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Mössbauer spectroscopy of iron chelated by deferoxamine
BIOCHIMICA ET BIOPHYSICA ACTA
245
BBA 26829
M(3SSBAUER SPECTROSCOPY OF I R O N C H E L A T E D BY D E F E R O X A M I N E
JAY. L. BOCK* AND GEORGE LANG
Nuclear Physics Division, United Kingdom Atomic Energy Research Establishment, Harwell, Didcot, Berhs (Great Britain) (Received November 5th, 1971)
SUMMARY
M6ssbauer spectra of Fe ~+ and Fe 3+ in the presence of deferoxamine, an ironchelating drug, were measured under several conditions of temperature and applied magnetic field. Well-resolved spectra of magnetically dilute Fe3+-deferoxamine samples were obtained at 4.2 °K in a weak or zero field, which indicated a single, highspin species with rhombic distortion. Spectra at slightly higher temperatures, or in stronger fields, were helpful in estimating the crystal-field parameters. Resolution was poor, however, at temperatures above 20 °K, or in samples with Fe 3+ concentration much above 5 raM. A computer program for simulating spectra in applied fields gave good fits using the following values: quadrupole interaction Q Vzz/4 = - 0 . 3 o m m . s -1, ~1/3 = o.46, crystal-field splitting D = o.5 cm -~, a s y m m e t r y parameter 2 = o.46. Spectra of a sample containing ferrous sulfate, deferoxamine, and sodium dithionite to prevent oxidation of the iron, were compared with a control containing no deferoxamine. No evidence was found for the specific binding of Fe 2+ to deferoxamine.
INTRODUCTION
A number of small molecules with extraordinary affinity for iron have been isolated from microorganisms. One of these, deferoxamine (originally called desferrioxamine B when derived from Streptomyces pilosus), is marketed in the form of its methanesulfonate ("Desferal") for use in the clinical treatment of iron poisoning. A number of its physical and chemical properties have been reported 1-4, including its Fe3+-affinity constant of IO ~1 and its atomic structure (Fig. I). I t remains, however, to obtain detailed information on the electronic structure of the binding site, which would be of intrinsic pharmacological relevance and also would serve as a model for other investigations of Fe 3+ in fields of low symmetry. Fortunately, deferoxamine is a very convenient subject for M6ssbauer spectroscopy, which has been found fruitful in studying similar molecules (ferrichrome A b y Wickman et al. 5, E D T A b y Lang et al."). The work presented here includes spectra of Fe3+-deferoxamine, taken at several con* Present address: Department of Biophysics, Albert Einstein College of Medicine, 13oo Morris Park Avenue, Bronx, New York 1o461, U.S.A.
Biochim. Biophys. Acta, 264 (1972) 245-251
246
J . L . BOCK, G. LANG
CO--NH
(iH2)5 /\
(CH2). 0 C c~ -...]e//O NH2--(CH2)5--N~o~"" "~O %../N
CO ~HI
(CH2)5
I
CH3 Fig. I. Atomic s t r u c t u r e of Fe3+-deferoxamine (ferrioxamine ]3). (Not i n t e n d e d to depict exact knowledge of t h e iron a t o m ' s stereochemistry.)
ditions of temperature and applied magnetic field, which are interpreted in terms of a simple spin-Hamiltonian for rhombically distorted Fe 3+. Evidence has been indecisive on whether deferoxamine binds iron in the ferrous state as well as the ferric. Schwarzenbach and Schwarzenbach 4 reported little presence at best of a Fe ~+ complex. Goodwin and Whitten 7 later reopened this question, finding that if deferoxamine is added to a ferrous sulfate solution, Fe 3~ complex will form unless the solution is air-free, or a strong reducing agent such as sodium dithionite is present. Under such reducing conditions, no interaction between Fe 2÷ and deferoxamine could be detected by optical spectroscopy. Because of the higher sensitivity of M6ssbauer spectroscopy, we have repeated the experiment using this technique. MATERIALS AND METHODS
Deferoxamine was obtained in the form of its methanesulfonate ("Desferal") from Ciba Laboratories Ltd., Horsham, Sussex. Iron metal enriched with 8o%~7Fe was obtained from the Atomic Energy Research Establishment, Harwell, Berks.
Preparation of Fe 3+ samples Enriched iron metal was dissolved in dilute sulfuric acid and oxidized with ammonium persulfate to give a solution containing 2.57 m g ' m l - l F e , 97% as Fe 3÷. In a typical sample o.i ml of this solution was added to 0.7 ml of o.I M citrate buffer containing 15 mg Desferal. This gave a sample of pH 6 with a sixfold excess of deferoxamine over its theoretical binding capacity with Fe 3+. One sample was prepared with glycerol replacing half the buffer volume so that it could be frozen to a glass to avoid precipitation of solute. A control was prepared with no Desferal. The samples were immediately frozen in polyethylene cells having i/8-inch-thiek compartments and I/32-inch-thick walls.
Preparation of Fe 2+ samples o.I ml of the 57Fe-enriched solution used above was added to 1.6 ml water containing 200 mg sodium dithionite. 50 mg Desferal was added to half of the resulting solution, with the remaining half serving as control. Biochim. Biophys. Acta, 264 (1972) 245-251
MOSSBAUER SPECTROSCOPY OF DEFEROXAMINE
247
Measurements
Spectra were measured on a 256-channel, constant-acceleration machine *, calibrated regularly with metallic iron. Runs at 4.2 °K were made with the sample immersed in liquid He. Between 4.2 and 77 °K the samples were in vacuum, clamped to a temperature-controlled Cu tongue. At higher temperatures the samples were in a cryostat cooled with liquid N2(77 °K), dry ice (195 °K) or freon (233 °K). Transverse fields were obtained either with a permanent magnet (0.55 kG) or a superconducting solenoid (lO-6O kG). Fields of o.I kG parallel to the v-beam were obtained by inducing persistent currents in superconducting Pb rings placed in Helmholtz fashion next to the sample. EXPERIMENTAL RESULTS
It was found that, even at low temperatures, well resolved spectra of Fe 3÷deferoxamine were only obtained with magnetically dilute samples containing about 4 #moles Fe in a volume of o.8 ml. At this Fe content excellent statistical accuracy was achieved in 48 h running time. All of the spectra shown in the figures are from the glycerol sample, which showed slightly narrower lines than the ordinary samples. Fig. 2a shows the zero-field spectrum of Fe3÷-deferoxamine at 4.2 °K. A similar solution, containing Fe 3÷ in citrate buffer but without deferoxamine is shown in 2b. The large change in the spectrum indicates that when the deferoxamine is present the iron binds to it, changing considerably its local environment. Our present theory is not adequate for calculating the zero-field spectra, but the comparisons with theory in the case of applied field spectra indicate that in Fe3÷-deferoxamine all iron atoms are in identical environments.
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Fig. 2. (a) Zero-field s p e c t r u m of Fe3+-deferoxamine in g l y c e r o l - w a t e r at 4.2 °K. (b) S p e c t r u m of Fe 3+ in citrate buffer u n d e r similar conditions w i t h no deferoxamine present.
Fig. 3 shows spectra at low temperatures in small applied fields. They are typical of a high-spin ferric material with rhombic distortion. A control spectrum (not shown), taken under the same conditions as 3a, was again very different and more complicated, justifying our assumption that we were looking at asingle, deferoxamine-bound species. The superposed solid lines in Fig. 3 are calculated spectra, made b y solving a spin-5/2 Hamiltonian as discussed below. The spectra show notable line-broadening, especially at 19.6 °K, from spin relaxation. It was hoped that at some higher temperature spin-relaxation would completely wash out the magnetic hyperfine structure to yield a simple quadrupole doublet. Biochim.
Biophys.
Acta,
264 (1972) 245-25I
248
j . L . BOCK, G. LANG
. . . .-I0 . . . . . . . . . .-5 .......
0
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VELOCITY (rnm/see tel Fe) Fig. 3. Dotted lines are spectra of Fe3+-deferoxamine taken at (a) 4.2 °K, 550 G transverse to the 7-beam, (b) 4.2 °K, IOO G parallel to 7-beam, (c) io.o °K, 55° G transverse, (d) 19.6 °K, 55o G transverse. Superposed solid lines are calculated spectra, as explained in the text. Magnetic effects persisted, however, at 77 a n d 195 °K, a n d only a completely featureless s p e c t r u m was o b t a i n e d at 233 °K. Hence we could n o t o b t a i n a n i n d e p e n d e n t check on our estimate of the q u a d r u p o l e splitting. Spectra t a k e n in high magnetic fields are shown in Fig. 4. Their usefulness in checking the theoretical model is described below.
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Fig. 4- Dotted lines are spectra of Fea+-deferoxamine at 4.2 °K, (a) in a 60 kG transverse field, (b) in a 2o kG transverse field, (c) in a IO kG transverse field. Solid lines are calculated spectra. A few of the spectra o b s e r v e d i n the Fe2+-deferoxamine s t u d y are shown in Fig. 5. All the spectra were q u a d r u p o l e doublets with no magnetic features, typical of highspin Fe ~+, b u t the wide a n d a s y m m e t r i c lines suggest a v a r i e t y of iron e n v i r o n m e n t s in the ferrous solutions, both with deferoxamine a n d in its absence. I n the first few m e a s u r e m e n t s , the samples exhibited spectra which differed b y more t h a n the exp e r i m e n t a l u n c e r t a i n t y . However, a d d i t i o n a l m e a s u r e m e n t s i n d i c a t e d that, with v a r i a t i o n of freezing conditions, each sample has a range of spectra a n d these ranges Biochim. Biophys. Acta, 264 (1972) 245-251
MOSSBAUER SPECTROSCOPY OF DEFEROXAMINE
i t
I
"2
2
0
249
4
VELOCITY (rnrn/sec tel Fe) Fig. 5. F e r r o u s s u l p h a t e s o l u t i o n spectra, (a) a n d (b), a n d s p e c t r a of a similar solution in w h i c h d e f e r o x a m i n e is p r e s e n t as well, (c) a n d (d). All were m e a s u r e d in zero field a t 77 °K. Vertical lines are d r a w n a t + 3.0 m m / s a n d - - o . 3 m m / s to facilitate c o m p a r i s o n . S p e c t r a (a) a n d (c) c o r r e s p o n d to q u i c k - f r e e z i n g of t h e s a m p l e , while for (b) t h e s a m p l e w a s frozen slowly. (d) w a s frozen q u i c k l y b u t s t o r e d for s e v e r a l w e e k s a t 77 ° K before t h e m e a s u r e m e n t was m a d e .
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Fig. 6. I l l u s t r a t i o n s h o w i n g t h e c o n t r i b u t i o n to a M 6 s s b a u e r s p e c t r u m f r o m t h e t h r e e electronic K r a m e r s d o u b l e t s . (d) is a r e p r o d u c t i o n of 3a ( t r a n s v e r s e field) a n d (h) is a r e p r o d u c t i o n of 3b ( l o n g i t u d i n a l field). T h e c u r v e s in (a), (b), (c), a n d (e), (f), (g) are c a l c u l a t e d s p e c t r a for t h e i n d i v i d u a l d o u b l e t s , in d e s c e n d i n g order of e n e r g y , w h i c h a d d to give t h e t o t a l c a l c u l a t e d s p e c t r u m . N o t e t h a t t h e e s s e n t i a l f e a t u r e of t h e longitudinal-field s p e c t r u m is t h a t , d u e to selection rules, t h e s e c o n d a n d fifth lines f r o m t h e m i d d l e d o u b l e t are effectively e l i m i n a t e d . Also o b s e r v e t h a t cont r i b u t i o n s f r o m t h e t o p d o u b l e t in t h e a c t u a l s p e c t r a are a l m o s t w a s h e d o u t b y s p i n relaxatiola.
Biochim. Biophys. Mcta, :264 (1972) 245-251
250
J . L . BOCK, G. LANG
overlap. The spectra of Fig. 5, taken at 77 °K, illustrate this. It was found that the width of the spectrum at 4.2 °K correlated with the width at 77 °K- Both samples had similar spectra at I95 °K, with broader and more asymmetric lines and a slightly reduced quadrupole splitting. DISCUSSION
The theory of Fe 3+ in rhombic environments has been discussed extensively elsewhere~,%%l°; ref. 6 should be consulted for the spirit of approach in the present study, as well as some description of the calculating methods. Here we will merely comment on the theory in light of problems raised by our present investigation. The system of iron nucleus plus d electrons may be described in terms of the following S ~ 5/2 spin Hamiltonian.
= D[S~ 2 + ~ (S, ~ -- Sy2)] + 2 $ H . S + Ao I.S + Q?~[Zz~-5- + ~3 - ( Z J - I ' 2 ) ] Here the first term expresses the splitting of the electronic levels which occurs even in zero magnetic field as a result of high-order spin-orbit interactions. The second term is the electronic Zeeman interaction. The third term expresses the magnetic interaction between electron spin and the magnetic moment of the iron nucleus. It is taken as isotropic because in this near-spherical electron distribution only Fermi contact interaction should be important. The fourth term describes the interaction of the nuclear quadrupole moment with the local electric-field gradient. The last term is the nuclear Zeeman term. The Hamiltonian has been written in terms of the excited state, ~TmFe. For the ground nucleus the magnitudes of the third and last terms are altered by the change in nuclear magnetic moment, while the quadrupole interaction disappears. In calculating the spectra shown in the figures we have assumed values D =0.5 cm 1, 2 = ~/3 = 0.46- For the excited nucleus Ao : 1.474 m m . s -1 in M6ssbauer energy units. This corresponds to a S = 5/2 saturation field of 542 kG. The strength of the quadrupole interaction is Q Vz~/4 = - o . 3 o m m . s -1 which would correspond to quadrupole splitting o.77 m m . s -1 in the absence of magnetic interactions. Spectra in small fields represent superposed contributions from the three electronic Kramers doublets of high-spin Fe a+ (see Fig. 6). At ;t = o,46 one would expect the bottom and upper doublets to give sharp spectra, while the middle doublet would give broad lines due to its moderately anisotropic g-tensor. Unfortunately, in actual spectra narrow lines come only from the bottom doublet. As seems often to be the case, the top doublet is made almost completely invisible by spin relaxation, and the middle doublet is broadened. The result is that it is very difficult unambiguously to fit measured spectra with computer simulations, both because several line positions are not precisely defined, and because intensities of lines with different widths must be compared. The situation is considerably improved by making high-field as well as low-field measurements. At 6o kG the overall width of the spectrum is relatively insensitive to the zero field splittings, so that the value of Ao may be determined. The overall width of the very low-field spectra (Fig. 3) then may be varied by changing 2. Finally, at fields of order IO kG (Fig. 4b, 4c) significant magnetically induced mixing Biochim. Biophys. Acta, 264 (1972) 245-251
MOSSBAUER SPECTROSCOPY OF DEFEROXAMINE
251
of the electronic doublets occurs, and the spectra are sensitive to D. D also affects the thermal population of each doublet, and hence the strength of its contribution to the total spectrum in each case. Since our calculated spectra show good qualitative agreement under various conditions of measurement, though they m a y not be the best fits possible, the parameters used should be considered good estimates of Fe3+-deferoxamine's actual structure. Verification by E S R and susceptibility studies would of course be desirable. Determination of parameters for iron in low-symmetry environments is unfortunately laborious with any present technique. M6ssbauer spectroscopy m a y perhaps prove the most straightforward when computer programs are developed that will automatically search parameter space for the best least-squares fit to a measured spectrum. As for the Fe 2÷ binding question, it m a y well be that deferoxamine did affect the spectra of the ferrous samples. But this need not imply formation of a complex; the deferoxamine could modify the structure of the ice and the water environment of Fe 2+ without binding taking place. In fact, the chance that specific binding would have so little effect on the spectra is rather small, and the evidence against a Fe 2+deferoxamine is strong. In conclusion, this study has revealed two interesting properties of deferoxamine: that its Fe 3+ complex apparently has a value of ). fairly distinct from those reported for m a n y similar substances, and that it has no interaction with Fe 2+ detectable by M6ssbauer resonance. M6ssbauer and E S R experiments will undoubtedly be used in the future to gain more data on deferoxamine and related compounds, such as the mycobactins and ferrichromes. Hopefully this information will serve as a basis for developing molecular orbital theories, permitting us to interpret the numbers in terms of chemical bonds. These efforts m a y be directed to the exciting goal of understanding complex biological phenomena, such as transport of iron by the human protein transferrin. ACKNOWLEDGEMENT
This investigation was supported in part b y P.H.S. Training Grant No. 5T5 GM 1674 from the National Institute of General Medical Sciences. REFERENCES i 2 3 4 5 6 7 8 9 io
H. Bickel, H. K e b e r l e a n d E. Vischer, Helv. Chim. Acta, 46 (1963) 1385. G. A. Snow, Biochem. J., I15 (1969) 199. H. Keberle, Ann. N. Y. Acad. Sci., 119 (1964) 758. G. S c h w a r z e n b a c h a n d K. S c h w a r z e n b a c h , Helv. Chim. Acta, 46 (1963) 139o. H. H. W i c k m a n , M. P. Klein, a n d D. A. Shirley, Phys. Rev., 154 (1966) 346. G. L a n g , R. Aasa, K. G a r b e t t a n d R. J. P. W i l l i a m s , J . Chem. Phys., 55 (1971) 4539. J. F. G o o d w i n a n d C. F. W h i t t e n , Nature, 205 (1965) 281. T. E. C r a n s h a w , N u d . Instrum. Methods, 3 ° (1964) IOI. H. H. W i c k m a n , M. P. K l e i n a n d D. A. Shirley, J. Chem. Phys., 42 (1965) 2113. G. L a n g , Q. Rev. Biophys., 3 (197o) I.
Biochim. Biophys. Acta, 264 (1972) 245-251
245
BBA 26829
M(3SSBAUER SPECTROSCOPY OF I R O N C H E L A T E D BY D E F E R O X A M I N E
JAY. L. BOCK* AND GEORGE LANG
Nuclear Physics Division, United Kingdom Atomic Energy Research Establishment, Harwell, Didcot, Berhs (Great Britain) (Received November 5th, 1971)
SUMMARY
M6ssbauer spectra of Fe ~+ and Fe 3+ in the presence of deferoxamine, an ironchelating drug, were measured under several conditions of temperature and applied magnetic field. Well-resolved spectra of magnetically dilute Fe3+-deferoxamine samples were obtained at 4.2 °K in a weak or zero field, which indicated a single, highspin species with rhombic distortion. Spectra at slightly higher temperatures, or in stronger fields, were helpful in estimating the crystal-field parameters. Resolution was poor, however, at temperatures above 20 °K, or in samples with Fe 3+ concentration much above 5 raM. A computer program for simulating spectra in applied fields gave good fits using the following values: quadrupole interaction Q Vzz/4 = - 0 . 3 o m m . s -1, ~1/3 = o.46, crystal-field splitting D = o.5 cm -~, a s y m m e t r y parameter 2 = o.46. Spectra of a sample containing ferrous sulfate, deferoxamine, and sodium dithionite to prevent oxidation of the iron, were compared with a control containing no deferoxamine. No evidence was found for the specific binding of Fe 2+ to deferoxamine.
INTRODUCTION
A number of small molecules with extraordinary affinity for iron have been isolated from microorganisms. One of these, deferoxamine (originally called desferrioxamine B when derived from Streptomyces pilosus), is marketed in the form of its methanesulfonate ("Desferal") for use in the clinical treatment of iron poisoning. A number of its physical and chemical properties have been reported 1-4, including its Fe3+-affinity constant of IO ~1 and its atomic structure (Fig. I). I t remains, however, to obtain detailed information on the electronic structure of the binding site, which would be of intrinsic pharmacological relevance and also would serve as a model for other investigations of Fe 3+ in fields of low symmetry. Fortunately, deferoxamine is a very convenient subject for M6ssbauer spectroscopy, which has been found fruitful in studying similar molecules (ferrichrome A b y Wickman et al. 5, E D T A b y Lang et al."). The work presented here includes spectra of Fe3+-deferoxamine, taken at several con* Present address: Department of Biophysics, Albert Einstein College of Medicine, 13oo Morris Park Avenue, Bronx, New York 1o461, U.S.A.
Biochim. Biophys. Acta, 264 (1972) 245-251
246
J . L . BOCK, G. LANG
CO--NH
(iH2)5 /\
(CH2). 0 C c~ -...]e//O NH2--(CH2)5--N~o~"" "~O %../N
CO ~HI
(CH2)5
I
CH3 Fig. I. Atomic s t r u c t u r e of Fe3+-deferoxamine (ferrioxamine ]3). (Not i n t e n d e d to depict exact knowledge of t h e iron a t o m ' s stereochemistry.)
ditions of temperature and applied magnetic field, which are interpreted in terms of a simple spin-Hamiltonian for rhombically distorted Fe 3+. Evidence has been indecisive on whether deferoxamine binds iron in the ferrous state as well as the ferric. Schwarzenbach and Schwarzenbach 4 reported little presence at best of a Fe ~+ complex. Goodwin and Whitten 7 later reopened this question, finding that if deferoxamine is added to a ferrous sulfate solution, Fe 3~ complex will form unless the solution is air-free, or a strong reducing agent such as sodium dithionite is present. Under such reducing conditions, no interaction between Fe 2÷ and deferoxamine could be detected by optical spectroscopy. Because of the higher sensitivity of M6ssbauer spectroscopy, we have repeated the experiment using this technique. MATERIALS AND METHODS
Deferoxamine was obtained in the form of its methanesulfonate ("Desferal") from Ciba Laboratories Ltd., Horsham, Sussex. Iron metal enriched with 8o%~7Fe was obtained from the Atomic Energy Research Establishment, Harwell, Berks.
Preparation of Fe 3+ samples Enriched iron metal was dissolved in dilute sulfuric acid and oxidized with ammonium persulfate to give a solution containing 2.57 m g ' m l - l F e , 97% as Fe 3÷. In a typical sample o.i ml of this solution was added to 0.7 ml of o.I M citrate buffer containing 15 mg Desferal. This gave a sample of pH 6 with a sixfold excess of deferoxamine over its theoretical binding capacity with Fe 3+. One sample was prepared with glycerol replacing half the buffer volume so that it could be frozen to a glass to avoid precipitation of solute. A control was prepared with no Desferal. The samples were immediately frozen in polyethylene cells having i/8-inch-thiek compartments and I/32-inch-thick walls.
Preparation of Fe 2+ samples o.I ml of the 57Fe-enriched solution used above was added to 1.6 ml water containing 200 mg sodium dithionite. 50 mg Desferal was added to half of the resulting solution, with the remaining half serving as control. Biochim. Biophys. Acta, 264 (1972) 245-251
MOSSBAUER SPECTROSCOPY OF DEFEROXAMINE
247
Measurements
Spectra were measured on a 256-channel, constant-acceleration machine *, calibrated regularly with metallic iron. Runs at 4.2 °K were made with the sample immersed in liquid He. Between 4.2 and 77 °K the samples were in vacuum, clamped to a temperature-controlled Cu tongue. At higher temperatures the samples were in a cryostat cooled with liquid N2(77 °K), dry ice (195 °K) or freon (233 °K). Transverse fields were obtained either with a permanent magnet (0.55 kG) or a superconducting solenoid (lO-6O kG). Fields of o.I kG parallel to the v-beam were obtained by inducing persistent currents in superconducting Pb rings placed in Helmholtz fashion next to the sample. EXPERIMENTAL RESULTS
It was found that, even at low temperatures, well resolved spectra of Fe 3÷deferoxamine were only obtained with magnetically dilute samples containing about 4 #moles Fe in a volume of o.8 ml. At this Fe content excellent statistical accuracy was achieved in 48 h running time. All of the spectra shown in the figures are from the glycerol sample, which showed slightly narrower lines than the ordinary samples. Fig. 2a shows the zero-field spectrum of Fe3÷-deferoxamine at 4.2 °K. A similar solution, containing Fe 3÷ in citrate buffer but without deferoxamine is shown in 2b. The large change in the spectrum indicates that when the deferoxamine is present the iron binds to it, changing considerably its local environment. Our present theory is not adequate for calculating the zero-field spectra, but the comparisons with theory in the case of applied field spectra indicate that in Fe3÷-deferoxamine all iron atoms are in identical environments.
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Fig. 2. (a) Zero-field s p e c t r u m of Fe3+-deferoxamine in g l y c e r o l - w a t e r at 4.2 °K. (b) S p e c t r u m of Fe 3+ in citrate buffer u n d e r similar conditions w i t h no deferoxamine present.
Fig. 3 shows spectra at low temperatures in small applied fields. They are typical of a high-spin ferric material with rhombic distortion. A control spectrum (not shown), taken under the same conditions as 3a, was again very different and more complicated, justifying our assumption that we were looking at asingle, deferoxamine-bound species. The superposed solid lines in Fig. 3 are calculated spectra, made b y solving a spin-5/2 Hamiltonian as discussed below. The spectra show notable line-broadening, especially at 19.6 °K, from spin relaxation. It was hoped that at some higher temperature spin-relaxation would completely wash out the magnetic hyperfine structure to yield a simple quadrupole doublet. Biochim.
Biophys.
Acta,
264 (1972) 245-25I
248
j . L . BOCK, G. LANG
. . . .-I0 . . . . . . . . . .-5 .......
0
5
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VELOCITY (rnm/see tel Fe) Fig. 3. Dotted lines are spectra of Fe3+-deferoxamine taken at (a) 4.2 °K, 550 G transverse to the 7-beam, (b) 4.2 °K, IOO G parallel to 7-beam, (c) io.o °K, 55° G transverse, (d) 19.6 °K, 55o G transverse. Superposed solid lines are calculated spectra, as explained in the text. Magnetic effects persisted, however, at 77 a n d 195 °K, a n d only a completely featureless s p e c t r u m was o b t a i n e d at 233 °K. Hence we could n o t o b t a i n a n i n d e p e n d e n t check on our estimate of the q u a d r u p o l e splitting. Spectra t a k e n in high magnetic fields are shown in Fig. 4. Their usefulness in checking the theoretical model is described below.
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Fig. 4- Dotted lines are spectra of Fea+-deferoxamine at 4.2 °K, (a) in a 60 kG transverse field, (b) in a 2o kG transverse field, (c) in a IO kG transverse field. Solid lines are calculated spectra. A few of the spectra o b s e r v e d i n the Fe2+-deferoxamine s t u d y are shown in Fig. 5. All the spectra were q u a d r u p o l e doublets with no magnetic features, typical of highspin Fe ~+, b u t the wide a n d a s y m m e t r i c lines suggest a v a r i e t y of iron e n v i r o n m e n t s in the ferrous solutions, both with deferoxamine a n d in its absence. I n the first few m e a s u r e m e n t s , the samples exhibited spectra which differed b y more t h a n the exp e r i m e n t a l u n c e r t a i n t y . However, a d d i t i o n a l m e a s u r e m e n t s i n d i c a t e d that, with v a r i a t i o n of freezing conditions, each sample has a range of spectra a n d these ranges Biochim. Biophys. Acta, 264 (1972) 245-251
MOSSBAUER SPECTROSCOPY OF DEFEROXAMINE
i t
I
"2
2
0
249
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VELOCITY (rnrn/sec tel Fe) Fig. 5. F e r r o u s s u l p h a t e s o l u t i o n spectra, (a) a n d (b), a n d s p e c t r a of a similar solution in w h i c h d e f e r o x a m i n e is p r e s e n t as well, (c) a n d (d). All were m e a s u r e d in zero field a t 77 °K. Vertical lines are d r a w n a t + 3.0 m m / s a n d - - o . 3 m m / s to facilitate c o m p a r i s o n . S p e c t r a (a) a n d (c) c o r r e s p o n d to q u i c k - f r e e z i n g of t h e s a m p l e , while for (b) t h e s a m p l e w a s frozen slowly. (d) w a s frozen q u i c k l y b u t s t o r e d for s e v e r a l w e e k s a t 77 ° K before t h e m e a s u r e m e n t was m a d e .
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Fig. 6. I l l u s t r a t i o n s h o w i n g t h e c o n t r i b u t i o n to a M 6 s s b a u e r s p e c t r u m f r o m t h e t h r e e electronic K r a m e r s d o u b l e t s . (d) is a r e p r o d u c t i o n of 3a ( t r a n s v e r s e field) a n d (h) is a r e p r o d u c t i o n of 3b ( l o n g i t u d i n a l field). T h e c u r v e s in (a), (b), (c), a n d (e), (f), (g) are c a l c u l a t e d s p e c t r a for t h e i n d i v i d u a l d o u b l e t s , in d e s c e n d i n g order of e n e r g y , w h i c h a d d to give t h e t o t a l c a l c u l a t e d s p e c t r u m . N o t e t h a t t h e e s s e n t i a l f e a t u r e of t h e longitudinal-field s p e c t r u m is t h a t , d u e to selection rules, t h e s e c o n d a n d fifth lines f r o m t h e m i d d l e d o u b l e t are effectively e l i m i n a t e d . Also o b s e r v e t h a t cont r i b u t i o n s f r o m t h e t o p d o u b l e t in t h e a c t u a l s p e c t r a are a l m o s t w a s h e d o u t b y s p i n relaxatiola.
Biochim. Biophys. Mcta, :264 (1972) 245-251
250
J . L . BOCK, G. LANG
overlap. The spectra of Fig. 5, taken at 77 °K, illustrate this. It was found that the width of the spectrum at 4.2 °K correlated with the width at 77 °K- Both samples had similar spectra at I95 °K, with broader and more asymmetric lines and a slightly reduced quadrupole splitting. DISCUSSION
The theory of Fe 3+ in rhombic environments has been discussed extensively elsewhere~,%%l°; ref. 6 should be consulted for the spirit of approach in the present study, as well as some description of the calculating methods. Here we will merely comment on the theory in light of problems raised by our present investigation. The system of iron nucleus plus d electrons may be described in terms of the following S ~ 5/2 spin Hamiltonian.
= D[S~ 2 + ~ (S, ~ -- Sy2)] + 2 $ H . S + Ao I.S + Q?~[Zz~-5- + ~3 - ( Z J - I ' 2 ) ] Here the first term expresses the splitting of the electronic levels which occurs even in zero magnetic field as a result of high-order spin-orbit interactions. The second term is the electronic Zeeman interaction. The third term expresses the magnetic interaction between electron spin and the magnetic moment of the iron nucleus. It is taken as isotropic because in this near-spherical electron distribution only Fermi contact interaction should be important. The fourth term describes the interaction of the nuclear quadrupole moment with the local electric-field gradient. The last term is the nuclear Zeeman term. The Hamiltonian has been written in terms of the excited state, ~TmFe. For the ground nucleus the magnitudes of the third and last terms are altered by the change in nuclear magnetic moment, while the quadrupole interaction disappears. In calculating the spectra shown in the figures we have assumed values D =0.5 cm 1, 2 = ~/3 = 0.46- For the excited nucleus Ao : 1.474 m m . s -1 in M6ssbauer energy units. This corresponds to a S = 5/2 saturation field of 542 kG. The strength of the quadrupole interaction is Q Vz~/4 = - o . 3 o m m . s -1 which would correspond to quadrupole splitting o.77 m m . s -1 in the absence of magnetic interactions. Spectra in small fields represent superposed contributions from the three electronic Kramers doublets of high-spin Fe a+ (see Fig. 6). At ;t = o,46 one would expect the bottom and upper doublets to give sharp spectra, while the middle doublet would give broad lines due to its moderately anisotropic g-tensor. Unfortunately, in actual spectra narrow lines come only from the bottom doublet. As seems often to be the case, the top doublet is made almost completely invisible by spin relaxation, and the middle doublet is broadened. The result is that it is very difficult unambiguously to fit measured spectra with computer simulations, both because several line positions are not precisely defined, and because intensities of lines with different widths must be compared. The situation is considerably improved by making high-field as well as low-field measurements. At 6o kG the overall width of the spectrum is relatively insensitive to the zero field splittings, so that the value of Ao may be determined. The overall width of the very low-field spectra (Fig. 3) then may be varied by changing 2. Finally, at fields of order IO kG (Fig. 4b, 4c) significant magnetically induced mixing Biochim. Biophys. Acta, 264 (1972) 245-251
MOSSBAUER SPECTROSCOPY OF DEFEROXAMINE
251
of the electronic doublets occurs, and the spectra are sensitive to D. D also affects the thermal population of each doublet, and hence the strength of its contribution to the total spectrum in each case. Since our calculated spectra show good qualitative agreement under various conditions of measurement, though they m a y not be the best fits possible, the parameters used should be considered good estimates of Fe3+-deferoxamine's actual structure. Verification by E S R and susceptibility studies would of course be desirable. Determination of parameters for iron in low-symmetry environments is unfortunately laborious with any present technique. M6ssbauer spectroscopy m a y perhaps prove the most straightforward when computer programs are developed that will automatically search parameter space for the best least-squares fit to a measured spectrum. As for the Fe 2÷ binding question, it m a y well be that deferoxamine did affect the spectra of the ferrous samples. But this need not imply formation of a complex; the deferoxamine could modify the structure of the ice and the water environment of Fe 2+ without binding taking place. In fact, the chance that specific binding would have so little effect on the spectra is rather small, and the evidence against a Fe 2+deferoxamine is strong. In conclusion, this study has revealed two interesting properties of deferoxamine: that its Fe 3+ complex apparently has a value of ). fairly distinct from those reported for m a n y similar substances, and that it has no interaction with Fe 2+ detectable by M6ssbauer resonance. M6ssbauer and E S R experiments will undoubtedly be used in the future to gain more data on deferoxamine and related compounds, such as the mycobactins and ferrichromes. Hopefully this information will serve as a basis for developing molecular orbital theories, permitting us to interpret the numbers in terms of chemical bonds. These efforts m a y be directed to the exciting goal of understanding complex biological phenomena, such as transport of iron by the human protein transferrin. ACKNOWLEDGEMENT
This investigation was supported in part b y P.H.S. Training Grant No. 5T5 GM 1674 from the National Institute of General Medical Sciences. REFERENCES i 2 3 4 5 6 7 8 9 io
H. Bickel, H. K e b e r l e a n d E. Vischer, Helv. Chim. Acta, 46 (1963) 1385. G. A. Snow, Biochem. J., I15 (1969) 199. H. Keberle, Ann. N. Y. Acad. Sci., 119 (1964) 758. G. S c h w a r z e n b a c h a n d K. S c h w a r z e n b a c h , Helv. Chim. Acta, 46 (1963) 139o. H. H. W i c k m a n , M. P. Klein, a n d D. A. Shirley, Phys. Rev., 154 (1966) 346. G. L a n g , R. Aasa, K. G a r b e t t a n d R. J. P. W i l l i a m s , J . Chem. Phys., 55 (1971) 4539. J. F. G o o d w i n a n d C. F. W h i t t e n , Nature, 205 (1965) 281. T. E. C r a n s h a w , N u d . Instrum. Methods, 3 ° (1964) IOI. H. H. W i c k m a n , M. P. K l e i n a n d D. A. Shirley, J. Chem. Phys., 42 (1965) 2113. G. L a n g , Q. Rev. Biophys., 3 (197o) I.
Biochim. Biophys. Acta, 264 (1972) 245-251