretical basis for the estimation of the distance between heme-iron and spin label in the same macro- molecule is presented by Leigh (19). The magnit.ude of the in- teraction is given by

where g is the electronic g factor of the observed spin, p and 7 are the effective magnetic moment and the relaxation time of the heme-iron, and r is t,he distance between the two spins. Since the label is moving, the original calculation which assumed im- mobilization is not strictly applicable to the present system. However, the calculation provides satisfactory results for the es- timation of the average distance between the spin label and iron in dissolved systems,2 as shown in the present manuscript. The electron spin relaxat.ion time of t,he heme-iron is estimated from the line width of the room temperature EPR spectrum of g = 6 iron signal in solution (23). The distances thus obtained are summarized in Table IV. These values for the iron-label dis- tance are slightly smaller than the value (12.5 A) previously obtained (5). This difference was found to be due to the con- tamination of a hemichrome form in the spin-labeled ferrihemo- globin previously prepared. Since the hemichrome compound is low spin and does not react with fluoride, the spin labels at- tached to the hemichrome heme show a higher resonance ampli- tude even in the presence of fluoride. In the present experiment, however, the hemichrome in the spin-labeled hemoglobin was completely removed after reduction of the hemoglobin with a methemoglobin reductase system. It is not clear at present whether the differences in the iron-label distance are due to the conformational difference between the dissolved and crystalline hemoglobin (5), or to the error which might be produced by the

* J. S. Leigh, Jr., personal communication.

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TARLII V

Magnetic moments and 7 values of hemoglobin derivatives

Hemoglobin derivative Spin

quantum number

s

Methemoglobin cyanide. ?!i Alkaline methemoglobin (high spin). 35 Acid methemoglobin (high spin). 55 Methemoglobin fluoride. 55

T Magnetic moments r

#B ns

1.73 0.002 5.80 0.2 5.80 0.2 5.92 0.5

I

a See Ref. 22.

TABLE VI

Fractions of the high spin form of jerrihemoglobin hydroxide al different zemperatures

Ferrihemoglobin hydroxide Fractions at various temperatures

Acid methemoglobin (spin label I method). .T 53

Acid methemoglobin (optical methoda)......... 30

Alkaline methemoglobin (optical methods).......... .,... 66

%

62 72 80

32 35 38

68 70 72

a See Ref. 26.

conformational difference between the cyanide and fluoride com- plexes.

.4s to the protein conformation of various hemoglobin deriva- tives, the present results as well as those obtained by McConnell et al. (24) who spin-labeled at the p-93 cysteine residues of hemo- globin, appear to suggest that the protein conformation in the vicinity of heme or the b-93 cysteine in various ferric hemoglobin derivatives are not identical.

High and Low Spin Forms of Alkaline Ferrihemoglobin-The central resonance amplitudes of the spin label signal in the al- kaline ferrihemoglobin show values intermediate between those of the completely high spin fluoride complex and the completely low spin cyanide complex (Fig. 10 and Table V). If the hemo- globin hydroxide is a purely high spin complex and if the environ- ments of the label are not altered the resonance amplitude of the alkaline hemoglobin is expected to be similar to that of the acid methemoglobin rather than that of the hemoglobin fluoride be- cause the magnetic moment as well as the electron-spin-relaxa- tion time of the high spin hydroxide is similar to those of the acid methemoglobin rather than to that of the fluoride complex (cf. Table V). However, since the hydroxide complex contains cer- tain amounts of low spin component, which has both a lower magnetic moment and shorter electron spin relaxation time, the resonance amplitude of the hydroxide is intermediate between those of high spin acid and methemoglobin and low spin cyanide hemoglobin. The values for the relaxation times of the heme- iron were estimated by measuring the solution EPR spectra of various derivatives of ferrihemoglobin at room temperature (23). If we assume that the electron spin relaxation time for the low spin hydroxide form is as small as that of cyanide complex and that acid methemoglobin is mostly high spin form, then the ratios of the high and low spin compounds at various temperatures can be estimated taking the cyanide and acid met forms as totally

low spin and high spin standards, respectively. The fraction of the high spin and low spin forms of the alkaline hemoglobin at different temperatures are calculated and summarized in Table VI. It is noteworthy that the fraction of the high spin and low spin complexes varies with temperature, clearly indicating that the alkaline hemoglobin is an equilibrium mixture of high and low spin states. These results, however, are significantly dif- ferent from those of George et al. (26) based 011 magnetic and spectroscopic measurements of alkaline hemoglobin. Although there is no clear explanation for these differences, the relative increase in the fraction of high spin alkaline hemoglobin obtained by the hemc spin label method are consistent with that of our room temperature EPR experiment 011 alkaline hemoglobin (23).

The present results indicate that the spin labeling of the heme group is a powerful method for probing the protein conformation in the vicinit,y of heme and for monitoring changes in the spin state of the heme-iron. An application of the method for ferrous hemoglobin will be reported in the following paper (20).

Acknowledgments-The author is indebted to Drs. 1). Chance, T. Yonetani, H. R. Drott, J. S. Leigh, G. Reed, and A. Coulson for their valuable discussions. He also thanks Drs. H. Akimoto and R. Hershberg for infrared measurements and helpful dis- cussions.

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Toshio AsakuraOF HEME-SPIN-LABELED FERRIHEMOGLOBIN

Heme-Spin Label Studies of Hemoglobin: I. PREPARATION AND PROPERTIES

1974, 249:4495-4503.J. Biol. Chem. 

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