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Electron spin resonance spectra of isolated ferrihemoglobin (α, β and γ) chains—An attempted correlation with optical absorption spectra
J. Mol. Riol. (1970) 50, 99-110
Electron Spin Resonance Spectra of Isolated Ferrihemoglobin (a, j? and y) Chains-An Attempted Correlation with Optical Absorption Spectra Y. HENRY
AND
R. BANERJEE
Innstitut de Biobgie Physico-Chirnique 13, rue Pierre et Marie Curie, Paris 5, France (Received 30 October 1969) The electron spin resonance spectra of the isolated ferri OL,j and y chains of human adult and foetal hemoglobins have been obtained (at 77°K) and compared with those of the products of their recombination, namely the respective methemoglobins. All the hemoproteins examined gave essentially similar spectra and g-values for a given liganded form (aquo, hydroxy, fluoride, azide); however, important differences between the aquo forms appeared on comparing the linewidths and signal heights per unit heme concentration. The aqua derivatives of a + sH and ~~+r~st gave high-spin signals (g = 56) with line widths of 100 + 5 and 85 f 5 gauss, respectively ; that of all other compounds examined (the intact methemoglobins, the 8’ and y+ chains with free or mercury-bound sulfhydryls) was 75 + 5 gauss. On comparing the spectra of the second group of hemoproteins (76 gauss line-width) it appears that the high spin contribution of y +rMB, Hb +A and Hb +Ft are nearly equal ; that of ,¶+rMs and y + sz is about 60% and that of /?+sx is only 27% of the high-spin contribution of Hb+A or Hb+Ft for equal heme concentration. It is inferred that the isolated y+sz and /!+sx chains are partly low-spin, the low-spin form being of a type non-observable under the experimental conditions (9.5 GHz md 77OK). This view gains support from the visible absorption spectra of the ferri chains. At O”C, unlike that of Hb +A, Hb +Ft and a + sz, the spectrum of y + sH was partly low-spin and that of j3+ sH much more so. The decreased high-spin content of these species seen from electron spin resonance at 77’K may be explained as due to the effect of low temperature on a thermal equilibrium mixture of high and low-spin types. The results are discussed in the context of chain interactions in the hemoglobins.
1. Introduction Measurement of electron spin resonance absorption haa recently found wide applications in the study of heme proteins. E.S.R. absorption of single crystals of some derivatives of two hemaproteins, namely metmyoglobin and methemoglobin, were made by Bennett, Ingram, George & Griffith (1955) and by Gibson, Ingram & Schonland (1958). These measurements, carried out at 20”K, led to the experimental determination of g-values with respect to the porphyrin plane of aquo, hydroxyde t Abbreviations used : zPMB,pPe,,,and yPMB,chains with thiol groupe transformed into paramercuribenzoate derivatives by reacting with p-hydroxy mercuribenzoate; asa, flsH and ask, chains with free thiol groups. ma=,wpMB,etc. : ferro chains. 01+sa, a +PMB,etc. : ferri ohains. Mb, myoglobin; Mb+, met- or ferrimyoglobin; HbA, edult human hemoglobin; HbFt, foetal human hemoglobin; Hb+A and Hb+Ft, ferrihemoglobins; E.S.R., electron spin resonance. 99
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Y. HENRY
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and azide derivatives. It was shown that the order of energy-level splittings is the same as that predicted on theoretical grounds for an octahedral complex. Ehrenberg (1962) has made measurements on frozen solutions of metmyoglobin derivatives at 77°K and upwards. For the “ionic” or “high-spin” aquo derivative Mb + -H,Ot, he observed an intense asymmetrio absorption with g = 6.8, and none in the g = 2 region, the latter can be observed only on single crystals. For “covalent” or “low-spin” hydroxide and azide complexes, three bands were resolved at g-values around 2, which wrrespond closely to the principal g-values determined by Gibson et al. (1958) on single crystals. The satisfactory accord of results in frozen solutions with those on single crystals have since favored the study of various hemoproteins, particularly with a view to correlating the spin-state with light absorption spectra in aqueous solutions. Williams (1966) and Brill & Williams (1961) by comparing the light absorption and magnetic moments of various ferric compounds, have suggested that the 480 to 506 rnp and 600 to 650 rnp absorption bands were of a high-spin type; these bands are believed to be charge-transfer bands. Refined methods are now available (Smith & Williams, 1968), which permit estimation of the percentage of Fe111 in different spin states (high or low) when they exist in equilibrium. The existence of such thermal equilibrium mixtures of high and low-spin forms has been demonstrated for alkaline horse methemoglobin by George, Beetlestone BEGriffith (1961) by a combination of different techniques. More recently, Rumen 6 Chance (1969), by a study of the optical absorption of lamprey hemoglobin at different temperatures have observed the appearance of low-spin bands as the temperature is decreased. As is well known, human hemoglobin (HbA) is composed of two pairs of unlike chains (u and fi). Very specific interactions between the chains, and particularly the unlike ones, are believed to govern the generation of heme-heme interaction as well as the lowering of over-all oxygen afllnity compared to that of the subunits. A convenient method is available since 1965 (Bucci & Fronticelli) for the preparation of the isolated chains of human hemoglobin in the reduced form. The ferri chains, very unstable in aqueous solution, have recently been prepared in a relatively stable form (Banerjee & Cassoly, 1969). It is thus possible to study the optical absorption and E.S.R. spectra of the isolated chains with a view to comparing the spin states of Fe111 of the chains with that obtained in the product of their association, Hb+. It was shown recently (Banerjee, Alpert, Leterrier & Williams, 1969) that the visible absorption spectrum of /l+ chains contains a substantial contribution of low-spin bands, even at 0°C. We describe in this paper the electron spin resonance spectra of the u, 6 and y ohains of human hemoglobins (HbA and HbFt); a correlation between the light absorption and E.S.R. spectra will also be attempted.
2. Experimental Procedure (a) Preparations Normal human hemoglobin (HbA) wss prepared from fresh hemolysates and crystallized once from 28 M-phosphate following Drabkin (1946). Human foetal hemoglobin (HbFt) wss isolated from fresh cord blood following the procedure of Allen, Schroeder & Balog (1968). The work reported in this paper was done with fraction Fu of Allen et al. (1968). M&hemoglobins (Hb+A, Hb +Ft) were prepared by adding potassium ferricyanide (5% in excess) to the respective deoxy derivative in Thunberg tubes under vacuum. The resulting solutions were dialysed against 2 x lo-’ M-NaCl and cleared by centrifugation.
ELECTRON
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OF HEME
PROTEINS
101
state from fresh normal human aPMB ad PPMB chains were prepared in the oxygenated hemolysates by the method of Bucci & Fronticelli (1965). YpMs chains were prepared from pure HbFt by the action of PMB at pH 4.7 as described by Kajita, Tsniguchi BE Shukuya (1969). The removal of PMB from apMB was always performed by dialysis against 0.06% thioglycollate at pH 7.5 as suggested by Bucci & Fronticelli (1966), and resulted in the regeneration of 0.95 to 1 free -SH group per heme. The same method applied to ypMB also gave good regeneration (0.95 to 1 free -SH per heme, ss against the expected value of I per heme). As would appear from the results below, some of the properties of the 5sn chain were found to depend on the method utilized for its preparation from /lPM,,. In order to have a comprehensive idea as to the c&use of these variations, several methods were used in parallel. The number of free -SH groups per heme as recovered by these methods involving the use of different reactants were as follows: 1 - dodecanthiol (De Renzo, Ioppolo, Amiconi, Antonini & Wyman, 1967) 1.7 to 1.9 -SH/heme; /Imercaptoethanol (Tyume, Benesoh & Benesch, 1966) 1.9 -SH/heme; thioglycollate (Bucci & Fronticelli, 1966) 1.35 to 1.55 -SH/heme; cyst&e (Reichlin et al., 1965) 1.22 -SH/heme. The free -SH groups in the different hemoproteins were estimated by the method of Bayer (1954). Analytical starch gel electrophoresis was performed by the method of Poulik (1957). The concentrations of the different hemoproteins were determined by spectrophotometry on the cyanomet derivatives (tmM = 11.5 at 540 mp). The oxidized a and /3chains were prepared in I.6 M-glycine as described earlier (Banerjee & Csssoly, 1969). The solutions were filtered immediately after oxidation, at 4°C; portions of the same solution were often used simultaneously for visible spectrophotometry as well as for E.S.R. work. For the latter, the required amounts of the stock solution were added to tubes containing the appropriate ligands and buffers, the liganded derivatives being immediately afterwards transferred to E.S.R. sample holders and frozen in liquid nitrogen. The y chains, both PMB bound and free, when oxidized in aqueous solution, were found to be stable. The ferri y chains and their derivatives were therefore prepared and studied in aqueous medium. The various liganded forms of the different hemoproteins were obtained under the following conditions: equo, pH 6.0; hydroxide, pH 9.5; ezide, 0.2 M-N~.N,, pH 6.5; fluoride, 0.2 to 0.4 M-KF, pH 6.5; cyanide, 0.2 M-KCN, pH 9. Independent spectrophotometric measurements showed that the ligand concentrations were saturating under the given conditions. The a and /3 chains contained, in addition, 1.5 M-glycine adjusted to the required pH. Hemoprotein concentrations used for E.S.R. spectra were between 6 x lo-’ M and 1-8x 10m3 M (heme). All speotrophotometric measurements were performed with a Gary 14 recording spectrophotometer equipped with a special thermostatic attachment for work near 0°C. (b) Electron spin resonunce spectrorrcopy The electron spin resonance spectra, were recorded with a Varian E3 spectrometer. The sample, contained in a quartz tube of 3 mm diameter, was maintained at 77°K in 8 V&an V 4546 dew&r adapted to the spectrometer cavity. The volume of the solution within the cavity was approximately 0.13 ml. A current of dry nitrogen was continuously passed into the sample holder and the wave guide in order to prevent the condensation of atmospheric oxygen and w&er. The microwave power was maintrtined at 20 mw; modulation amplitude w&8 5 gauss. After recording each sample spectrum, a blank was run with the sample holder containing the solvent (buffer salts, glycine, ligands) and the net spectrum wss obtained by graphical subtraction. The stable free radical, l,l- diphenyl-2-picrylhydrazyl was always used ae a marker. The resonating field corresponding to the same radical was determined independently by use of a proton resonance fluxmeter.
3. Results (a) Optical ldworptim
spectra
The optical absorption spectra of the aqua and hydroxy derivatives of a+ and fi+ chains of human hemoglobin have been described earlier by Bmerjee & Casaoly
102
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BANERJEE
(1969) and by Banerjee et at. (1969). The main features of the aquo spectra may be summarized by saying that those of a+an and afpMB resemble closely that of Hb + under the same conditions. On the other hand, the p+sH spectrum shows substantial contribution (at 0°C) of two low-spin bands (maximum at about 535 rnp and 570 mp) with simultaneous decrease in the intensity of the charge-transfer (high-spin) bands at 500 rnp and 630 m,u. It was also shown that the effect of reacting the free
OlI
(J ! ’ I I I I 450 480 510 540 570 600 630 6iO I
I
I
I
1
I]
‘-J 480 510 540 570 600 630 Wavelength (mp)
FIQ. 1. (a) and (b) Visible absorption spectra of the ferri chains y&, and y.$.. at 20°C, pH 6.0 (a) and pH 10.0 (b). Concentrations were normalized for the chains on the cyano-met derivatives ( CrnM at 540 rnp taken as 11.5). yp+aa8spectra are represented by the solid curves and coincide with the spectra of methemoglobin. ye+Hspectra are represented by the dotted curves.
5.8 2.56 2.17 1.83
(weak) (90) (160) (150)
no resonance
gl= 2.81 (136) g2= 2.19 (135) g3= 1.68 (176)
g = 5-8 (55) = 1.99 (46) g g = 2.01 (46)
g = g,= ga= g,=
g = 6.8 (75f5)
5.8 2.56 2.17 1.83
(weak) (100) (120) (120)
no resonance
gl= 2.80 (110) ga= 2.20 (110) g,= 1.68 (176)
g = 5.8 (55) = 1.99 (50) g g = 2.01 (50)
g = gl= ga= gas
g = 5.8 (75f5)
Hb+A (4 (1)
2.80 2.25 1.70 -
-
-
2.61 2.19 132
5.8
Mb+ (5) (3)
gx= 2.82 gI= 2.20 gn= 1.70
-
gx= 2.6 g,= 2.3 ge= 1.7
5.9
Hb+A (c) (2)
found for someferrihemoproteins
-
2.80 2.18 1.68
6 2
6 2
8 2.85 2.22 1.62
Hb+A (8) (4)
refers to sample solutions frozen at 77’K. refers to monocry&alline samples at 20’K. refers to this work. refers to work by Gibson, Ingram 8z Schonland (1958). refers to work by Ehrenberg (1962). refers to work by Rein & Ristau (1965). Signals corresponding to g-values g N 6 and g, have the shape of the derivative of a gaussian curve, although very asymmetrio; see Fig. 2; the g-values calculated correspond to the magnetic field at the half-point between maximum and minimum and the line-widths (given, in gauss, between brackets) have been estimated also between maximum and minimum. Signals corresponding to g-values g N 2, g, and g3 have roughly a “bell-shape”; the g-values calculated correspond to the magnetic field at the maximum and the line-widths (given, in gauss, between brackets) have been estimated at the half maximum amplitude. For the aquo derivatives the line-widths are given with an uncertainty; this has been estimated from more than three independent experiments and more than twelve spectra for each species.
(8) (c) (1) (2) (3) (4)
no resonance
CNno resonance
g1 = 2.81 (96) ga= 2.20 (90) g3= 1.69 (165)
g,= 2.81 (110) g*= 2.19 (110) g3= 1.68 (170)
F-
Ni-
(weak) (110) (120) (130)
g = 6-8 (50) = 1.99 (60) g g = 2.01 (50)
5.8 2.56 2.16 1.83
g = gl= ga= g3=
(weak) (100) (120) (130)
g = gl= ga= g3=
5.8 2.56 2.17 1.83
g =5.8 (100&5)
g = 6.8 (85f5)
ah (s) (1)
g = 5.8 (50) g = 1.99 (60) g = 2.01 (50)
HO-
Ha0
Derivatives
g-Values and e&mated line-width
TABLE I
104
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HENRY
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BANERJEE
-SH group in jIBa by PMR was to reduce the intensity of the low-spin bands and to restore partly that of the charge-transfer bands. The hydroxy spectrum of CC+~~is very similar to that of Hb+ : that of pSH is characterized by a slightly increased absorption at 640 rnp (j3 band) and a shift of the a band (578 rnp) to a shorter wavelength (by 40 A). Thia band haa a lower intensity for /I,‘, compared with that obtained with Hb + or /Ir&,. This particular property is clearly visible in the hydroxy spectrum of y&r. It may be noted that, aa in the cam of fisH chain, the reaction of the free thiol groups of ysa by PMR restores the high-spin oharaoter (Fig. I(a) and (b)). (b) Electron spin resonance spectra (i) The methemoglobins The E.S.R. spectra of liganded forms of both Hb +A and Hb +Ft were found to have the same shape aa those reported by Ehrenberg (1962) for the corresponding derivatives of Mb + . Table 1 gives the measured g-values and an estimation of the line-widths. From this Table, a comparison of the g-values measured in the course of the present work may be made with those previously reported. (ii) The a-chain The E.S.R. spectra (Fig. 2) of the various liganded derivatives of a.& and aGMMB are I
I
I
/ Gain x 1
FIG. B = The Gain
1
I
/
I
/
F-
2. Eleatron spin resonanoe epeotrs of various derivetivez of a&, in frozen solutions 2-0037 corresponda to reeonanoe of l,l-diphenyl-2-picrylhydrazyl. heme concentration wee 1.2 X 10ea III and the ligmd conaentration was 0.2 P. waz x 1 for F- derivative. x 4 for HzO, HO-, NC derivatives.
at 77°K.
aim&r to those of the corresponding derivatives of Hb + . However it is important to note some significant differences in line-widths of the high-spin signals of aqua derivatives. Those of a& and a&= are respectively 100 f 5 gauss and 85 f 6 gauss, oompared to 75 f 5 gauss line-widths for all other aqua ferrihemoproteina here studied. Also included in the Table 1 are the g-values of the j?& chain, which, a~ will be seen, are also very similar to those of Hb + .
ELECTRON
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RESONANCE
OF
HEME
PROTEINS
106
/ Gain x 4
Gain x4
k
in various ligended forms, in Fra. 3. Eleotron spin reaonanoe speotre for fl& (1-dodeoanthiol) frozen solutions at 77°K. Q = 2.0037 corresponds to IWOIUUO~ of l,l-diphenyl-2picrylhydrazyl. Fluoride derivative: heme conoentretion, 5 x lo-’ M; ligaud concentration, O-4 Y; reaeiver gain, x 2; the high field spectrum has been repeeted again with a x 20 gain. 1.3 x lo-” M; ligand concentration Other derivetives (HaO, HO-, N,): heme oonoentretion, (N;), 0.2 M; receiver gain x 4.
TABLET
g-Value8 and c8titnated line-widths found for @& chain8 derivatives
&(
1 -dodeoanthiol)
g = g;= g;= g;=
HO-
F-
N-
(7bf5) (60) (36) (46)
g = S-8 (76&S)
g = 6.8 g; = 2.40 (60) g;= 2.22 (overlap) g;= 1.92 (46)
(weak) g,= 2.64 (90) gl= 2.18 (overlap) g,= 1.84 (86)
g = 6-S (weak) $I= 2.66 (90) $a= 2.17 (120) g3= 1.84 (110)
g =5.8 g;= 2.40 (60) g; = 2.23 (36) g;= 1.92 (40)
(65) g = 2.01 (40) g = 1.99 (46)
g = 6.8 (66) g = 2.01 (40) g = 1.99 (45)
g;= 2.39 (SO) g;= 2.23 (overlap) g; = 1.92 (40)
CNSee comment on Table 1; bends referred 88 81. s; ad 8;.
6.8 2.39 2.23 1.92
/I&, obtained by other methods, see text
gl= 2.80 (110) gz = 2.20 (overlap) g3= 1+37(verybroad)
no r8sonanoe to in the text aa “atypical
gl= 2.80 (96) gg= 2-20 (110) g,= 1.68 (160) no reaonBnoe ” are designated
in this Table 2
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Y.
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(iii) The /Muin In view of the unusual features of the optical absorption spectrum of /I,‘,, its E.S.R. absorption was studied with great care, on material obtained from nine independent preparations. The conversion of &,, to j&u was attained by several different methods as described in the Experimental Procedure section. The E.S.R. spectra of /3& by all methods other than the one involving treatment with I-dodecanthiol were identical in profile with those of Hb+. On the other hand, /?.& (l-dodecanthiol) always presented one distinctive feature, namely the presence of three low-spin bands with g = l-92, 2.23, 2.39, irrespective of the particular liganded species. The above is illustrated in Figure 3, which shows the persistence of the above mentioned low-spin bands in addition to those (high-spin or low-spin) that would be expected for the particular liganded species. These “atypical” low-spin bands disappeared totally when &nOa was reacted again with PMB prior to oxidation; they also disappeared almost completely by combining the p&r chain with an equivalent amount of c&. The reaction with CN- or reduction with dithionite resulted in the complete disappearance of resonance, as normally expected. However the “atypical” bands could be observed, end were the only ones present when a frozen solution of oxygenated &n chain was examined ; the same was true when carbon monoxide was passed into the oxy- chain prior to freezing. Table 2 summarizes the g-values obtained with the /3.& chain. (iv) The y-chain The E.S.R. spectra of y&z and y& were identical in shape with each other and with those of Hb+. However analysis of bands by double integration revealed important qmmtitative differences which will be discussed further below. Table 3 records the g-values and an estimation of line-widths observed with y&z and y&r chains. TABLE
3
g- Values and estimated line-widths found for yp+MBannE“/s, chains Derivatives
J&to HO-
F-
&CN-
YPXB g = 5.8 g = gl= g,= g,=
(75 f
6.8 (weak) 2.66 2.17 l-83
rat 5)
g = 5.8 g = gl= ga= g3=
(75 f
5.8 (weak) 2.66 2.17 I.83
g = 1.99
g = 5.8 (60) g = 2.01 g = I.99
gl= 2.80 ga= 2.19 gs= 1.68
g1= 2.81 g2= 2-18 g3= 1.68
; 1 y1
(55)
no resonfbnce
no msonancs
See~comments on Table 1.
5)
ELECTRON
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RESONANCE
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HEME
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107
4. Discussion We shall attempt, in course of this discussion, to correlate the optical absorption and E.S.R. spectra of the hemoproteins examined. Before doing so, it is necessary to arrive at an unambiguous description of the E.S.R. spectrum of /3& since, as has been mentioned earlier, the E.S.R. spectra of this species prepared by the l-dodecanthiol procedure was different from that obtained for /3,& prepared by all other reagents. (a) Origin of the atypical low-spin electron spin resonance bands of,?& (I-dodecunthiol) The g-values of the atypical bands of /?& prepared by use of 1-dodecanthiol are identical with those obtained by Hollocher (1966) and Hollocher & Buckley (1966) by treating oxy- or methemoglobin with heat, urea or acid under defined conditions. These authors have attributed the same bands to the formation of a hemichrome, the nature of which was not investigated. Recently, Rachmilewitz, Peisach, Bradley t Blumberg (1969) have reported bands with similar g-values in the E.S.R. spectra of inclusion bodies adhering to ghost erythrocytes of subjects having hemoglobin H (/?*) disease. The similarity of the spectra in the three cases would indeed suggest that samples of p.& (1 -dodecanthiol) in the frozen state contain appreciable quantities of a hemichrome of some type. However, several independent and concordant experimental facts tend to show that the observed “hemichrome” is caused by freezing and does not exist prior to this step. Some of this evidence is summarized below. (i) Careful examination of the visible absorption spectrum of freshly prepared /3,,0, (1-dodecanthiol) failed to show the presence of appreciable amounts of a hemichrome. Slight discrepancies in the ratio of the intensities of the a and p bands (576 and 542 rnp) could be accounted for by a small (less than 5%) fraction of ferri-groups. The reduction of such a solution by dithionite showed the total absence of a hemochrome (shoulder at 528 mp). Yet, the same solution, when frozen, showed the atypical low-spin E.S.R. bands of about the same intensity as that found with /3& (I-dodecanthiol) at equivalent concentration. (ii) The combination of /I,+, (1-dodecanthiol) with an equivalent amount of a& results in the disappearance of the low-spin bands in the visible as well as in the E.S.R. spectra. It has beenshown earlier (Banerjee & Cassoly, 1969) that the product is native methemoglobin. (iii) Oxyhemoglobin reconstituted from asHOa and psuOa (I-dodecanthiol) was studied by De Renzo et al. (1967) who have reported oxygen-binding parameters very close to those of native hemoglobin. It would seem possible that the emulsification step involved in the l-dodecanthiol procedure renders the /3 chain somewhat fragile to freezing, without visibly affecting in other ways the properties in aqueous solution. So far as the E.S.R. spectra are concerned, we conclude that the atypical low-spin bands observed in p&r (1-dodecanthiol) are not a characteristic feature of the species, since they are absent in the spectra of samples obtained by other methods. An earlier suggestion (Banerjee et al., 1969) relating these bands with the low-spin bands of optical spectrum must now therefore be revised. (b) Correlation of optical absorption and electron spin resonance spectra An important fact to consider is that the visible absorption spectrum of aquo- /?& is the same whatever be the method utilized for the regeneration of free -SH groups.
108
Y.
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As has been said before, they show, at 0°C substantial contribution of low-spm bands. The effect of lowering the temperature (observed between 15°C and 0%) is to increase the intensity of the low-spin both in && and in ys+II.These facts auggest a thermal equilibrium of spin states. The proportion of low-spin may therefore be expected to increase much further at 77”K, at which temperature the E.S.R. spectra were obtained. The absence of low-spin bands in the E.S.R. spectra of the aquo derivatives of /3&r and y.&.r suggest that the low-spin component may be of a type non-observable at 95 GHz band and at 77°K. In fact, Ehrenberg (1962) has noted some low-spin compounds such as Mb +CN and Hb +CN belonging to this category. The principal g-values of these compounds are believed to be around 1*0,2.2 and 3.0 but the absorption lines are very broad; actually we did not find any absorption band at 9.15 GHz and 77°K for any of the cyanide derivatives studied. We have therefore undertaken a quantitative analysis of the E.S.R. spectra with a view to correlating the chemically estimated concentration of the absorbing species with the integrated area covered by the absorption bands. The concentrations were normalized for all the hemoproteins (with or without PMB) as the met-cyanide derivative (z,~ = 115 at 540 mp) , a procedure for which some experimental justifioation has been advanced elsewhere (Banerjee et al., 1969). Double integration of the corrected spectra was found on trial to involve significant (more than 10%) uncertainty. We have therefore preferred to compare the heights of the g = 5.8 signal (between maximum and minimum) after graphical substraction of a blank at these points. In fact, the height of the g = 59 signal ean be reasonably assumed as proportional to the concentration of a given hemoprotein multiplied by the fraction of high-spin to total under given conditions. We are unaware of any theoretical objections to extending this comparison between different hemoproteins provided the fundamental condition, i.e. equality of band line-widths, is fulfilled. It will be seen from Tables 1, 2, and 3 that the line-widths of the aquo derivatives of a+A, m+Ft, B&p t%.m Y& and y&s are the same (75 & 5 gauss). On the other hand, a& and a& in the aquo form, having much larger line-widths, cannot be compared to any of the other ferrihemoproteins here studied. Is the fact that the hemoproteins of the first group (75 gauss line-widths) are all tetramers, but that a& and a&B are both monomers, of any significance ? Experimental results (Fig. 4(a) and (b)) show that for Hb +A and Hb +Ft at pH 6, the values of the g = 5.8 signal height per unit concentration are close at various protein concentrations; and that the values for Hb +Ft and Hb +A are the same within 10%. The value of the quantity signal height per unit concentration of y& at g = 5.8 is slightly higher (117% of that of Hb +Ft) ; this may be due to this particular compound having nearly lOOo/o iron in a high-spin state compared to about 90% of the same for Hb+Ft. The optical absorption spectra of yp+MB,Hb+A and Hb+Ft are very similar at 20°C and were not analyzed for very fine differences. On the other hand, the absorption spectra of /3i&, and y&, at 0°C were decidedly partly low-spin, and that of t$& much more so. Despite the fact that the signal heights per unit concentration compared in Figure 4(a) and (b) were obtained at 77°K it appears in a general way that the order of high-spin contribution y&e II Hb+A N Hb+Ft > ,6P+MB N ysf;I > && is maintained. The ratio of the values of g = 5.8 signal height per unit concentration for /?& to that for pgfhiBwas found to be 52%) the ratio for p& to Hb +A being 27% ;
ELECTRON
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HEME
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109
(b) Fxa. 4. (a) and (b) Amplitude of the derivative of g = 5-8 absorption of aqua derivative of verious ferrihemoproteins plotted against heme conoentretion. The amplitude was taken between maximum end minimum of the signal, the amplitude of blank at these points being subtraoted. The heme concentration wes normalized on the cyano-met derivatives ( smmM= 11-5 at 640 mr). The receiver gain was always x 4. The semple holder in the cavity was always the same, mlthough the quartz tubes of 3 mm diameter were not normalized. The experimental points for each curve were obtained in two or more independent experiments. (a) n , Hb+A; A, /I&,; 0, g,‘, (/I-mercaptoethanol), see text. (b) n , Hb+Ft; A, rp+aw; 0, y,&s; the dotted line corresponds to experiments on Hb+A in (a).
the ratio for y&/y&e was similarly found to be 60%. These results bring strong evidence in support of the view that at 77X, the aqua derivatives of /3& and y.& are 73% and 40% low-spin. The result of a+++ and a+-y+ chain interaction is thus to swing the spin equilibrium of fi + and y + chain iron from partial low-spin state to high-spin state. Isolated chains have until now been prepared only for the two systems HbA and HbFt; atudy of the constituent chains of other hemoglobins, when available, will show whether the above phenomenon is general. Nevertheless, it is already relevent to point out that the partial low-spin character is observed, in both of the systems examined, in the “non-a” chains. A common structural feature of all such chains in mammalian hemoglobins so far studied is the presence of a cysteine residue next to the heme-binding hi&idine. The role of this apparently non-replaceable residue in the functional properties of hemoglabin is not clear. Further studies along these lines may, it is hoped, help to define their role in the structural context of the chain-mediated heme-heme interaction.
110
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This work was supported by grants from the Centre National de la Recherche Scientifique (ERA no. 37) and from the Delegation G&&ale de la Recherche Scientifique et Technique. We thank Miss F. Stetzkowski for useful technical help. We are indebted to Prof. Douzou for kindly giving access to his E.S.R. spectrometer, and to Dr R. J. P. Williams for helpful discussions. We also thank Professor Varangot and Miss Garcia of the Mater&e PortRoyal, Paris, for facilities for the collection of cord blood. REFERENCES Allen, D. W., Schroeder, W. A. & Balog, J. (1958). J. Amer. Chem. Sot. 80, 1628. Banerjee, R., Alpert, Y., Leterrier, F. & Williams, R. J. P. (1969). Biochemistry, 8, 2862. Banerjee, R. & Cassoly, R. (1969). J. Mol. Biol. 42, 337. Bennett, J. E., Ingram, D. J. E., George, P. & Griffith, J. S. (1955). Nature, 176, 394. Boyer, P. D. (1954). J. Amer. Ghem. Sot. 76, 4331. Brill, A. S. & Williams, R. J. P. (1961). Biochem. J. 78, 246. Bucci, E. & Fronticelli, C. (1965). J. Biol. Chem. 240, PC 551. De Renzo, E. C., Ioppolo, C., Amiconi, G., Antonini E. & Wyman, J. (1967). J. BioZ. Chem. 242, 4850.
Drabkin, D. (1946). J. Biol. Chem. 164, 703. Ehrenberg, A. (1962). Arkivftir Kemi, 19, 119. George, P., Beetle&one, 5. & Griffith, J. S. (1961). In Haematin Enzymes, ed. by J. E. Falk, R. Lemberg & R. H. Morton, p.105. Oxford: Pergamon Press. Gibson, J. F., Ingram, D. 5. E. & Schonland, D. (1958). Disc. Fu~aday. Sot. 26, 72. Hollocher, T. C. (1966). J. BioZ. Chem. 241, 1958. Hollocher, T. C. & Buckley, L. M. (1966). J. Biol. Chem. 241, 2976. Kajita, A., Taniguchi, K. & Shukuya, R. (1969). Biochim. biophy8. Acta, 175, 41. Poulik, M. D. (1957). Nature, 180, 1477. Rachmilewitz, E. A., Peieach, J., Bradley, T. B. 8z Blumberg, W. E. (1969). Nature, 222, 248. Reiohlin, M., Bucci, E., Fronticelli, C.,-Wyman, J., Antonini, E. & Rossi-Fanelli, A. (1965). J. Mol.
BioZ.
12, 774.
Rein, H. & R&au, 0. (1965). Rumen, N. M. & Chance, B. Smith, D. W. & Williams, R. Tyuma, J., Benesch, R. E. & Williams, R. J. P. (1955). &SC.
Biochim. biophye. Acta, 94, 516. (1969). Biochim. biophys. Acta, 175, 242. J. P. (1968). Biochem. J. 110, 297. Benesch, R. (1966). Biochemistry, 5, 295. Faraday Sot. 20, 291.
Electron Spin Resonance Spectra of Isolated Ferrihemoglobin (a, j? and y) Chains-An Attempted Correlation with Optical Absorption Spectra Y. HENRY
AND
R. BANERJEE
Innstitut de Biobgie Physico-Chirnique 13, rue Pierre et Marie Curie, Paris 5, France (Received 30 October 1969) The electron spin resonance spectra of the isolated ferri OL,j and y chains of human adult and foetal hemoglobins have been obtained (at 77°K) and compared with those of the products of their recombination, namely the respective methemoglobins. All the hemoproteins examined gave essentially similar spectra and g-values for a given liganded form (aquo, hydroxy, fluoride, azide); however, important differences between the aquo forms appeared on comparing the linewidths and signal heights per unit heme concentration. The aqua derivatives of a + sH and ~~+r~st gave high-spin signals (g = 56) with line widths of 100 + 5 and 85 f 5 gauss, respectively ; that of all other compounds examined (the intact methemoglobins, the 8’ and y+ chains with free or mercury-bound sulfhydryls) was 75 + 5 gauss. On comparing the spectra of the second group of hemoproteins (76 gauss line-width) it appears that the high spin contribution of y +rMB, Hb +A and Hb +Ft are nearly equal ; that of ,¶+rMs and y + sz is about 60% and that of /?+sx is only 27% of the high-spin contribution of Hb+A or Hb+Ft for equal heme concentration. It is inferred that the isolated y+sz and /!+sx chains are partly low-spin, the low-spin form being of a type non-observable under the experimental conditions (9.5 GHz md 77OK). This view gains support from the visible absorption spectra of the ferri chains. At O”C, unlike that of Hb +A, Hb +Ft and a + sz, the spectrum of y + sH was partly low-spin and that of j3+ sH much more so. The decreased high-spin content of these species seen from electron spin resonance at 77’K may be explained as due to the effect of low temperature on a thermal equilibrium mixture of high and low-spin types. The results are discussed in the context of chain interactions in the hemoglobins.
1. Introduction Measurement of electron spin resonance absorption haa recently found wide applications in the study of heme proteins. E.S.R. absorption of single crystals of some derivatives of two hemaproteins, namely metmyoglobin and methemoglobin, were made by Bennett, Ingram, George & Griffith (1955) and by Gibson, Ingram & Schonland (1958). These measurements, carried out at 20”K, led to the experimental determination of g-values with respect to the porphyrin plane of aquo, hydroxyde t Abbreviations used : zPMB,pPe,,,and yPMB,chains with thiol groupe transformed into paramercuribenzoate derivatives by reacting with p-hydroxy mercuribenzoate; asa, flsH and ask, chains with free thiol groups. ma=,wpMB,etc. : ferro chains. 01+sa, a +PMB,etc. : ferri ohains. Mb, myoglobin; Mb+, met- or ferrimyoglobin; HbA, edult human hemoglobin; HbFt, foetal human hemoglobin; Hb+A and Hb+Ft, ferrihemoglobins; E.S.R., electron spin resonance. 99
100
Y. HENRY
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and azide derivatives. It was shown that the order of energy-level splittings is the same as that predicted on theoretical grounds for an octahedral complex. Ehrenberg (1962) has made measurements on frozen solutions of metmyoglobin derivatives at 77°K and upwards. For the “ionic” or “high-spin” aquo derivative Mb + -H,Ot, he observed an intense asymmetrio absorption with g = 6.8, and none in the g = 2 region, the latter can be observed only on single crystals. For “covalent” or “low-spin” hydroxide and azide complexes, three bands were resolved at g-values around 2, which wrrespond closely to the principal g-values determined by Gibson et al. (1958) on single crystals. The satisfactory accord of results in frozen solutions with those on single crystals have since favored the study of various hemoproteins, particularly with a view to correlating the spin-state with light absorption spectra in aqueous solutions. Williams (1966) and Brill & Williams (1961) by comparing the light absorption and magnetic moments of various ferric compounds, have suggested that the 480 to 506 rnp and 600 to 650 rnp absorption bands were of a high-spin type; these bands are believed to be charge-transfer bands. Refined methods are now available (Smith & Williams, 1968), which permit estimation of the percentage of Fe111 in different spin states (high or low) when they exist in equilibrium. The existence of such thermal equilibrium mixtures of high and low-spin forms has been demonstrated for alkaline horse methemoglobin by George, Beetlestone BEGriffith (1961) by a combination of different techniques. More recently, Rumen 6 Chance (1969), by a study of the optical absorption of lamprey hemoglobin at different temperatures have observed the appearance of low-spin bands as the temperature is decreased. As is well known, human hemoglobin (HbA) is composed of two pairs of unlike chains (u and fi). Very specific interactions between the chains, and particularly the unlike ones, are believed to govern the generation of heme-heme interaction as well as the lowering of over-all oxygen afllnity compared to that of the subunits. A convenient method is available since 1965 (Bucci & Fronticelli) for the preparation of the isolated chains of human hemoglobin in the reduced form. The ferri chains, very unstable in aqueous solution, have recently been prepared in a relatively stable form (Banerjee & Cassoly, 1969). It is thus possible to study the optical absorption and E.S.R. spectra of the isolated chains with a view to comparing the spin states of Fe111 of the chains with that obtained in the product of their association, Hb+. It was shown recently (Banerjee, Alpert, Leterrier & Williams, 1969) that the visible absorption spectrum of /l+ chains contains a substantial contribution of low-spin bands, even at 0°C. We describe in this paper the electron spin resonance spectra of the u, 6 and y ohains of human hemoglobins (HbA and HbFt); a correlation between the light absorption and E.S.R. spectra will also be attempted.
2. Experimental Procedure (a) Preparations Normal human hemoglobin (HbA) wss prepared from fresh hemolysates and crystallized once from 28 M-phosphate following Drabkin (1946). Human foetal hemoglobin (HbFt) wss isolated from fresh cord blood following the procedure of Allen, Schroeder & Balog (1968). The work reported in this paper was done with fraction Fu of Allen et al. (1968). M&hemoglobins (Hb+A, Hb +Ft) were prepared by adding potassium ferricyanide (5% in excess) to the respective deoxy derivative in Thunberg tubes under vacuum. The resulting solutions were dialysed against 2 x lo-’ M-NaCl and cleared by centrifugation.
ELECTRON
SPIN
RESONANCE
OF HEME
PROTEINS
101
state from fresh normal human aPMB ad PPMB chains were prepared in the oxygenated hemolysates by the method of Bucci & Fronticelli (1965). YpMs chains were prepared from pure HbFt by the action of PMB at pH 4.7 as described by Kajita, Tsniguchi BE Shukuya (1969). The removal of PMB from apMB was always performed by dialysis against 0.06% thioglycollate at pH 7.5 as suggested by Bucci & Fronticelli (1966), and resulted in the regeneration of 0.95 to 1 free -SH group per heme. The same method applied to ypMB also gave good regeneration (0.95 to 1 free -SH per heme, ss against the expected value of I per heme). As would appear from the results below, some of the properties of the 5sn chain were found to depend on the method utilized for its preparation from /lPM,,. In order to have a comprehensive idea as to the c&use of these variations, several methods were used in parallel. The number of free -SH groups per heme as recovered by these methods involving the use of different reactants were as follows: 1 - dodecanthiol (De Renzo, Ioppolo, Amiconi, Antonini & Wyman, 1967) 1.7 to 1.9 -SH/heme; /Imercaptoethanol (Tyume, Benesoh & Benesch, 1966) 1.9 -SH/heme; thioglycollate (Bucci & Fronticelli, 1966) 1.35 to 1.55 -SH/heme; cyst&e (Reichlin et al., 1965) 1.22 -SH/heme. The free -SH groups in the different hemoproteins were estimated by the method of Bayer (1954). Analytical starch gel electrophoresis was performed by the method of Poulik (1957). The concentrations of the different hemoproteins were determined by spectrophotometry on the cyanomet derivatives (tmM = 11.5 at 540 mp). The oxidized a and /3chains were prepared in I.6 M-glycine as described earlier (Banerjee & Csssoly, 1969). The solutions were filtered immediately after oxidation, at 4°C; portions of the same solution were often used simultaneously for visible spectrophotometry as well as for E.S.R. work. For the latter, the required amounts of the stock solution were added to tubes containing the appropriate ligands and buffers, the liganded derivatives being immediately afterwards transferred to E.S.R. sample holders and frozen in liquid nitrogen. The y chains, both PMB bound and free, when oxidized in aqueous solution, were found to be stable. The ferri y chains and their derivatives were therefore prepared and studied in aqueous medium. The various liganded forms of the different hemoproteins were obtained under the following conditions: equo, pH 6.0; hydroxide, pH 9.5; ezide, 0.2 M-N~.N,, pH 6.5; fluoride, 0.2 to 0.4 M-KF, pH 6.5; cyanide, 0.2 M-KCN, pH 9. Independent spectrophotometric measurements showed that the ligand concentrations were saturating under the given conditions. The a and /3 chains contained, in addition, 1.5 M-glycine adjusted to the required pH. Hemoprotein concentrations used for E.S.R. spectra were between 6 x lo-’ M and 1-8x 10m3 M (heme). All speotrophotometric measurements were performed with a Gary 14 recording spectrophotometer equipped with a special thermostatic attachment for work near 0°C. (b) Electron spin resonunce spectrorrcopy The electron spin resonance spectra, were recorded with a Varian E3 spectrometer. The sample, contained in a quartz tube of 3 mm diameter, was maintained at 77°K in 8 V&an V 4546 dew&r adapted to the spectrometer cavity. The volume of the solution within the cavity was approximately 0.13 ml. A current of dry nitrogen was continuously passed into the sample holder and the wave guide in order to prevent the condensation of atmospheric oxygen and w&er. The microwave power was maintrtined at 20 mw; modulation amplitude w&8 5 gauss. After recording each sample spectrum, a blank was run with the sample holder containing the solvent (buffer salts, glycine, ligands) and the net spectrum wss obtained by graphical subtraction. The stable free radical, l,l- diphenyl-2-picrylhydrazyl was always used ae a marker. The resonating field corresponding to the same radical was determined independently by use of a proton resonance fluxmeter.
3. Results (a) Optical ldworptim
spectra
The optical absorption spectra of the aqua and hydroxy derivatives of a+ and fi+ chains of human hemoglobin have been described earlier by Bmerjee & Casaoly
102
Y.
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BANERJEE
(1969) and by Banerjee et at. (1969). The main features of the aquo spectra may be summarized by saying that those of a+an and afpMB resemble closely that of Hb + under the same conditions. On the other hand, the p+sH spectrum shows substantial contribution (at 0°C) of two low-spin bands (maximum at about 535 rnp and 570 mp) with simultaneous decrease in the intensity of the charge-transfer (high-spin) bands at 500 rnp and 630 m,u. It was also shown that the effect of reacting the free
OlI
(J ! ’ I I I I 450 480 510 540 570 600 630 6iO I
I
I
I
1
I]
‘-J 480 510 540 570 600 630 Wavelength (mp)
FIQ. 1. (a) and (b) Visible absorption spectra of the ferri chains y&, and y.$.. at 20°C, pH 6.0 (a) and pH 10.0 (b). Concentrations were normalized for the chains on the cyano-met derivatives ( CrnM at 540 rnp taken as 11.5). yp+aa8spectra are represented by the solid curves and coincide with the spectra of methemoglobin. ye+Hspectra are represented by the dotted curves.
5.8 2.56 2.17 1.83
(weak) (90) (160) (150)
no resonance
gl= 2.81 (136) g2= 2.19 (135) g3= 1.68 (176)
g = 5-8 (55) = 1.99 (46) g g = 2.01 (46)
g = g,= ga= g,=
g = 6.8 (75f5)
5.8 2.56 2.17 1.83
(weak) (100) (120) (120)
no resonance
gl= 2.80 (110) ga= 2.20 (110) g,= 1.68 (176)
g = 5.8 (55) = 1.99 (50) g g = 2.01 (50)
g = gl= ga= gas
g = 5.8 (75f5)
Hb+A (4 (1)
2.80 2.25 1.70 -
-
-
2.61 2.19 132
5.8
Mb+ (5) (3)
gx= 2.82 gI= 2.20 gn= 1.70
-
gx= 2.6 g,= 2.3 ge= 1.7
5.9
Hb+A (c) (2)
found for someferrihemoproteins
-
2.80 2.18 1.68
6 2
6 2
8 2.85 2.22 1.62
Hb+A (8) (4)
refers to sample solutions frozen at 77’K. refers to monocry&alline samples at 20’K. refers to this work. refers to work by Gibson, Ingram 8z Schonland (1958). refers to work by Ehrenberg (1962). refers to work by Rein & Ristau (1965). Signals corresponding to g-values g N 6 and g, have the shape of the derivative of a gaussian curve, although very asymmetrio; see Fig. 2; the g-values calculated correspond to the magnetic field at the half-point between maximum and minimum and the line-widths (given, in gauss, between brackets) have been estimated also between maximum and minimum. Signals corresponding to g-values g N 2, g, and g3 have roughly a “bell-shape”; the g-values calculated correspond to the magnetic field at the maximum and the line-widths (given, in gauss, between brackets) have been estimated at the half maximum amplitude. For the aquo derivatives the line-widths are given with an uncertainty; this has been estimated from more than three independent experiments and more than twelve spectra for each species.
(8) (c) (1) (2) (3) (4)
no resonance
CNno resonance
g1 = 2.81 (96) ga= 2.20 (90) g3= 1.69 (165)
g,= 2.81 (110) g*= 2.19 (110) g3= 1.68 (170)
F-
Ni-
(weak) (110) (120) (130)
g = 6-8 (50) = 1.99 (60) g g = 2.01 (50)
5.8 2.56 2.16 1.83
g = gl= ga= g3=
(weak) (100) (120) (130)
g = gl= ga= g3=
5.8 2.56 2.17 1.83
g =5.8 (100&5)
g = 6.8 (85f5)
ah (s) (1)
g = 5.8 (50) g = 1.99 (60) g = 2.01 (50)
HO-
Ha0
Derivatives
g-Values and e&mated line-width
TABLE I
104
Y.
HENRY
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BANERJEE
-SH group in jIBa by PMR was to reduce the intensity of the low-spin bands and to restore partly that of the charge-transfer bands. The hydroxy spectrum of CC+~~is very similar to that of Hb+ : that of pSH is characterized by a slightly increased absorption at 640 rnp (j3 band) and a shift of the a band (578 rnp) to a shorter wavelength (by 40 A). Thia band haa a lower intensity for /I,‘, compared with that obtained with Hb + or /Ir&,. This particular property is clearly visible in the hydroxy spectrum of y&r. It may be noted that, aa in the cam of fisH chain, the reaction of the free thiol groups of ysa by PMR restores the high-spin oharaoter (Fig. I(a) and (b)). (b) Electron spin resonance spectra (i) The methemoglobins The E.S.R. spectra of liganded forms of both Hb +A and Hb +Ft were found to have the same shape aa those reported by Ehrenberg (1962) for the corresponding derivatives of Mb + . Table 1 gives the measured g-values and an estimation of the line-widths. From this Table, a comparison of the g-values measured in the course of the present work may be made with those previously reported. (ii) The a-chain The E.S.R. spectra (Fig. 2) of the various liganded derivatives of a.& and aGMMB are I
I
I
/ Gain x 1
FIG. B = The Gain
1
I
/
I
/
F-
2. Eleatron spin resonanoe epeotrs of various derivetivez of a&, in frozen solutions 2-0037 corresponda to reeonanoe of l,l-diphenyl-2-picrylhydrazyl. heme concentration wee 1.2 X 10ea III and the ligmd conaentration was 0.2 P. waz x 1 for F- derivative. x 4 for HzO, HO-, NC derivatives.
at 77°K.
aim&r to those of the corresponding derivatives of Hb + . However it is important to note some significant differences in line-widths of the high-spin signals of aqua derivatives. Those of a& and a&= are respectively 100 f 5 gauss and 85 f 6 gauss, oompared to 75 f 5 gauss line-widths for all other aqua ferrihemoproteina here studied. Also included in the Table 1 are the g-values of the j?& chain, which, a~ will be seen, are also very similar to those of Hb + .
ELECTRON
SPIN
RESONANCE
OF
HEME
PROTEINS
106
/ Gain x 4
Gain x4
k
in various ligended forms, in Fra. 3. Eleotron spin reaonanoe speotre for fl& (1-dodeoanthiol) frozen solutions at 77°K. Q = 2.0037 corresponds to IWOIUUO~ of l,l-diphenyl-2picrylhydrazyl. Fluoride derivative: heme conoentretion, 5 x lo-’ M; ligaud concentration, O-4 Y; reaeiver gain, x 2; the high field spectrum has been repeeted again with a x 20 gain. 1.3 x lo-” M; ligand concentration Other derivetives (HaO, HO-, N,): heme oonoentretion, (N;), 0.2 M; receiver gain x 4.
TABLET
g-Value8 and c8titnated line-widths found for @& chain8 derivatives
&(
1 -dodeoanthiol)
g = g;= g;= g;=
HO-
F-
N-
(7bf5) (60) (36) (46)
g = S-8 (76&S)
g = 6.8 g; = 2.40 (60) g;= 2.22 (overlap) g;= 1.92 (46)
(weak) g,= 2.64 (90) gl= 2.18 (overlap) g,= 1.84 (86)
g = 6-S (weak) $I= 2.66 (90) $a= 2.17 (120) g3= 1.84 (110)
g =5.8 g;= 2.40 (60) g; = 2.23 (36) g;= 1.92 (40)
(65) g = 2.01 (40) g = 1.99 (46)
g = 6.8 (66) g = 2.01 (40) g = 1.99 (45)
g;= 2.39 (SO) g;= 2.23 (overlap) g; = 1.92 (40)
CNSee comment on Table 1; bends referred 88 81. s; ad 8;.
6.8 2.39 2.23 1.92
/I&, obtained by other methods, see text
gl= 2.80 (110) gz = 2.20 (overlap) g3= 1+37(verybroad)
no r8sonanoe to in the text aa “atypical
gl= 2.80 (96) gg= 2-20 (110) g,= 1.68 (160) no reaonBnoe ” are designated
in this Table 2
106
Y.
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(iii) The /Muin In view of the unusual features of the optical absorption spectrum of /I,‘,, its E.S.R. absorption was studied with great care, on material obtained from nine independent preparations. The conversion of &,, to j&u was attained by several different methods as described in the Experimental Procedure section. The E.S.R. spectra of /3& by all methods other than the one involving treatment with I-dodecanthiol were identical in profile with those of Hb+. On the other hand, /?.& (l-dodecanthiol) always presented one distinctive feature, namely the presence of three low-spin bands with g = l-92, 2.23, 2.39, irrespective of the particular liganded species. The above is illustrated in Figure 3, which shows the persistence of the above mentioned low-spin bands in addition to those (high-spin or low-spin) that would be expected for the particular liganded species. These “atypical” low-spin bands disappeared totally when &nOa was reacted again with PMB prior to oxidation; they also disappeared almost completely by combining the p&r chain with an equivalent amount of c&. The reaction with CN- or reduction with dithionite resulted in the complete disappearance of resonance, as normally expected. However the “atypical” bands could be observed, end were the only ones present when a frozen solution of oxygenated &n chain was examined ; the same was true when carbon monoxide was passed into the oxy- chain prior to freezing. Table 2 summarizes the g-values obtained with the /3.& chain. (iv) The y-chain The E.S.R. spectra of y&z and y& were identical in shape with each other and with those of Hb+. However analysis of bands by double integration revealed important qmmtitative differences which will be discussed further below. Table 3 records the g-values and an estimation of line-widths observed with y&z and y&r chains. TABLE
3
g- Values and estimated line-widths found for yp+MBannE“/s, chains Derivatives
J&to HO-
F-
&CN-
YPXB g = 5.8 g = gl= g,= g,=
(75 f
6.8 (weak) 2.66 2.17 l-83
rat 5)
g = 5.8 g = gl= ga= g3=
(75 f
5.8 (weak) 2.66 2.17 I.83
g = 1.99
g = 5.8 (60) g = 2.01 g = I.99
gl= 2.80 ga= 2.19 gs= 1.68
g1= 2.81 g2= 2-18 g3= 1.68
; 1 y1
(55)
no resonfbnce
no msonancs
See~comments on Table 1.
5)
ELECTRON
SPIN
RESONANCE
OF
HEME
PROTEINS
107
4. Discussion We shall attempt, in course of this discussion, to correlate the optical absorption and E.S.R. spectra of the hemoproteins examined. Before doing so, it is necessary to arrive at an unambiguous description of the E.S.R. spectrum of /3& since, as has been mentioned earlier, the E.S.R. spectra of this species prepared by the l-dodecanthiol procedure was different from that obtained for /3,& prepared by all other reagents. (a) Origin of the atypical low-spin electron spin resonance bands of,?& (I-dodecunthiol) The g-values of the atypical bands of /?& prepared by use of 1-dodecanthiol are identical with those obtained by Hollocher (1966) and Hollocher & Buckley (1966) by treating oxy- or methemoglobin with heat, urea or acid under defined conditions. These authors have attributed the same bands to the formation of a hemichrome, the nature of which was not investigated. Recently, Rachmilewitz, Peisach, Bradley t Blumberg (1969) have reported bands with similar g-values in the E.S.R. spectra of inclusion bodies adhering to ghost erythrocytes of subjects having hemoglobin H (/?*) disease. The similarity of the spectra in the three cases would indeed suggest that samples of p.& (1 -dodecanthiol) in the frozen state contain appreciable quantities of a hemichrome of some type. However, several independent and concordant experimental facts tend to show that the observed “hemichrome” is caused by freezing and does not exist prior to this step. Some of this evidence is summarized below. (i) Careful examination of the visible absorption spectrum of freshly prepared /3,,0, (1-dodecanthiol) failed to show the presence of appreciable amounts of a hemichrome. Slight discrepancies in the ratio of the intensities of the a and p bands (576 and 542 rnp) could be accounted for by a small (less than 5%) fraction of ferri-groups. The reduction of such a solution by dithionite showed the total absence of a hemochrome (shoulder at 528 mp). Yet, the same solution, when frozen, showed the atypical low-spin E.S.R. bands of about the same intensity as that found with /3& (I-dodecanthiol) at equivalent concentration. (ii) The combination of /I,+, (1-dodecanthiol) with an equivalent amount of a& results in the disappearance of the low-spin bands in the visible as well as in the E.S.R. spectra. It has beenshown earlier (Banerjee & Cassoly, 1969) that the product is native methemoglobin. (iii) Oxyhemoglobin reconstituted from asHOa and psuOa (I-dodecanthiol) was studied by De Renzo et al. (1967) who have reported oxygen-binding parameters very close to those of native hemoglobin. It would seem possible that the emulsification step involved in the l-dodecanthiol procedure renders the /3 chain somewhat fragile to freezing, without visibly affecting in other ways the properties in aqueous solution. So far as the E.S.R. spectra are concerned, we conclude that the atypical low-spin bands observed in p&r (1-dodecanthiol) are not a characteristic feature of the species, since they are absent in the spectra of samples obtained by other methods. An earlier suggestion (Banerjee et al., 1969) relating these bands with the low-spin bands of optical spectrum must now therefore be revised. (b) Correlation of optical absorption and electron spin resonance spectra An important fact to consider is that the visible absorption spectrum of aquo- /?& is the same whatever be the method utilized for the regeneration of free -SH groups.
108
Y.
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R.
BANERJEE
As has been said before, they show, at 0°C substantial contribution of low-spm bands. The effect of lowering the temperature (observed between 15°C and 0%) is to increase the intensity of the low-spin both in && and in ys+II.These facts auggest a thermal equilibrium of spin states. The proportion of low-spin may therefore be expected to increase much further at 77”K, at which temperature the E.S.R. spectra were obtained. The absence of low-spin bands in the E.S.R. spectra of the aquo derivatives of /3&r and y.&.r suggest that the low-spin component may be of a type non-observable at 95 GHz band and at 77°K. In fact, Ehrenberg (1962) has noted some low-spin compounds such as Mb +CN and Hb +CN belonging to this category. The principal g-values of these compounds are believed to be around 1*0,2.2 and 3.0 but the absorption lines are very broad; actually we did not find any absorption band at 9.15 GHz and 77°K for any of the cyanide derivatives studied. We have therefore undertaken a quantitative analysis of the E.S.R. spectra with a view to correlating the chemically estimated concentration of the absorbing species with the integrated area covered by the absorption bands. The concentrations were normalized for all the hemoproteins (with or without PMB) as the met-cyanide derivative (z,~ = 115 at 540 mp) , a procedure for which some experimental justifioation has been advanced elsewhere (Banerjee et al., 1969). Double integration of the corrected spectra was found on trial to involve significant (more than 10%) uncertainty. We have therefore preferred to compare the heights of the g = 5.8 signal (between maximum and minimum) after graphical substraction of a blank at these points. In fact, the height of the g = 59 signal ean be reasonably assumed as proportional to the concentration of a given hemoprotein multiplied by the fraction of high-spin to total under given conditions. We are unaware of any theoretical objections to extending this comparison between different hemoproteins provided the fundamental condition, i.e. equality of band line-widths, is fulfilled. It will be seen from Tables 1, 2, and 3 that the line-widths of the aquo derivatives of a+A, m+Ft, B&p t%.m Y& and y&s are the same (75 & 5 gauss). On the other hand, a& and a& in the aquo form, having much larger line-widths, cannot be compared to any of the other ferrihemoproteins here studied. Is the fact that the hemoproteins of the first group (75 gauss line-widths) are all tetramers, but that a& and a&B are both monomers, of any significance ? Experimental results (Fig. 4(a) and (b)) show that for Hb +A and Hb +Ft at pH 6, the values of the g = 5.8 signal height per unit concentration are close at various protein concentrations; and that the values for Hb +Ft and Hb +A are the same within 10%. The value of the quantity signal height per unit concentration of y& at g = 5.8 is slightly higher (117% of that of Hb +Ft) ; this may be due to this particular compound having nearly lOOo/o iron in a high-spin state compared to about 90% of the same for Hb+Ft. The optical absorption spectra of yp+MB,Hb+A and Hb+Ft are very similar at 20°C and were not analyzed for very fine differences. On the other hand, the absorption spectra of /3i&, and y&, at 0°C were decidedly partly low-spin, and that of t$& much more so. Despite the fact that the signal heights per unit concentration compared in Figure 4(a) and (b) were obtained at 77°K it appears in a general way that the order of high-spin contribution y&e II Hb+A N Hb+Ft > ,6P+MB N ysf;I > && is maintained. The ratio of the values of g = 5.8 signal height per unit concentration for /?& to that for pgfhiBwas found to be 52%) the ratio for p& to Hb +A being 27% ;
ELECTRON
SPIN
RESONANCE
OF
HEME
PROTEINS
109
(b) Fxa. 4. (a) and (b) Amplitude of the derivative of g = 5-8 absorption of aqua derivative of verious ferrihemoproteins plotted against heme conoentretion. The amplitude was taken between maximum end minimum of the signal, the amplitude of blank at these points being subtraoted. The heme concentration wes normalized on the cyano-met derivatives ( smmM= 11-5 at 640 mr). The receiver gain was always x 4. The semple holder in the cavity was always the same, mlthough the quartz tubes of 3 mm diameter were not normalized. The experimental points for each curve were obtained in two or more independent experiments. (a) n , Hb+A; A, /I&,; 0, g,‘, (/I-mercaptoethanol), see text. (b) n , Hb+Ft; A, rp+aw; 0, y,&s; the dotted line corresponds to experiments on Hb+A in (a).
the ratio for y&/y&e was similarly found to be 60%. These results bring strong evidence in support of the view that at 77X, the aqua derivatives of /3& and y.& are 73% and 40% low-spin. The result of a+++ and a+-y+ chain interaction is thus to swing the spin equilibrium of fi + and y + chain iron from partial low-spin state to high-spin state. Isolated chains have until now been prepared only for the two systems HbA and HbFt; atudy of the constituent chains of other hemoglobins, when available, will show whether the above phenomenon is general. Nevertheless, it is already relevent to point out that the partial low-spin character is observed, in both of the systems examined, in the “non-a” chains. A common structural feature of all such chains in mammalian hemoglobins so far studied is the presence of a cysteine residue next to the heme-binding hi&idine. The role of this apparently non-replaceable residue in the functional properties of hemoglabin is not clear. Further studies along these lines may, it is hoped, help to define their role in the structural context of the chain-mediated heme-heme interaction.
110
Y. HENRY
AND
R. BANERJEE
This work was supported by grants from the Centre National de la Recherche Scientifique (ERA no. 37) and from the Delegation G&&ale de la Recherche Scientifique et Technique. We thank Miss F. Stetzkowski for useful technical help. We are indebted to Prof. Douzou for kindly giving access to his E.S.R. spectrometer, and to Dr R. J. P. Williams for helpful discussions. We also thank Professor Varangot and Miss Garcia of the Mater&e PortRoyal, Paris, for facilities for the collection of cord blood. REFERENCES Allen, D. W., Schroeder, W. A. & Balog, J. (1958). J. Amer. Chem. Sot. 80, 1628. Banerjee, R., Alpert, Y., Leterrier, F. & Williams, R. J. P. (1969). Biochemistry, 8, 2862. Banerjee, R. & Cassoly, R. (1969). J. Mol. Biol. 42, 337. Bennett, J. E., Ingram, D. J. E., George, P. & Griffith, J. S. (1955). Nature, 176, 394. Boyer, P. D. (1954). J. Amer. Ghem. Sot. 76, 4331. Brill, A. S. & Williams, R. J. P. (1961). Biochem. J. 78, 246. Bucci, E. & Fronticelli, C. (1965). J. Biol. Chem. 240, PC 551. De Renzo, E. C., Ioppolo, C., Amiconi, G., Antonini E. & Wyman, J. (1967). J. BioZ. Chem. 242, 4850.
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