Biomacromolecules 2003, 4, 1630-1635
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Rotational Correlation Times of 3-Carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy Spin Label with Respect to Heme and Nonheme Proteins S. Cavalu*,† and G. Damian‡ Faculty of Medicine and Pharmacy, Department of Biophysics, University of Oradea, P-ta 1 Decembrie No. 10, Oradea, 3700, Romania, and Faculty of Physics, Babes-Bolyai University, Ro-3400 Cluj-Napoca, Romania Received March 26, 2003; Revised Manuscript Received August 26, 2003
Noncovalent spin labeled proteins (ovalbumin, bovine serum albumin, hemoglobin, and cytochrome c) were investigated in order to follow the different type of interactions between the nitroxide radical of 3-carbamoyl2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy spin label and functional groups of heme and nonheme proteins as well as the pH influence on molecular motion of the label with respect to these proteins. EPR spectra were recorded at room temperature and the computer simulation analysis of spectra was made in order to obtain the magnetic parameters. Noncovalent labeling of proteins can give valuable information on the magnetic interaction between the label molecule and the paramagnetic center of the proteins. The relevance of this interaction can be obtained from line shape analysis: computer simulations for nonheme proteins assume a Gaussian line shape, whereas for heme proteins, a weighted sum of Lorentzian and Gaussian components is assumed. In the framework of the “moderate jump diffusion” model for rotational diffusion, the rotational correlation time is strongly influenced by pH, because of the electrostatic interactions and hydrogen bonding. Introduction The successful application of spin labeling to protein structure investigations is limited by the possibility to chemically change specific side chains in proteins. However, useful information on protein properties can be obtained by noncovalent spin labeling if the affinity of the protein for the label molecules is great enough to affect their motional freedom.1-5 The rate of rotation (or tumbling) of the spin label influences the line shape of its EPR spectrum. Therefore, the EPR signal of a spin label covalently or noncovalently bonded to a biomolecule can yield a range of information about its structural environment in conventional ESR and allows for full spectral coverage.6,7 At the same time, EPR has been an invaluable tool for probing microscopic molecular motions in a variety of systems, including isotropic solvents,8 liquid crystals,9 model membranes, and biomolecules.10-12 In the present work, noncovalent spin labeled bovine serum albumin (BSA), ovalbumin, bovine hemoglobin (BH), and cytochrome c, with Tempyo spin label (3-carbamoyl-2,2,5,5tetramethyl-3-pyrrolin-1-yloxy) were investigated both in liquid and lyophilized samples, in the pH range 2.5-11, to obtain useful information related to the interaction between the nitroxide group and the functional site of the proteins. Interactions of the spin label with heme or nonheme proteins might affect the spin label spectra, and at the same time, it is well-known that the pH strongly influences the * To whom correspondence should be addressed. E-mail: scavalu@ rdslink.ro. † University of Oradea. ‡ Babes-Bolyai University.
conformation of proteins leading to significant changes in the type and degree of these interactions. In this pH range, we followed the effect of protein conformational changes on the interactions between the nitroxide and the functional groups of proteins and also the pH influence on molecular motion emphasized by the EPR spectra of the spin label. All structural and functional properties of proteins derive from the chemical properties of the polypeptide side-chain. The ionizable groups of the side-chains of charged amino acids are often involved in biochemical transactions (binding, catalysis). Therefore, pH usually has rather dramatic effects on the function of proteins. Polar residues are both buried as well as on the surface of the protein. They either form hydrogen bonds with other polar residues in the protein or with water, but nonpolar residues do not interact favorably with water. Materials and Methods Powdered bovine serum albumin, ovalbumin, hemoglobin (>95% methemoglobin) and cytochrome c from Sigma Chemicals, were used without further purification. Proteins were hydrated in phosphate buffer physiological saline at a final concentration of 10-3 M. Tempyo spin-label (3carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy), from SIGMA Chemicals (Figure 1), was added to the liquid samples of each protein in a final concentration of 10-3 M (protein/spin label molar ratio 1:1) and the pH values were adjusted to the desired value in the range 2.5-11. A total of 5 mL was taken from each sample and was lyophilized for 30 h at -5 °C and used for the EPR measurement, at room temperature.
10.1021/bm034093z CCC: $25.00 © 2003 American Chemical Society Published on Web 10/16/2003
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in which
Figure 1. Chemical structure of the Tempyo spin label.
EPR spectra for both liquid and lyophilized samples were recorded at room temperature with a JEOL-JES-3B spectrometer, operating in X-band (9.5 GHz), equipped with a computer acquisition system. Samples were placed in quartz capillary tubes. The spectrometer settings were as follows: modulation frequency 100 kHz, field modulation 1 G, microwave power 20mW. The computer simulation analysis of spectra, for obtaining the magnetic characteristic parameters, was made by using a program that is available to the public through the Internet (http://epr.niehs.nih.gov) The line shape of an EPR spectrum depends on, among other factors, the orientation of the paramagnetic center with respect to the applied magnetic field. In a powder, or a frozen aqueous solution, the paramagnetic centers will be fixed with a random distribution of orientations, and in the case of the anisotropic g factor and hyperfine interactions, this will lead to a broadened EPR spectrum, because all orientations contribute equally. In the liquid state, however, the paramagnetic centers are not fixed but undergo rotational fluctuation. In the case of fast rotation, the anisotropic interactions are thereby averaged to zero, giving rise to sharp EPR lines. If the velocity of the rotational motion decreases, the EPR spectrum will approach that of the powder spectrum. Therefore, a rotational correlation time for a paramagnetic molecule can also be determined by EPR. For isotropic motion in the rapid tumbling limit, the spectra will be isotropic with the averages of the principal components of the g values and hyperfine splitting factor, aN. The rate of the isotropic motion determines the relative widths of resonances and the width, ∆Hm, of an individual (hyperfine) line, in the first approximation can be written as a function of the z component of the nitrogen nuclear spin number (m ) -1, 0, 1):13 ∆Hm ) A + Bm + Cm2
(1)
where the A coefficient includes other contributions than motion. The terms B and C are functions related to the rotational correlational time (τ) and can be defined as a function of peak to peak line width of the central line, ∆H0 [G], and the amplitudes of the mth line Im:11,12 1 B ) ∆H0 2
(x x ) I0 I1
I0 ) 0.103ωe[∆g∆aN + I-1
(x x ) [
1 C ) ∆H0 2
I0 + I1
[
]
3 3(δg)(δaN)]τB 1 + (1 + ω2e τ2B)-1 (2) 4 I0 - 2 ) 1.181 × 106[(∆aN)2 + I-1
]
3 1 3(δaN)2]τc 1 - (1 + ω2c τ2c )-1 - (1 + ω2e τ2c )-1 (3) 8 8
1 1 ∆aN ) aNzz - (aNxx + aNyy),δaN ) (aNxx - aNyy) 2 2
(4)
1 1 ∆g ) gzz - (gxx - gyy), δg ) (gxx - gyy) 2 2
(5)
and ωN ) 8.8 × 10-6〈aN〉, aN is the isotropic hyperfine splitting and ωe is the ESR spectrometer frequency in angular units. In the range from 5 × 10-11 to 10-9 s (motion in the rapid tumbling limit) and a magnetic field above 3300 G, ∆g and ∆aN vanish, and the correlation times τB and τC are directly related to the B and C coefficients by the following simple relations:4 τB ) τz ) K1B
(6)
τC ) τx,y ) K2C
(7)
where K1 ) 1.27 × 10-9 and K2 ) 1.19 × 10-9. The average correlation times is τ ) (τBτC)1/2
(8)
The slow motion of the spin probe lead to a broadening of the EPR lines. In this case, the rotational correlation time, τ, is larger than 10-9 s, and thus, eq 8 is not applicable. The isotropic nitrogen hyperfine splitting changes to a powder like spectrum, with the peak-to-peak distance between the external peaks of the spectrum (2a′zz(N)) depending on the magnitude of the rotational correlation time, τ. Another line shape theory for slow isotropic Brownian rotational diffusion of spin-labeled proteins has been developed by Freed and co-workers.8 Thus, the correlation time can be evaluated from the ratio of the observed splitting between the derivate extrema a′zz and principal value azz, determined from the rigid matrix spectrum:4,6
( )
τ)R 1-
a′zz azz
β
(9)
The R and β parameters are empirical constants depending on the kind of the diffusion process and are tabulated in e.g. Poole and Farach.14 For a small spin probe, the intermediate jump diffusion is preferable.14 Results and Discussion In the low concentration liquid state of proteins solution, the Tempyo spin label, which is a relatively small molecule, gives rise to a spectrum with narrow lines and constant hyperfine splitting, typical for fast isotropic rotational motion (Figure 2) with very low rate of migration between protein molecule and water. For this kind of rotation, the rotational correlation time can be estimated from the intensity ratio of the low-field and high-field N lines using a semiempirical formula (eq 8). For the Tempyo spin label in nonheme proteins (BSA and ovalbumin) aqueous solution, the calculated rotational correlation times was 2.5 × 10-10 s, whereas
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Figure 2. EPR spectrum of the Tempyo spin label at low pH.
Figure 4. EPR spectra, experimental (solid line) and simulated (dotted line), of the Tempyo spin labeled proteins at pH ) 6.7.
Figure 3. EPR spectra, experimental (solid line) and simulated (dotted line), of the Tempyo spin label proteins at pH ) 2.5.
with respect to heme protein (BH and cytochrome c) solutions, the respective value was around 2. × 10-10, which is consistent with fast rotation as expected for a small molecule. No detectable changes were observed in the EPR spectra of the aqueous solution of the Tempyo spin label without proteins, at different pH values. The characteristic EPR spectra of a Tempyo spin label in lyophilized samples is due primarily to anisotropy in the nitrogen hyperfine coupling typical for slow rotation. For slow rotations, the EPR spectrum of spin labels, depends in a much more complicated fashion on the combined influences of molecular motion and magnetic interactions. Figures 3-5 display the experimental and simulated spectra for Tempyo spin label with respect to BSA, ovalbumin, BH, and cytochrome c, lyophilized at pH ) 2.5, 6.7, and 11, respectively. To find the magnetic parameters, the experimental EPR spectra were simulated. The best fit of experimental EPR spectra of the Tempyo spin label in lyophilized BSA and ovalbumin was obtained assuming a single paramagnetic species and a Gaussian line shape corresponding to static dipolar interactions. The magnetic parameters are listed in Tables 1 and 2, respectively. The main feature of the EPR spectra of the Tempyo spin label in lyophilized haemoglobin and cytochrome c exhibits the characteristics to the slow motion of the spin label but with more broadening of the line shape. The simulation of the experimental EPR spectra can be obtained by assuming the presence of two functional groups in heme proteins,
Figure 5. EPR spectra, experimental (solid line) and simulated (dotted line), of the Tempyo spin labeled proteins at pH ) 11.
associated with two nonequivalent paramagnetic species.15 Computer simulations, indicate a weighted sum of mixed Gaussian line shapes (static dipolar interactions) and Lorentzian line shapes (spin-spin interactions). To support this affimation, in Figures 6 and 7 are presented the EPR spectra of the Tempyo spin label in lyophylized BH at low and high pH, in which are displayed the contributions of each species. Generally, the broadening of the Gaussian line shape is due to the static dipolar interactions of the spin label molecules, whereas the broadening of the Lorentzian line shape is due to spin-spin interactions.16-17 From the computer analysis of spectra, we suggest that at low pH (pH ) 2.5) the main contributions are due to species with a Gausian line shape (∼80%). This contribution decreases to ∼50% at high pH (pH ) 11). The first species, with Gaussian line shape and poor resolved hyperfine splitting, is not influenced by the
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Table 1. Magnetic Parameters Values of the Tempyo Spin Label in Lyophilized Bovine Serum Albumin at Various pH Values pH 11 6.7 2.5
gxx
gyy 10-4
2.0103 ( 4.3 × 2.0011 ( 4.3 × 10-4 2.0124 ( 4.3 × 10-4
gzz 10-4
2.0084 ( 4.3 × 2.0074 ( 4.3 × 10-4 2.0066 ( 4.3 × 10-4
10-4
2.0044 ( 4.3 × 2.0050 ( 4.3 × 10-4 2.0054 ( 4.3 × 10-4
Axx(G)
Ayy(G)
Azz(G)
4.7 ( 0.2 6.2 ( 0.3 8.3 ( 0.4
8.5 ( 0.4 6.6 ( 0.3 5.3 ( 0.3
35.5 ( 1 33.2 ( 0.9 36.0 ( 1.1
Table 2. Magnetic Parameters Values of the Tempyo Spin Label in Lyophilized Ovalbumin at Various pH Values pH
gxx
gyy
gzz
Axx(G)
Ayy(G)
Azz(G)
11 6.7 2.5
2.0111 ( 4.1 × 10-4 2.0012 ( 4.1 × 10-4 2.0014 ( 4.1 × 10-4
2.0069 ( 4.1 × 10-4 2.0063 ( 4.1 × 10-4 2.0098 ( 4.1 × 10-4
2.0041 ( 4.1 × 10-4 2.0053 ( 4.1 × 10-4 2.0055 ( 4.1 × 10-4
4.3 ( 0.2 7.3 ( 0.3 4.6 ( 0.2
7.7 ( 0.3 7.2 ( 0.2 9.7 ( 0.4
34.1 ( 0.9 32.8 ( 0.8 35.6 ( 1.1
Figure 6. Experimental EPR spectrum and its subspectra of the Tempyo spin label in lyophilized hemoglobin at pH ) 2.5.
Figure 7. experimental EPR spectrum and its subspectra of Tempyo spin label in lyophilized hemoglobin at pH ) 11.
presence of the heme iron, and therefore, we assume to be located far from the heme group. The second species with a Lorentzian line shape and a well resolved hypefine structure is located, probably, near the heme group, giving rise to a spin-spin interaction between the nitroxide radical and the paramagnetic iron of the heme group. Our results are in
accordance with covalently labeled methemoglobin and other porphyrins in frozen samples under 50 K.18,19 In these previous studies, spectra of covalently labeled methemoglobin were analyzed by using perturbation calculations in order to estimate the iron to nitroxyl distances, and it was suggested that plausible distances are in the range of 14.517.5 Å. The g tensor and A tensor components used in the simulation for the best fit values of the simulation of the effective powder spectrum are presented in Tables 3 and 4 (for Tempyo-BH and Tempyo-cytochrome c, respectively). Because of increased spatial restrictions of the protein structure in the vicinity of label, by lyophilization, the mobility of the spin label is slow on the EPR time scale (∼5 × 106 s-1), leading to a broadening of the EPR lines, with the peak-to-peak distance between the external peaks of the spectrum (2a′zz(N)) depending on the magnitude of the rotational correlation time, τ. Generally, a broadening of the peaks in an EPR spectrum is indicative of immobilization of the spin label, whereas sharpening of the peaks points to an increase in label mobility. By comparison of the apparent nitrogen hyperfine splitting (termed a′zz(N)) with the nitrogen hyperfine splitting obtained from their rigid limit values (a′zz(N)), the rotational correlation times can be calculated using eq 9. The values of R and β coefficients depend on the motional model. The study of the influence of the different diffusional models on the spectral line shape in the regime of the slow motional spin label by high EPR fields has showed that jump diffusion mainly affects the line widths at the same motional rates.20 In our calculations, the intermediate jump diffusion model was considered, with coefficients values of R ) 5.4 × 10-10 s and β ) -1.36.21 In Figure 8 are plotted the average of the correlation time for differents values of the pH. As shown in the figure, the pH influences the rotational correlation time of Tempyo with respect to all these proteins. In the acid pH range, the NH2 groups of the label molecule as well as those of the amino acids residues are protonated. The fact that τ shows greater values in this range followed by a significant decrease in the basic pH range indicates a low mobility of spin label in acid environment, whereas an increasing of mobility can be noticed in the basic pH range. From the pH dependence of correlation time (involving the mobility of the label as well), we assume that in an acid environment the mobilities of spin label molecules are reduced because if the formation of the hydrogen bonds between the NH2 group of the spin label and the side chains of neighboring amino acids. In the case of BSA, one can correlate this observation with the fact that
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Table 3. Magnetic Parameters Values of the Tempyo Spin Label in Lyophilized Hemoglobin at Various pH Valuesa pH 11 6.7 2.5 a
gxx
gyy 10-4
2.0124 ( 4.3 × 2.0112 ( 4.3 × 10-4 2.0011 ( 4.2 × 10-4 2.0122 ( 4.2 × 10-4 2.0127 ( 4.2 × 10-4 2.0117 ( 4.2 × 10-4
gzz 10-4
2.0077 ( 4.2 × 2.0050 ( 4.2 × 10-4 2.0077 ( 4.2 × 10-4 2.0048 ( 4.2 × 10-4 2.0082 ( 4.2 × 10-4 2.0029 ( 4.2 × 10-4
10-4
2.0037 ( 4.2 × 2.0061 ( 4.2 × 10-4 2.0042 ( 4.2 × 10-4 2.0056 ( 4.2 × 10-4 2.0041 ( 4.2 × 10-4 2.0051 ( 4.2 × 10-4
Axx(G)
Ayy(G)
Azz(G)
4.8 ( 0.2 6.4 ( 0.3 3.3 ( 0.2 7.4 ( 0.3 4.1 ( 0.2 7.1 ( 0.3
8.7 ( 0.4 7.8 ( 0.7 8.2 ( 0.4 8.9 ( 0.4 5.4 ( 0.2 9.4 ( 0.4
34.0 ( 1 (L) 33.2 ( 0.9 (G) 33.7 ( 0.9 (L) 35.8 ( 1.1 (G) 35.6 ( 1.1 (L) 36.5 ( 1.1 (G)
(L), Lorentzian line shape; (G), Gaussian line shape.
Table 4. Magnetic Parameters Values of the Tempyo Spin Label in Lyophilized Cytochrome c at Various pH Valuesa pH 11 6.7 2.5 a
gxx
gyy
gzz
Axx(G)
Ayy(G)
Azz(G)
2.0155 ( 4.2 × 10-4 2.0124 ( 4.2 × 10-4 2.0141 ( 4.2 × 10-4 2.0136 ( 4.2 × 10-4 2.0183 ( 4.2 × 10-4 2.0124 ( 4.2 × 10-4
2.0109 ( 4.2 × 10-4 2.0140 ( 4.2 × 10-4 2.0118 ( 4.2 × 10-4 2.0113 ( 4.2 × 10-4 2.0092 ( 4.2 × 10-4 2.0129 ( 4.2 × 10-4
2.0056 ( 4.2 × 10-4 2.0087 ( 4.2 × 10-4 2.0049 ( 4.2 × 10-4 2.0057 ( 4.2 × 10-4 2.0025 ( 4.2 × 10-4 2.0087 ( 4.2 × 10-4
4.8 ( 0.2 6.4 ( 0.3 6.0 ( 0.3 5.2 ( 0.2 3.3 ( 0.2 7.8 ( 0.4
8.7 ( 0.4 7.8 ( 0.3 5.9 ( 0.3 3.7 ( 0.2 7.1 ( 0.4 6.4 ( 0.3
33.0 ( 0.9 (L) 34.2 ( 0.9 (G) 33.0 ( 0.9(L) 35.3 ( 1 (G) 32.1 ( 0.8 (L) 33.4 ( 0.9 (G)
(L), Lorentzian line shape; (G), Gaussian line shape.
of proteins can give valuable information on the magnetic interactions between the label molecule and the paramagnetic center of the proteins. The relevance of this interaction can be obtained from line shape analysis: computer simulations for a nonheme protein assume a Gaussian line shape, whereas for a heme protein, a weighted sum of Lorentzian and Gaussian components is assumed. The contribution of the each line shape to experimental spectrum depends on the pH. We can conclude that, on averrage, τcyto < τHb < τovalb < τBSA. References and Notes Figure 8. Correlation times (τ) as a function of pH for Tempyo spin label in lyophilized cytochrome c (9), hemoglobin (b), ovalbumin (2), and bovine serum albumin (1).
serum albumin undergoes reversible isomerization in the pH range 2.7-7 from the expanded form characterized by 35% R-helix content to normal the form characterized by 55% R-helix content accompanied by a decrease in β-sheet.22,23 It is well-known that the β-sheet conformation favors the formation of hydrogen bonding. By comparing the results in Figure 8, we can notice that the mobility of Tempyo is greater with respect to that of the heme proteins, which is not surprising if we take into account that hydrogen bonding opportunities depend on the β-sheet content: in hemoglobin, the β-sheets represent 50%, whereas in BSA, the percentage varies from 70% to 45%, depending on pH. On averrage, τcyto < τHb < τovalb < τBSA. As shown in Figure 8, the mobility of Tempyo increases in an acid environment, followed by a slow decrease. We suggest that in the basic pH range, where the label is not subject to strong electrostatic interactions, dipolar and spinspin interactions are manifested almost with the same contribution in the brodening of the spectrum. Conclusions EPR spectroscopy is very useful to study the mobility of nitroxide radicals with respect to heme or nonheme proteins in different environmental conditions. Noncovalent labeling
(1) Morrisett, J. D.; Wien, R. W.; McConnell, H. M. The use of spin labels for measuring distances in biological systems. Ann. N.Y. Acad. Sci. 1973, 222, 149-162. (2) Marsh, D. In Spectroscopy and Dynamics of Molecular Biological Systems; Bayley, P. M., Dale, R. E., Eds.; Academic Press: London, 1985; pp 209-238. (3) Jost, P.; Griffith, O. H. Electron spin resonance and the spin labeling method. In Methods in Pharmacology; Chignell C., Ed.; Appleton: New York, 1972. (4) Morrisett, J. D.; Pownall, H. J.; Gotto, A. M. Bovine serum albumin, Study of the fatty acid and steroid binding sites using spin labeled lipids. J. Biol. Chem. 1975, 250, 2487-2494. (5) Morrisett, J. D. Spin labeled enzymes. In Spin Labeling-Theory and Application; Berliner, J., Ed.; Academic Press: New York, 1975. (6) Frajer, P. Electron Spin Resonance Spectroscopy Labeling in Peptide and Protein Analysis. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; John Wiley & Sons Ltd.: New York, 2000). (7) Biswas, R.; Kuhne, H.; Brudvig, G. W.; Gopalan, V. Use of EPR spectroscopy to study macromolecular structure and function. Sci. Prog. 2001, 84 (1), 45-68. (8) Hwang, J. S.; Mason, R. P.; Hwang, L.-P.; Freed, J. H. Electron Spin Resonance Studies of Anisotropic Rotational Reorientation and Slow Tumbling in Liquid and Frozen Media. III. Perdeuterated 2,2,6,6,-Tetramethyl-4-piperidone N-Oxide and an Analysis of Fluctuating Torques. J. Phys. Chem. 1975, 79, 489-511. (9) Meirovitch, E.; Igner, D.; Moro, G.; Freed, J. H. Electron-spin relaxation and ordering in smectic and supercooled nematic liquid crystals. J. Chem. Phys. 1982, 77, 3915-3938. (10) Tanaka, H.; Freed, J. H. Electron spin resonance studies on ordering and rotational diffusion in oriented phosphatidylcholine multilayers: evidence for a new chain-ordering transition. J. Phys. Chem. 1984, 88, 6633-6643. (11) Marsh, D.; Horvath, L. I. Spin label studies of structure and dynamics of lipide and proteins in membranes. In AdVance EPR-Application in Biology and Biochemistry; Hoff, A. J., Ed.; Elsevier: Amsterdam, 1989. (12) Schreier, S.; Polnaszek, C. F.; Smith, I. C. P. Spin labels in membranes. Biochim. Biophys. Acta 1978, 515, 375-436.
Rotational Correlation Times of Nitroxide Radical (13) Goldman, S. A.; Bruno, G. V.; Polnaszek, C. F.; Freed, J. H. An ESR study of anisotropic rotational reorientation and slow tumbling in liquid and frozen media. J. Chem. Phys. 1972, 56, 716-735. (14) Poole, C. P., Jr.; Farach, H. A. In Theory of Magnetic Resonance; John Wiley & Sons: New York, 1987; pp 319-321. (15) Schneider, D. J.; Freed, J. H. In Spin Labeling Theory and Applications; Berliner, L. J., Reuben, J., Eds.; Plenum Press: New York, 1989; pp 1-76. (16) Marsh, D. In Spin Labeling Theory and Applications; Berliner, L. J., Reuben, J., Eds.; Plenum Press: New York, 1989; pp 255-303. (17) Berliner, L. J. Spin Labeling, Theory and Application; Adademic Press: New York, 1976. (18) Budker, V.; Du, J.-L.; Seiter, M.; Eaton, G. R.; Eaton, S. S.; Electron-electron spin-spin interaction in spin labeled low-spin methemoglobin. Biophys. J. 1995, 68, 2531-2542.
Biomacromolecules, Vol. 4, No. 6, 2003 1635 (19) Rakowsky, M. H.; More, M. K.; Kulikov, A. V.; Eaton, G. R.; Eaton, S. S. Time-Domain Electron Paramagnetic Resonance as a Probe of Electron-Electron Spin-Spin Interaction in Spin-Labeled Low-Spin Iron Porphyrins. J. Am. Chem. Soc. 1995, 117, 2049-2057. (20) Earle, K. A.; Budil, D. E.; Freed, J. H. 250-GHz EPR of Nitroxide in the Slow-Motional Regime: Models of Rotational Diffusion. J. Phys. Chem. 1993, 97, 13289-13297. (21) Eaton, G. R.; Eaton, S. S. Interaction of spin labels with transition metals. Coord. Chem. ReV. 1978, 26, 207-262. (22) Foster, J. F. In Albumin Structure, Function and Uses; Rosenoer, V. M., Oratz, M., Rothschild, M. A., Eds.; Pergamon: Oxford, 1977; pp 53-84. (23) Carter, D. C.; Ho, J. X. Structure of Bovine Serum Albumine. AdV. Protein Chem. 1994, 45, 153-203.
BM034093Z
