3526
J . Phys. Chem. 1992, 96, 3526-3531
the formation of CO: one alternative involves the direct photoreduction of C 0 2 to CO (eq 5 ) , and the second possibility includes CO elimination from the intermediate zinc formate as follows ZnCOOH
-
ZnOH
+ CO
(8)
Conclusion The efficient photofmation of C 0 2 in semiconductor particulate systems can be achieved under control of pH 7 by using quantized ZnS (ZnS-0) in the presence of SH- as photocatalysts and NaH2P02as an electron donor. A large quantity of HC02accompanied by a small quantity of CO is formed, and simultaneous large quantity of H2 evolution as a result of water photoreduction observed. The remarkable efficiency can be obtained by controlling the concentration of the electron donor of NaH2P02 and the sulfur vacancies suppressor of Na2S. Total quantum yield for the formation of HCOy and H2as high as 0.84 was attained in the presence of H2P02- (0.70 M) and SH- (0.24 M). The fact that the singleelectron transfer to C02can be achieved effectively in the semiconductor particulate system as well as
electrochemical reductions suggests that development of artificial photosynthetic semiconductor systems should become promising in view of the simplicity of the system regarding semiconductor particles as micro-photocells. Improvement in selectivity of photoproducts and development of semiconducting materials sensitive to visible light could further lead to effective photofmtion of C 0 2 by solar light.
Acknowledgment. We appreciate the reviewer’s comments suggesting the alternate mechanisms, Le., the quenching mode by SH- and the contribution of particle size to the difference in activity between ZnS-0 and ZnS-100.This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (No. 022031 19). The research was also conducted as a theme of the Research Skiety for C02Fixation sponsored by Institute of Laser Technology under the commission of the Kansai Electric Power Co., Inc. Registry No. C02, 124-38-9; ZnS, 1314-98-3; H 2 0 , 7732-18-5; NaH2P02, 7681-53-0; Na2S, 1313-82-2; H2P02-, 15460-68-1; SH-, 15035-72-0; H i , 1333-74-0; CO, 630-08-0; HCOO-, 71-47-6.
Measurement of Distance Distribution between Spin Labels in Spin-Labeled Hemoglobin Using an Electron Spin Echo Method A. Raitsimring,*?+J. Peisach, H. Caroline Lee, Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
and X. Chen Department of Chemistry, University of Houston, Houston, Texas 77204 (Received: November 15, 1991) One of the varieties of electron spin echo methods, that utilizing the “2 + 1“ pulse train, was used to determine the distribution of distance between a pair of nitroxide radicals in spin-labeled, tetrameric hemoglobin. The method allows one to obtain the spin echo kinetics determined solely by the dipoldipole interaction between a pair of labels located within a protein molecule and to suppress the dipoledipole interaction of the pair with other pairs in the bulk medium. The kinetic behavior was simulated using various distribution functions. The agreement between experimental and simulated kinetics was found for a spin-label distance distribution function centered at 35 A and with a half-width of 3 A.
Introduction A problem of particular interest in the study of biomolecules is the determination of distances between structural components. Distance measurements have been performed using a variety of methods including X-ray crystallography, fluorescence quenching, Mossbauer spectroscopy, electron or X-ray scattering, and a number of magnetic resonance techniques (ESR, NMR, ENDOR).l-’ Application of electron spin resonance (ESR) methods for distance measurements can be used when the biomolecule of interest contains either natural (metals and free radicals) or artificial (metals and spin labels) paramagnetic centers. The distance dependence of the dipole-dipole interaction between paramagnetic centers has allowed for the use of the EPR technique for measurements of distances between paramagnetic metal ions5 and the determination of local concentrations of spin labels.s The dipole-dipole interaction contributes to the broadening of the continuous-wave (CW) ESR spectrum and the alteration of relaxation parameters of individual paramagnetic centers. C W ESR methods have not been used to measure distances exceeding 10-20 A. However, pulsed ESR methods have been developed recently9 that allow for the determination of distances between *To whom correspondence should be addressed. ‘Onleave from Institute of Chemical Kinetics and Combustion S B AS USSR, Novosibirsk, 630090.
0022-365419212096-3526$03.00/0
paramagnetic centers up to 100-150 A. These new methods which employ the “2 1 pulse train procedure have been successfully applied to the investigation of the distribution of paramagnetic centers in irradiated soliddoand catalysts containing paramagnetic metal ions.” However these methods have never been used to solve structural problems in systems such as biological materials where the state of aggregation depends strongly on the local environment of the material of interest. In this work we apply the ‘2 + 1” pulse train procedure to determine the distance distribution between nitroxide spin labels tethered to the cysteine sulfhydryls a t position 93 on each of the
+
( I ) Fermi, G.; Perutz. M. F. J. Mol. Biol. 1977, 114, 421.
(2)O H a r a , P.;Yeh, S.M.; Meares, C. F.; Berson, R. Biochemistry 1981, 20, 4704.
(3)Belonogova, 0.V.;Lichtenstein, G.R.; Goldanskii, V. I. Dokl. Akad. USSR 1918, 241, 219. (4) Nicolson, G. L.; Yanamigachi, R.; Yanamigachi, H. J . Cell Biol. 1975, 66, 263. ( 5 ) More, K. H.; Eaton, G.R.; Eaton, S.J . Magn. Reson. 1985,63, 151. (6)Zweir, J. L.; Wooten, J. B.; Cohen, J. S.Biochemistry 1981, 20, 3505. (7) Mustafi, D.;Sachleben, J. R.; Wells, G. B.; Makinen, M. W.J . Am. Chem. SOC.1990, 112, 2558. ( 8 ) (a) Zweir, J. L. J . Biol. Chem. 1983, 258, 13759. (b) Kulikov, A. V.; Cherepanova, E. S.;Lichtenshtein, G.R. Eiol. Membr. 1989, 6, 1085. (9)Raitsimring, A. M.;Salichov, K. M. Bull. Magn. Reson. 1985, 7, 184. (IO) Kurshev, V. V.; Raitsimring, A . M.; Ichikava, Ts.J. Phys. Chem. 1991, 95, 3563. ( I I ) Levi, Z.; Raitsimring, A.; Goldfarb, D. J . Phys. Chem. 1991, 95, 7830. Nauk.
0 1992 American Chemical Society
The Journal of Physical Chemistry, Vol. 96, No. 8, 1992 3521
Spin-Labeled Hemoglobin
O 400
\
. .
I
,
0 200
8
0
t
27
7
Figure 1. Scheme of the ‘2 + I ” pulse train. The primary ESE signal is induced by the first and the third pulses, separated by a time interval T. The signal amplitude is measured as a function of the second pulse position, t . Other echo signals are not shown.
,3-chains of tetrameric hemoglobin A and demonstrate the features of application of these methods to protein samples. ”ry
To select for the dipoledipole interaction between paramagnetic centers, we used a recently developed “2 + 1” pulse train in electron spin echo (ESE) spcctroscopy.I2 This ESE method employs three pulses of the same carrier frequency. Two of them, the first and the third, induce a primary ESE signal. The dependence of the amplitude, U(t),of the primary ESE signal on the position of the additional second pulse, 1, at fixed interval T between second and third pulses is studied. The scheme of this train is presented in Figure 1. The variation of U(t) is induced only by an instantaneous diffusion mechanismI3 and depends directly on the magnitude of the dipoledipole interaction between paramagnetic centers as well as on the ESR line shape and the duration and amplitude of pulses. The interaction between spins may be investigated by means of a usual two-pulse train? but the use of such a method is hampered by the presence of other relaxation mechanisms (e.g., electron-nuclear or spin-lattice interactions) acting simultaneously. In the case of the application of the “2 1” pulse train, no other relaxation processes contribute to the variation in amplitude of the primary spin echo signal. Furthermore, as will be shown later, this method allows one to distinguish the different types of dip o l d i p o l e interactions in the sample under investigation. A detailed description of the spin system evolution under the “2 1” pulse train has been given before.12 In this present work we make use only of the final results, which describe the dependencies of U(t)for different spatial distributions of paramagnetic centers. In the present system, spin-labeled hemoglobin, as is known a priori, there are two types of spin distributions. The first is a pair distribution of spin labels in the hemoglobin tetramer, and the second is the distribution of these pairs throughout the volume of the sample. As was shown in ref 12, the spin echo signal variation U(t) in this case can be presented as a product:
+
+
Here U2(t)results from the dipole-dipole interaction in a pair of spins; U l ( t )is the interaction of a given pair with other paramagnetic centers in the bulk medium. The variation of ESE signal amplitude for the random spatial distribution of spins is described by the following expression:
I r
U(t) = U , ( t ) = (sin 8(1)Cf2)S(3))d,) exp --98;.Srihon q = (S(2)(1
where
(e-)
J
(2)
- 2s’)) )dw)
where wo is the microwave pulse carrier frequency, hl is the pulse amplitude, and t, is the pulse duration. For spins with Zeeman frequency w equal to the microwave pulse carrier frequency wo (resonant spins), 6w = 0, and 80 = ?hitpis the angle a resonant spin is turned by the kth pulse. It is thus seen from expression 2 that as r changes, the signal Ul(t) either decays or increases depending on the sign of q. For specially adjusted pulse parameters the value of q equals zero. Under these conditions, the ESE signal does not depend on t , i.e., U l ( t )= constant. This phenomenon of suppression of dipole-dipole interactions was demonstrated before.I2 Figure 2 depicts the dependence of q on pulse parameters for spin-labeled hemoglobin. The removal of the dipole-dipole interaction from ESE kinetics with pulses of equal duration (‘,= 16 ns) is shown to take place at Bo = 0 . 6 5 ~ .Therefore, the “2 1” pulse train allows for rather simple regulation of the manifestation of the dipoledipole interaction between spins in the bulk medium. The kinetics observed in the various cases of pair distribution are considered here. The dipole-dipole interaction between a pair of spins fixed in space gives the following expression for the variation of ESE signal amplitude:
+
where A = (y2h/Z)( 1 - 3 cos2 a), r is the spin separation in the pair, and 0 is the angle between r and the direction of external magnetic field Ho. In the case of a pair arrangement with an intrapair distance distribution Ar),as in spin-labeled hemoglobin, the overall time evolution of the ESE signal intensity is given by
represents averaging over the ESR line shape, C is
(12) Kurshev, V . V.; Raitsimring, A. M.; Tsvetkov, Yu. D. J . Magn. Reson. 1989, 81, 441. (13) Klauder, J. R.; Anderson, P. W. Phys. Reu. 1962, 125, 912.
(14) Abragam, A. The Principles of Nuclear Magnetism; Clarendon: Oxford, 1961.
3528 The Journal of Physical Chemistry, Vol. 96, No. 8, 199I2 with
Raitsimring et al. 0800
, /’
,
(5’)
Relation 5’ also describes the signal variation in pairs with a constant intraspin distance r and an arbitrary orientation relative to external magnetic field Hoe Expressions 3-5’ allow one to calculate the optimal experimental conditions for investigating of intrapair dipole-dipole interaction. Thus xrsin2
sin 9 d 9 = 1
at Ax
+
U,
-
\
t
>> 1
for sufficiently large values of 1, T , and T - t , where the ESE signal intensity reaches the limit
U(t(r)
06004
(1 - S3) - S(2)+ S(3)S(2)),, w )/ ( I - s ( 3 ) ) g ( 4 (6)
which is determined by the fraction of excited spins, is time independent, and does not approach zero as in the case of a random spin distribution. The relative variation of signal amplitude, measured experimentally, is between unity and the value of U,. Figure 2 depicts the set of curves (1 - U,) for different microwave pulse parameters available experimentally. The relative signal decay due to intrapair dipole-dipole interaction with some pulse parameters can reach approximately 30%. This set of pulses, however, does not switch off the dipoledipole interaction between spins in the bulk medium. To suppress the spin echo signal decay due to dipole-dipole interaction between spins distributed throughout the sample volume but retain ESE signal variation due to intrapair interaction, it is necessary to choose another set of pulse parameters, e.g., the aforementioned train of pulses of equal duration. Under these conditions the value of (1 - U,)is approximately 1.5 times smaller than that of the previous case. Note also that U, is not maximal, but an asymptotic value of the signal decay because of an oscillating behavior of U(t1r). In the cases where U , ( t )and Uz(t)are comparable, or when U l ( t )can be removed from U(t) by means of a different, nonpulse-adjusted method, it is preferable to use such pulse parameters to accentuate the variation of ESE signal due to intrapair interactions as much as possible. This procedure has been used to investigate the spatial distribution of radicals in y-irradiated alcohols.1° This system allows for the transformation of a complex radical distribution into a random radical distribution without changing radical concentration. Therefore, as a result, it is possible to remove U,(r) from a total signal decay. Otherwise, it is better to carry out the selection of U2(t)with pulse adjustment although some loss of the effect of the intrapair interaction may result. Finally, we note some features of the behavior of Uz(r)for the condition of suppression of bulk dipole-dipole interaction. An inspection of the expression 4 shows, U(Olr) = U(slr),Le., the amplitudes of ESR signal at the upper and lower limits of the time interval are the same and their normalized values equal unity. The kinetics are symmetric relative to the center of the time interval r / 2 . If y 2 h r / r 3>> 1, the limit of decrease in signal amplitude, as was noted previously, is equal to U,. The set of U(t(r)calculated at different values of r are shown in Figure 3. Experimental Section We have used three types of samples for this investigation. The first, which served as a model system for a randomly distributed paramagnetic centers, contained the spin label 3-(2-iodoacetamido)proxil from Sigma in water/ethylene glycol (1 :1, v/v). The concentration of label in these solutions varied from 3 X 10’’ to ~ . second sample was spin-labeled tetrameric 2.4 X lo’*~ m - The hemoglobin. Hemoglobin A was prepared as previously described.I5 Spin-labeled hemoglobin A was prepared according to a modification of published procedures.16J7 A 20-fold excess (IS) Magliozzo. R. S.;McCracken, J.; Peisach. J . Biochemisrry 1987. 26, 1923.
o
loo
2cc
JCO
400
eoa
700
600
500
t i rs
Figure 3. Signal kinetics Li(fJr)at various intrapair separation r a n d fixed pulsed parameters: tp(*) = = 16 ns, h , ( * ) / h , ( j=) 1; 8, = 0 . 6 5 ~T, = 800 ns. Curve 1, r = 28, 2-34, 3-40 A.
fi3)
of 3-(2-iodoacetamido)proxilwas added to a solution of carbomonoxy hemoglobin A in 10 mM potassium phosphate buffer, pH 7.8. The solution was allowed to stand in air at 4 OC for 63 h. Unreacted spin label was removed by chromatography on Sephadex G-25 at pH 7.8. The labeled hemoglobin was rechromatographed on CM-cellulose at pH 7.8. The purified labeled hemoglobin was concentrated and saturated with carbon monoxide immediately before transferring to EPR tubes and freezing in liquid nitrogen. Samples for ESE measurements contained 2.54 mM heme (0.64 mM tetramer). The spin concentration was found to be 1.33 f 0.13 mM by ESR, based on a comparison with a solution of spin label of known concentration. The third sample was spin-labeled hemoglobin in 50% (v/v) ethylene glycol/water. The concentration of heme and spin-label samples was the same as above. ESR measurements were performed at 77 K using a Varian model E-12 spectrometer. The ESR spectra of the spin label were typical for nitroxide radicals in frozen solution.’* However, the line intensity ratio of ESR spectra of labeled hemoglobin was slightly different (, 2, 8 X 10” cm-3, 3, 2.4 X 10l8cm-3.
1171 Figure 6. Moduli of slopes of the curves shown in Figure 5 vs 171. PC concentration,calculated from the tangent of the slope (eq 2), equals 2.7 x ~m-~. 1
mn
I
/ I
t
1.400
4
1.200
/ ...:
0.600
’.....
c
3
c
-0.800 0
200 400 600 800 1000 1200 1400 1600 1800 2000 2200 t /ns
Figure 5. Logarithm of the signal kinetics U ( t ) for spin labels (C = 2.4 X 10l8cm-’) in frozen water/ethylene glycol solution at various Bo. t i 2 ) = t k 3 ) = 16 ns, hl(z)/hl(3)= 1. Curve 1, Bo = 0.8s. The variation of microwave power of following curves is 2, 1 dB, 3, 2 dB, 4, 6 dB, 5, 12 dB, 6, 15 dB.
the same pulse durations the primary ESE signal has a typical antisymmetrical shape for do = A, which made it possible to determine h, with an accuracy of 2%. Further hl variations were induced by a calibrated attenuator fixed between the microwave source (traveling wave tube) and cavity. The signal-to-noise ratio was improved by signal averaging. The amount of signal accumulation was chosen to achieve an accuracy in measurements equal to I-2%. Such accuracy allows the final effect of the dipoledipole intrapair interaction to be determined with an error of 10-15%. The measurements were performed between 32 and 700 ns for spin-labeled hemoglobin samples and approximately 2200 ns for spin labels in water/ethylene glycol solutions.
Data Analysis and Results We have studied the spin echo signal kinetics of a spin label in a frozen glass. Figure 4 depicts the dependence of the logarithm of ESE signal amplitude on t . The three curves correspond to three different radical concentrations and a constant value of 9. The set of data was obtained at t i 1 ) = t i 3 ) = 32 ns, tJ2) = 16 ns, and h l ( 2 ) / h l (=3 )3. As seen in Figure 4, in accordance with expression 2, the logarithm of the signal amplitude depends linearly on 1, and the slopes are proportional to the radical concentration. The concentration of radicals calcualated from the tangent of slopes using expression 2 agreed well with that calculated based on sample preparation. Analogous measurements were performed using the second set of pulse parameters (Le., using pulses of equal duration and amplitude). Figure 5 depicts In U ( t ) at different pulse amplitudes. It can be seen that the ESE signal either decays or increases at different h, depending on the sign of 7 (see Figure 2). The dependence of the slopes of the curves shown in Figure (20) Umanscii, B.; Raitsimring, A.; Salichov, K . Fiz. Tuerdogo Tela 1974, 16, 756.
ma......__
_.
__
,
0
100
200
300
t
/
460
500
600
700
ns
Figure 7. Signal kinetics U ( t ) for spin-labeled hemoglobin in buffer = t J 3 ) = 16 ns, hl(z)/hl(3)= 1 . Points, solution at various Bo. ti2) experiment; solid and dashed curves, simulation. 1, Bo = 0.82*, 2,0.65*, 3,0.4r. Simulation was done in accordance with expressions 1-5’ with local concentration equal to 4 X 10” cm-) andflr), shown in Figure 9. Solid curves were calculated withflr) (2); dashed curve withf(r) (1). Curves 1 and 3 are shifted by +0.1 and -0.1, respectively.
5 on 9 is demonstrated in Figure 6 . This dependence is found to be linear. The tangent of the slope gives the dipolar broadening and the spin concentration in the sample. As in the preceding case the difference in concentration between that calculated based on sample preparation and determined by this type of analysis does not exceed 10%. Thus the experimental data obtained for the samples with randomly distributed labels demonstrated the experimental accuracy of the determination of hl and the angles of resonant spin rotation and showed the possibility of suppressing the dipoledipole interactions between spins in the bulk medium. On the basis of these experiments, we expected that in spinlabeled hemoglobin samples the decay induced by the bulk spin interactions would be comparably small in the case when pulse parameters were adjusted for maximal expression of dipoledipole intrapair interaction, because the average spin concentration was only 8 X IO” ~ m - ~However . the first experiments showed that the ESE signal decay under this experimental condition essentially exceeded the expected one. It meant that at the time of sample freezing the protein molecules did not distribute randomly, thereby producing aggregates with high local spin concentrations. Our estimation showed that the local concentration of spins was ap~ . same spin-labeled hemoglobin proximately (3-5) X 10I8~ m - The which was prepared in a water/ethylene glycol mixture demonstrated a decay which approximately corresponded to the expected one for a randomly distributed system. However, as was known from the literature,2’ ethylene glycol could induce the dissociation (21) Brill, A. S.; Sandberg, H . E.; Turley, P. A. In Probes of Sirucrure and Function of Macromolecules and Membranes. VII. Probes of Enzymes and Hemoproieins; Chance, B., Yonetani, T.,Mildvan, A. S., Eds.; Academic:
New York, 1971.
3530 The Journal of Physical Chemistry, Vol. 96, No. 8, I992
Raitsimring et al.
? 0
\ A L
v L
0 7W --
24
26
28
30
32
34
36
38
40
42
44
46
48
r/A Figure 9. Spin label pair distribution function within tetrameric hemoglobin. 1, the random positioning of each label on a sphere ( r = 6 A), the distance between spheres’ centers equal to 34 A. 2, The same conditions with restriction of z coordinate by f3 A.
of tetramer into dimers; therefore the loss of information about intrapair distribution might occur here as well. these kinetics to obtain information about the spatial distribution We were therefore faced with a dilemma: in samples where of the pair of labels in the tetrameric molecule. tetramers existed, the random distribution was unachievable, and, on the other hand, when a random distribution of spin-labeled Discussion hemoglobin was attained, protein dissociation took place. The The methods of analyzing spin echo kinetics to obtain the solution of this problem was to perform experiments using spinintrapair distribution function were developed previously? In this labeled hemoglobin samples in aqueous buffer, while suppressing present case we used one of them, the so-called solution of the the bulk dipoledipole interaction. direct problem,23which is performed in the following way. Based Figure 7 shows the ESE signal kinetics, measured with pulses on some structural model, the distribution functionflr) is found, of equal duration and different values of hi. Curve 2 of this figure and then the spin echo kinetics are simulated and compared with corresponds to pulse parameters, 7 = 0 and U , ( f )= constant, Le., experimental ones. The spin label is known to be bound to both this curve shows the ESE signal variation due only to the intrapair @ 93 cysteine residues of the hemoglobin tetramer. The distance interaction. between the points of covalent attachment of the label to the The analogous dependencies were measured for spin-labeled polypeptide is 34 A,24and the distance from the point of bonding hemoglobin in water/ethylene glycol solutions, and one of these of the attached label to the location of the spin is 6 A. It is obvious (at 7 = 0) is shown in Figure 8 along with curve 2 from the that the label molecule cannot occupy any random position within preceding figure. The comparison of kinetics corresponding to the protein molecule due to steric barriers. However, computer intrapair interaction shows that the behaviors are similar, but the modeling showed (we have used a modeling program “Quanta, relative decay in the first case is approximately 2 times that of Polygen COT.”), that there are many ways in which the label may the second one. This means that some of the hemoglobin tetramers locate within the protein. Therefore, considering the structural dissociated into dimers and the amount of dissociated molecules constraints and taking into account experimental uncertainty, we is approximately half the total spin-labeled hemoglobin tetramer limited ourselves to very simple models. The first one assumed in the sample. a random distribution of the label on a sphere with radius of 6 As seen from Figure 7, curve 2, the maximal value of ESE A. The spatial distribution function of the labels (this is a pair signal decay is 0.22 f 0.03, which is close to U , (0.19, see Figure distribution function, flr)) was calculated by a Monte Carlo 2). The additional experimental error here was caused by the error method. This procedure is routine, and we emphasize only the in determining h , and by the ESE signal modulation. As was main points. mentioned in the Experimental Section, the accuracy of hi deThe coordinates of a point on a sphere, x , y , z , are given by termination is equal to 2%, and the dependence of q on h, in the the following: vicinity of the zero value is rather sharp. Therefore the suppression of bulk dipole-dipole interaction may not be complete. As is seen from Figure 7 the kinetics of ESR signal variation are modulated with a frequency of about 14 MHz. The modulation of ESE signal for “2 1” pulse train is determined by the same phenomena as for primary and stimulated echoes, namely, the electron-nuclear interaction. The features of modulation where y is a random number in the 0-1 interval, r is the radius. phenomena with the “2 + 1” pulse train were considered in a The distance between labels is given by rk = ( ( x k ( I )- xk(2))2+ previous where the methods of suppression of modulation Cyk(])- y k ( 2 ) )+ 2 ( z k ( I) z k ( 2 )+ 6)2)1/2, where d is the distance were shown. Under our experimental conditions, however, it was between cys sulfhydryl residues in the adjoining @-chainsof the impossible to adjust the pulses in such a way as to simultaneously hemoglobin tetramer; (1) and (2) are the label’s number. The avoid both the modulation and the bulk dipole-dipole interaction. step of the histogram of distribution was chosen to be 1 A. The The analysis of kinetics, shown in Figure 7 allows one to eshistogram accumulation was repeated until the signal-tenoise ratio timate the local concentration of paramagnetic centers in spinwas approximately 1%. labeled hemoglobin samples by means of a simulation (see DisFigure 9 depicts the distribution function obtained from these cussion). The simulated local concentration was 4 X 10l8 ~ m - ~ , calculations. Then, using expressions 4 and 5’ we calculated the approximately 5 times higher than the average concentration. ESE signal variation, which is shown with the experimental data As a result of this study the ESE signal kinetics caused only in Figure 7. The calculated curve is placed somewhat higher than by the dipole-dipole interaction between labels in spin-labeled hemoglobin was obtained. Let us now consider the analysis of
+
(22) Kurshev, V.; Astashkin, A.; Raitsimring, A. Zh. Sfrucr. Khim. 1988,
29, 73.
(23) Raitsimring, A.; Tregub, V . Chem. Phys. 1983, 77, 123. (24) Fermi, G.;Perutz, M. F.; Shaanan, B.; Fourme, R. The Protein Data
Bank: A Computer Based Archival File For Macromolecular Structures. J . Mol. Biol. 1984, 175, 159.
3531
J . Phys. Chem. 1992,96, 3531-3536 the experimental one. To obtain better agreement between these curves, we calculated a truncated distribution function with a restriction upon the position of spin on the sphere by some sphere layer. The function for which the best agreement was obtained and the kinetics calculated with this function are shown in Figure 9 and 7. Using this function we also calcuated the spin echo kinetics in Figure 7 at different h l , having varied the local concentration of spins. The best agreement between experiment and simulation was achieved at local spin concentration corresponding to 4 X 101*cm-3. The results allow us to conclude that the distance distribution of spin labels is within the limit by 28-41 A and one of the possible distribution functions may be the one shown in Figure 9, curve 2.
Conclusion In this work we have shown the features of an ESE technique applied to the investigation of the structure of proteins containing
more than a single paramagnetic center. Comparison of the previous radical spatial distribution investigation^^,^^,^^ and this one reveals that the main problem here is to prepare sample with random distribution of protein molecules without the loss of the investigated properties. However, even in those instances where protein aggregation does take place, it is possible to make distance measurements by the method set forth in this study. By reducing the protein aggregation effect the quality of the analysis would be improved.
Acknowledgment. This work was supported by N I H Grants GM-40168 and RR-02583 to J.P. We thank Prof. L. Kevan and Drs. R. S. Magliozzo, M. Colaneri, and C. Bender for their constant help and valuable discussions. A.R. is very grateful to Feng Jiang and H. McManus for their help in solving computer problems. Registry No. Carbomonoxy hemoglobin A, 9072-24-6; cysteine, 5290-4; 3-(2-iodoacetamido)proxil, 27048-01-7.
Simultaneous EPR and Electrochemical Measurements on Polyaniiine in Ambient Temperature Molten Salts J. Tang, R. D. Allendoerfer, and R. A. Osteryoung* Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14214 (Received: October 9, 1991; In Final Form: December 16, 1991)
Simultaneous EPR and electrochemical measurements have been carried out on polyaniline (PAn) prepared by monomer oxidation in an acidic aqueous solution and investigated in an ambient temperature ionic liquid, which consists of a mixture chloride. The maximum EPR response was found at the point where of aluminum chloride and 1-methyl-3-ethylimidazolium half the total observed charge had been passed in both cyclic voltammetry and potential step experiments. A one-to-one relationship between the number of spins observed and the number of electrons removed was found to about 25% of full oxidation. Experiments are explained in terms of two unresolved one-electron steps, with a thermodynamiccomproportionation equilibrium among the neutral, polaron, and bipolaron states. The equilibrium constant K,, of the reduced form, a,and of the oxidized form, j3, changes with the conductivity and ionic environment of the film. The bipolaron is favored in the initial doping process, and the polaron is dominant in the final doping stage. The EPR response of the polaron decays with a half-life between 8 and 17 s.
Introduction There has been considerable interest in the electrochemical behavior of polyaniline (PAn) in recent y e a r ~ . l - ~One view of the first oxidation peak on the cyclic voltammogram of PAn in acidic aqueous solution is that it involves a single reversible one-electron step," followed by chemical steps such as dimerization and disproportionation. Another view is that the first peak results from two unresolved one-electron steps,*" followed by thermodynamic equilibrium (1) Diaz, A. F.;Logan, J. A. J. Elecfroanal. Chem. 1980, 111, 1 1 1. (2)Genies, E. M.;Tsintavis, C . J. Elecrroanal. Chem. 1986, 200, 127. (3) Kobayashi. T.; Yoneyama. H.; Tamura, H.J. Elecfroanal. Chem. 1984, 177, 281. (4)Stilwell, D. E.;Park, S. M. J. Elecfrochem. SOC.1989, 136, 427. ( 5 ) Cushman. R.J.: McManus. P. M.: Yann. S. C. J. Elecrroanal. Chem. 1986,. 291, 335. ( 6 ) Focke, W. W.; Wnek, G. E. J. Elecfroanal. Chem. 1988, 256, 343. (7) Watanabe, A.; Mori, K.;Iwasaki. Y.; Nakamura, Y.; Niizuma, S. Macromolecules 1987, 20, 1793. (8) Chance, R. R.;Burdeaux, D. S.;Wolf, J. R.; Shacklette, L. W.; Silky, R.; Themans, B.; Andre, J. M.; Bredas, J. L. Synfh. Mer. 1986, I S , 105. (9)Genies, E. M.; Lapkowski, M. Synfh. M e f . 1988, 24, 61.
-
0022-365419212096-3531$03.00/0
among Ro,R I , and R2, which can be described as
Ro 3 RI'+ + eRl'+ s R;+ Ro
+ e-
El0
(1)
Ez0
(2)
+ R2*+ ~t 2R1'+
(3) where E l 0 and E2' are the standard potentials of the first and second oxidation step, respectively. Most workers treat the PAn film as uniform; that is, it is assumed to exist in one stable conformation regardless of its oxidation or reduction state. Recently, however, Albery and co-workers" have suggested that the PAn film can exist in two forms, a and 6. The a form is stable at reducing potentials, but is metastable once the film becomes conducting. The j3 form is the stable conformation when the polymer is conductive. The charged species in most conducting polymers are polarons or bipolar on^.^^.'^ In many polymers polarons are dominant (10)Lapkowski, M.; Genies, E. M. J. Elecfroanal.Chem. 1990,284, 127. ( I I ) A l k r y , W. J.; Chen, Z.; Horrocks, B. R.; Mount, A. R.; Wilson, P. Bloor, D.; Monkman, A. T.; Elliott, C. M. Faraday Discuss. Chem. SOC. 1989, 88, 247.
J.;
0 1992 American Chemical Society
