ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 137, 51-58 (197b, The Magnetic Susceptibility of Hemin 303-4.5”K SAMUEL SULLIVAN, PETER HAMBRIGHT,’ BILLY JOE EVANS, ARTHUR THORPE, AND JOHN A. WEAVER2 The Departments of Chemistry and Physics, Howard University, Washington, D.C. %OOOl Received August 25, 1969; accepted December 13, 1969 The magnetic susceptibility of hemin has been measured from 303-4.5”K. The magnetic moments are in agreement with the predictions of Harris at 273 and 4.5”K, whereas the value at 4°K is roughly 9y0 below the t,heoretical estimate. The hemin data do not fit the Griffith-Kot,ani equation, and t,he interpretation of susceptibility measurements using this equation has not been successful in determining the zero- field splitting parameter, D, for hemins. Magnetic susceptibility measurements have assumed an increasingly important role in elucidating the behavior of biological systems and molecules (1,Z). Susceptibility work has been done on transplantable hepa- toma and normal liver tissues (3), on cells (4, 5), eggs (6), and in vitro studies with iron loaded rats (7). The majority of work has involved isolated biological compounds, such as copper proteins (8, 9), non-heme iron pro- teins [ferredoxins (lo), ferroverdin (11)) ferritin (12)) conalbumen (13)) transferrin (14)], and especially heme proteins, such as hemoglobin (15)) myoglobin (16)) cyto- chromes (17), catalase (18), and peroxi- dases (19). A step lower in molecular complexity are the magnetic studies on metalloporphyrins (20), particularly the ferric porphyrins. These compounds exhibit magnetic proper- ties characteristic of three kinds of spin states which depend upon the coordination about the ferric ion. In pseudooctahedral environments with two high field nitrog- enous bases, the compounds show low spin rlj behavior (21) ; in five coordinate pseudo- square-pyramidal fields with various anions, the complexes approximate high spin cl5 moments (22, 23); whereas in the recently studied oxybridged dimers, room tempera- 1 For further communications. 2 AC%PWF Graduate Fellow, 1969-1970. ture moments of 1.7 BM which decrease wiylth temperature have been found (24). Accurate susceptibility measurements are useful in determining the effects of the ligands on the electronic state of the ferric ion in porphyrins. These data will, in turn, be useful in attempts to understand some of the differences in the biological activities of heme-type molecules having different axial ligands. Because of its biological activity, relatively simple structure, and easy labora- tory synthesis of a variety of derivatives, we have been investigating the properties of the rather thoroughly characterized chloro de- rivative of Fe(II1) protoporphyrin(IX), hemin. The detailed X-ray structure (25), along with the far-infrared (26), Mossbauer (27, 28, 29), and electron spin resonance parameters (30), have been obtained. In particular, the isothermal magnetic sus- ceptibility of hemin has been measured by many workers, giving moments of 6.1-6.3 (31), 5.83 (32), 5.79 (22), 5.94 (23), and 5.93 BM (33). The usefulnessof isothermal susceptibility measurements is limited since the relative importance of the intra- and intermolecular interactions, along with an imprecise knowledge of the diamagnetic corrections, can often lead to magnetic moments that result in incorrect interpreta- tions as to the electronic state of the metal ion. For example, the apparently high mo- 51
Transcript
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 137, 51-58 (197b,
The Magnetic Susceptibility of Hemin 303-4.5”K
SAMUEL SULLIVAN, PETER HAMBRIGHT,’ BILLY JOE EVANS, ARTHUR THORPE, AND JOHN A. WEAVER2
The Departments of Chemistry and Physics, Howard University, Washington, D.C. %OOOl
Received August 25, 1969; accepted December 13, 1969
The magnetic susceptibility of hemin has been measured from 303-4.5”K. The magnetic moments are in agreement with the predictions of Harris at 273 and 4.5”K, whereas the value at 4°K is roughly 9y0 below the t,heoretical estimate. The hemin data do not fit the Griffith-Kot,ani equation, and t,he interpretation of susceptibility measurements using this equation has not been successful in determining the zero- field splitting parameter, D, for hemins.
Magnetic susceptibility measurements have assumed an increasingly important role in elucidating the behavior of biological systems and molecules (1,Z). Susceptibility work has been done on transplantable hepa- toma and normal liver tissues (3), on cells (4, 5), eggs (6), and in vitro studies with iron loaded rats (7). The majority of work has involved isolated biological compounds, such as copper proteins (8, 9), non-heme iron pro- teins [ferredoxins (lo), ferroverdin (11)) ferritin (12)) conalbumen (13)) transferrin (14)], and especially heme proteins, such as hemoglobin (15)) myoglobin (16)) cyto- chromes (17), catalase (18), and peroxi- dases (19).
A step lower in molecular complexity are the magnetic studies on metalloporphyrins (20), particularly the ferric porphyrins. These compounds exhibit magnetic proper- ties characteristic of three kinds of spin states which depend upon the coordination about the ferric ion. In pseudooctahedral environments with two high field nitrog- enous bases, the compounds show low spin rlj behavior (21) ; in five coordinate pseudo- square-pyramidal fields with various anions, the complexes approximate high spin cl5 moments (22, 23); whereas in the recently studied oxybridged dimers, room tempera-
1 For further communications. 2 AC%PWF Graduate Fellow, 1969-1970.
ture moments of 1.7 BM which decrease wiylth temperature have been found (24).
Accurate susceptibility measurements are useful in determining the effects of the ligands on the electronic state of the ferric ion in porphyrins. These data will, in turn, be useful in attempts to understand some of the differences in the biological activities of heme-type molecules having different axial ligands. Because of its biological activity, relatively simple structure, and easy labora- tory synthesis of a variety of derivatives, we have been investigating the properties of the rather thoroughly characterized chloro de- rivative of Fe(II1) protoporphyrin(IX), hemin. The detailed X-ray structure (25), along with the far-infrared (26), Mossbauer (27, 28, 29), and electron spin resonance parameters (30), have been obtained. In particular, the isothermal magnetic sus- ceptibility of hemin has been measured by many workers, giving moments of 6.1-6.3 (31), 5.83 (32), 5.79 (22), 5.94 (23), and 5.93 BM (33). The usefulness of isothermal susceptibility measurements is limited since the relative importance of the intra- and intermolecular interactions, along with an imprecise knowledge of the diamagnetic corrections, can often lead to magnetic moments that result in incorrect interpreta- tions as to the electronic state of the metal ion. For example, the apparently high mo-
51
52 SULLIVAN ET AL.
ment of ferrohemoglobin, which was initially interpreted as indicating heme-heme inter- actions (15), was found to be due simply to the use of an improper diamagnetic correc- tion for globin (34).
Several groups (20, 35, 36, 37) have demonstrated that hemin follows a Curie law from 300-77”K, and the most recent measurements are in good agreement (5.93, 5.97, and 5.92 BM). Schoffa and Scheler (35) found that hemin deviated markedly from Curie law behavior above 300°K. We will demonstrate that this deviation could arise from the decomposition of hemin at these temperatures.
The considerable interest in the magnetic susceptibility of hemin arises primarily out of the suggestion by Kotani (38), Griffith (39), and Weissbluth (40) that the zero- field splitting parameter, D, can be obtained from low temperature magnetic suscepti- bility measurements. There have been many experiments conducted in recent years in attempts to understand the systematics of the spin Hamiltonian parameter D for S- state ions, for example, Watanabe (41) and Germanier et al. (42). These studies, plus those of Nicholson and Burns (43), have cast serious doubt on present ideas regarding the nature and origin of the zero-field splitting for S-state ions. Recently, however, Harris (44) has used a strong crystal-field model, with spin-orbit coupling, to successfully account for the observed D values in a number of iron porphyrins. In addition, the temperature dependence of the magnetic moments of hemin and possible mixing of the six CA states by an external magnetic field were also calculated. The agreement of the above calculations with the existing experi- mental data stimulated us to make a detailed determination of the magnetic moment of hemin from 303 to 4.5”K in order to test, first of all, the Kotani relation for deducing D from susceptibility measurements and, secondly, to test the predictions of Harris’s calculations. If the Kotani-Griffith relation can be shown to reproduce the susceptibility data, with values of D in agreement with the directly determined far-infrared values, then the more difficult and expensive far-infrared measurements can be supplanted by the
more easily performed and relatively inex- pensive susceptibility techniques. Further- more, by comparing the temperature de- pendence of the magnetic moments with the calculations of Harris, especially in the low temperature region, there may result some new contributions to our understanding the systematics of the zero-field splitting param- eter.
EXPERlMENTAL PROCEDURE
Recrystallized hemin was obtained from Gen- eral Biochemicals and recrystallized twice again by the method of Fischer (45), with the omission of the ethanol wash. The microanalyses were done by Aldridge Associates and Co., Washington, D. C. (theoretical: C = 62.61’%, H = 4.96%, N = 8.60%; observed: C = 62.44%, H = 5.11%, N = 8.61%).
The susceptibility apparatus and method used in the present investigation have been described elsewhere (20, 46). The sample was checked for ferromagnetic contamination by making suscepti- bility measurements in magnetic fields between l,OOC-5,500 Oe. An approximately constant value of 2.06 X 10m5 erg/Oe’ was measured at room tem- perature for each field, while at the liquid nitrogen temperature a value of 8.56 X 10-S erg/Oe” was obtained. This implies the absence of any ferro- magnetic impurities in sufficient amounts to affect the results of the experiment.
Measurements of susceptibility as a function of temperature were made using 0.814 f 0.002 mg of the bulk sample in a I-kOe field. An Ag-Au thermocouple in a helium gas atmosphere was em- ployed to measure the temperature between 16 and 300°K. A carbon resistor was used for measure- ments below 16°K. Two independent sets bf data were collected for each of the two temperature regions, 30s77°K and 774.5”K. Consistent result,s were obtained in all cases. The field strength of 1 kOe is much lower than those used by previous workers (37). This has important consequences regarding some suggestions that have been ad- vanced to explain unexpected behavior in the tem- perature dependence of the susceptibility as determined by others and will be discussed below.
RESULTS
The gram susceptibilities and effective observed moments of hemin are given in Table I. Figure 1 shows the results of the susceptibility measurements from 77 to 300°K. In this temperature interval, the magnetic moment is expected to be tempera- ture independent, and the data are plotted
MAGXETIC SUSCXPTIBLLITY OF HEMIN 53
as xp vs. l/T in accordance with the Curie equation
netic moment, k is Boltzman’s constant, T the absolute temperature, B the Bohr magneton, and JI the molecular weight of hemin. xs and XDiam are the total and the diamagnetic susceptibilities, respectively. The points fall on a straight line, confirming the magnetic moment of hemin to be tem- perature independent in this range. The ob- served moment found by least-square analysis \vas 5% f 0.03 B11, which is in the usual range for (15 high spin Pe(II1) com- plexes, with a BS5,2 ground state. The mo- ment agrees within the experimental uncer- tainty with the previous measurements of 5.93 BI\I (20), 5.97 Bhl (35), and 5.92 B:\I (37). XDiarll xvas equal to -1.2 X 1OP cgs units.
If D is of the order of IO%, the suscepti- bility is expected to deviate from a Curie law behavior at temperatures significantly lower than 77°K. Figure 2 shows the devia- tions from the Curie Law below 30°K. The moments at lower temperatures are listed in Table I and wcrc reduced using Equation 1, where
/.L = 2.84 (X&T)? (2)
The diamagnetic susceptibility of hemin is negligible in comparison to the paramag- netic term and was neglected.
Griffith (39) and Kotani (38) have shown
FIG. 1. Gram susceptibility of hemin as a function of l/l’. 303-77°K.
54 SULLIVAN ET AL.
5.6 -
5.4 -
4
5.2 -
434 -
4.2 1 I I I
0 0.05 0.I YT “K-’
0.2
FIG. 2. Y versus l/Z for the zero-field sDlitting parameters D = 6, 10, 14, and 22 cm-l, I_
from Equation 3. The shaded triangles are the observed data.
that the temperature dependence of the effective magnetic moment of a high spin ferrihemoprotein can be expressed as
19 + 16/s + exp (-2X)(9 - 11/z)
+ exp (-66x)(25 - 5/2) (3)
“= l+exp(--2x)+exp(-62) ’
where P is given by Equation 2, and x = D/kT, with D the zero-field splitting param- eter. George and Beetlestone (47) have used Equation 3 over a small temperature range with ferrimyoglobin, and Kotani and co- workers (48) have applied this equation to several high spin hemoproteins.
mental data are represented fairly well above 7°K by D = 10 cm-‘, serious devia- tions occur below this temperature. The ob- served moments fall off faster with l/T be- low 7°K than Equation 3 predicts for values of D, from 6 to 26 cm-l. The same behavior is apparent in the data presented on frozen solutions of ferric myoglobin and its fluoride derivative (48) and in the recent results of Maricondi and co-workers (37) on hemin. The latter authors found what they believed to be an approximate fit of Equation 3 with a D of 11.8 cm-l.
DISCUSSION
Figure 2 shows the comparison of our x versus l/T data on hemin with the corre- sponding theoretical curves calculated from Equation 3 using various values of D. AD of 6.8 cm-l (9.8”K) for hemin was determined by Richards and co-workers (26) from far infrared measurements. While our experi-
Harris (44) has calculated the magnetic moments of hemin to be 5.89 BM at 293”K, 5.88 BM at 77”K, and 5.05 BM at 4°K. We observed moments of 5.86 f 0.05 at 293 and 77°K and 4.58 f 0.05 at 4°K. The high temperature data are in excellent agreement with the theoretically calculated moments.
MAGNETIC SUSCEPTIBILITY OF HEMIN 55
It is worthy of note that both the experi- mental and theoretical values for the mag- netic moment at 293 and 4°K are lower than the spin-only value of 5.92 BM. There is serious disagreement, however, at 4°K. The experimental moment is about 9% lower than the theoretical value of Harris. In view of the close agreement between Harris’s calculations and other observed properties of the porphyrins, it is difficult to explain the divergence between theory and experiment at low temperatures.
Of greater importance, though, is our in- ability to fit the Kotani-Griffith equation to the low temperature data for any value of D in the temperature range studied. The func- tional dependence of the observed suscepti- bility on temperature is significantly differ- ent from that predicted by the Kotani- Griffith equation. This same lack of agree- ment between the experimental data and the Kotani-Griffith relation has been observed for numerous other porphyrins and hemo- proteins (37, 4S). Even if we accept the possibility of reasonable fit of the Kotani- Griffith equation to our data above 7”K, the resulting D value of 10 cm-’ is about 50% larger than the more reliable infrared deter- mined (26) value of 6.8 cm-‘. Therefore, in addition to the conclusions drawn by Mari- condi et al. (37) that the zero-field splitting parameter for hemin, derived from sus- ceptibility data, is apparently incorrect, we are forced to conclude that the Kotani- Griffith equation does not reproduce the susceptibility data over the most pertinent temperature region.
There are several nonmutually exclusive ways of explaining the deviation of the low temperature susceptibility from a Curie law. One of these is, of course, the changes in the population of the different states in the 6A manifold. This is accounted for by the Kotani-Griffith relation. There are still some unresolved questions regarding the validity of this equation under conditions of nonaxial symmetry, but it is felt that this effect alone cannot produce the large deviations that we observe. Another is the possibility of inter- molecular spin coupling. In the case of some approximate antiparallel arrangement of adjacent spins, we would expect the mag-
netic moment to decrease faster than pre- dicted by the Kotani-Grifhth equation. In fact Fig. 3 shows a fit to our data of the purely empirical relation
This equation gives a satisfactory represen- tation of our data throughout the entire temperature interval from 304 to 4.4’K, with C = 7.09 X 10da and c = 3.1. Gen- erally, such a Curie-Weiss law implies the presence of intermolecular spin-spin inter- actions and eventual magnetic ordering at some nonzero temperature; nonetheless, we do not mean to say that there is any long- range magnetic order in hemin at these tem- peratures. A third possibility for explaining the deviations from a Curie law, as well as deviations from the Kotani-Griffith equa- tion, would be the simultaneous occurrence of changes in the population of the states in the ‘jA manifold and local (or long-range) antiferromagnetic spin coupling.
JJaricondi et al. (37) have suggested, on the basis of the Harris calculations (44), that the difference between their susceptibility determined zero-field splittings and the infrared determinations is due to magnetic field mixing of the six states in the 6A mani- fold. A magnetic field strength of IS kOe was used in their studies; we have used a field strength of only 1 kOe and observe the same low temperature moments and deviations of the data from the Kotani-Griflith equation as they noted. Thus, magnetic field mixing cannot be the primary source of the lack of agreement between theory and experiment. Therefore, either the Griffith-Kotani equa- tion is inherently incapable of reproducing the susceptibility data (even when the re- sulting D is incorrect) due to the neglect of certain low-order crystal field components, or there must be some kind of intermolecular spin interactions present in hemin.
Of the two final possibilities suggested in the last paragraph, the former will have to await further theoretical investigations be- fore any conclusive statements can be made. There is evidence both in favor of and against the latter suggestion. Electron spin resonance data on hemin have indicated the
56 SULLII’AN ET AL.
FIG. 3. Gram susceptibility of hemin versus l/T from 303-4.4”K. E = 0 are the observed data. The E = 3.1 data are from Equation 4.
possibility of magnetic interactions at low temperatures (30). The asymmetric quadru- pole doublets in the Mossbauer spectra of hemin have also been attributed to the pres- ence of magnetic ordering (27). The tem- perature dependence of the line shape could be explained quite satisfactorily on the assumption of changes in the population of the states in 6A, accompanied by simul- taneous magnetic ordering (as suggested above). A recent interpretation of the Moss- bauer data (29) is that the asymmetric line shapes and their temperature dependence are due to slow spin-spin relaxation. This possi- bility has not been fully explored.
One source of difficulty is the fact that the asymmetry is present in dry, powdered hemin as well as in frozen solutions. This result makes spin-lattice relaxation unlikely. Even in the case of spin-spin relaxation, one would expect to see a difference in the spec- trum on going from powders to frozen solu- tions. This was not observed. As the concen- tration of hemin in solution is progressively decreased, it is expected that the spin-lattice relaxation time will increase, and the Moss- bauer quadrupole doublet will broaden and
eventually produce well resolved magnetic hyperfine structure. This experiment has not been done. The insensitivity of the Moss- bauer spectra to the state of aggregation of hemin seems to suggest that the origin of the asymmetric line shapes and their tempera- ture dependence lay in intramolecular phenomena.
Of some significance, however, is the fact that certain unexplained breaks in the sus- ceptibility curves of the ferric porphyrins occur in precisely the same temperature interval as that in which the line shapes of the Mossbauer spectra change from the high temperature asymmetric doublets to the low temperature symmetric doublets (29). These changes in the Mossbauer spectra are inter- preted (29) as being consistent with the assumptions involved in the derivation of the Kotani-Griffith equation, and zero-field splitting parameters are deduced from the data that are in good agreement with the infrared data. These breaks in the suscepti- bility represent serious deviations from the explicit equation, the Griffith-Kotani equa- tion, for the temperature dependence of the susceptibility. We are presently measuring
?VIAGNETIC SUSCEPTIBILITY OF HEMIN 57
the susceptibility of those prophyrins studied by Moss et al. (29) to determine if the breaks in the x vs T curve do indeed occur for all these samples in the same temperature interval as the changes in the shape of Mossbauer spectra occur.
Schoffa and Scheler (35) observed that the susceptibility of hemin from 300-500°K did not obey the Curie law, which was followed between 77 and 3OO”Ii. The susceptibilities in the high temperature range were much higher than the Curie values. They at- tributed these deviations to populations of higher multiplet states with increasing tem- perature, a phenomenon shown by several lanthanide ions. Using a differential thermal analysis apparatus, hemin in air shows a single endotherm at 650°K. This peak is not due to melting or a phase change, but pre- sumably to decarboxylations and/or de- composition of the phorphyrin. The exo- therm temperature is more characteristic of the apparatus than the porphyrin, and other porphyrins can be decarboxylated at lower temperatures. Equation 1 shows that the ob- served susceptibility is inversely propor- tional to the molecular weight, and we feel that t#he high temperature deviations in susceptibility might have arisen from the physical decomposition of hemin under the measurement conditions, and not from higher state population.
SUM&‘lARY
It has been shown that the Griffith- Kotani equation does not account satisfac- t#orily for the low temperature susceptibility of hemin. Similar deviations at low tempera- tures of the experimental data from the theoretical curves are present in the data of several other workers (37, 48), though they failed to attach any significance to them. We believe these deviations to be real and to have important consequences regarding the validity of the Kotani-Griffith equation as applied to hemin and other related com- pounds. The agreement between theory and experiment for the bromo derivative of hemin (37) can be regarded as fortuitous in view of the general lack of agreement for most other hemins. The theoretical predic- tions of Harris (44), insofar as the magnetic
moments are concerned, are in good agree- ment with our data except at the lowest temperatures, at which divergences occur.
We have, however, been able to fit the susceptibility data to a Curie-Weiss law rather well over the entire temperature range of 303 to 4.5%. This result raised the question again of some kind of magnetic ordering in hemin. There is presently a great deal of controversy about this possibility, and its final resolution will have to a&& further susceptibility and RIossbauer meas- urements strongly directed toward this particular problem.
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