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[26] Detection and quantitation of free cytochrome P-450 and cytochrome P-450 complexes by EPR spectroscopy
252
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[26]
[26] D e t e c t i o n a n d Q u a n t i t a t i o n o f F r e e C y t o c h r o m e P-450 and Cytochrome P-450 Complexes by EPR Spectroscopy By NANETTE R. ORME-JOHNSON and W. H. ORME-JOHNSON
Cytochromes P-450 in the oxidized (Fe 3÷) state exhibit electron paramagnetic resonance (EPR) spectra at low temperature, both in the free form and when bound to substrates, some intermediates and products, or certain inhibitors. The EPR 1"2 spectra fall into two categories, those arising from a high-spin heme species (S = 5/2) with absorption maxima at approximately g = 8, 4, and 1.8 and those arising from a low-spin heme species (S = 1/2) with absorption maxima at approximately g = 2.4, 2.25, and 1.9; which form is observed depends on the state of ligation of the cytochrome. Figure 1 shows high-spin and low-spin signals from P-450eam. 3 In general, free (uncomplexed) cytochrome P-450 is the low-spin state. 4 This is true for example of P-450cam from P s e u d o m o n a s p u t i d a , 3 the P-450s from liver microsomes, 5 the P450 in placental microsomes, e and the P-450 (P-45011~) from adrenal mitochondria involved in the conversion of deoxycorticosterone to corticosterone. 7 In general, the binding of amines or other nitrogen containing ligands to P-450's produces low-spin species while the binding of subtrates (or some inhibitors) produces high-spin species, s A 1 G. Palmer, this series, Vol. 10, p. 598. 2 j. A. Fee, this series, Vol. 49 [20]. 3 R. Tsai, C. A. Yu, I. C. Gunsalus, J. Peisach, W. Blumberg, W. H. Orme-Johnson, and H. Beinert, Prec. Natl. Acad. Sci. U.S,A. 66, 1157 (1970). * An apparent exception to this is the P-450 (P-450~c) isolated from adrenal mitochondria and involved in the conversion of cholesterol to pregnenolone. This cytochrome is predominantly high spin as isolated; however, the isolated preparations contain at least a stoichiometric amount of cholesterol so that this high-spin species may be the P-450~ccholesterol complex. 5 A. Y. H. Yu, K. W. Junk, and M. J. Coon, J. Biol. Chem. 44, 3714 (1969). e j. A. Canick, K. J. Ryan, N. R. Orme-Johnson, W. H. Orme-Johnson, and A. C. Brownie, Meeting of the Endocrine Society, 58th, 1976, San Francisco, Calif., p. 113. 7 C. R. Jefcoate, R. Hume, and G. S. Boyd, FEBS Lett. 9, 41 (1970). s Even when an excess of a ligand is present, there may be an equilibrium between the high-spin and low-spin forms so that measuring the amount of P-450 in the predominant spin form will not yield the total amount. However, in the cases for which this phenomenon has been quantitated, the amount in the alternate spin state is less than 10% of the total amount. Therefore, quantitation of the predominant form yields numbers that are very close indeed to the true value, and, quantitation of both spin forms gives the true value [S. G. Sligar, Biochemistry 15, 5399 (1976)].
[26]
253
DETECTION AND QUANTITATION OF CYTOCHROME P-450
79* K 245
15 ° K tge
I
2~15226 1~t911/~
450
PHE-
.IM ) y_)/ f~S----
P450 ÷ CAMPHOR W
FIG. 1. Electron paramagnetic spectra of Pseudomonas putida P-450 at 79 ° and 15°K. Top: 1.1 rnM P-450 untreated; center: after addition of 0.25 volume of a 6 mM solution of 1phenylimidazole; bottom: after addition of 0.25 volume of a 6 mM solution of D-(+)camphor. The conditions of EPR spectroscopy were at 79 ° and 15°K, respectively: microwave power, 27 and 0.27 mW; scanning rate 160 and 625 G/min; modulation amplitude 6 G and time constant 0.25 sec throughout. The amplification of the center and bottom curves, recorded at 79°K, is 1.25 times that of the top curve to correct for dilution and the amplification of the bottom curve recorded at 15°K is 3.1 times that of the others. From R. Tsai, C. A. Yu, I. C. Gunsalus, J. Peisach, W. Blumberg, W. H. Orme-Johnson, and H. Beinert, Pro('. Natl. Acad. Sci. U.S.A. 66, 1157 (1970).
few specific examples may hint at the enormous amount of information available from EPR spectra. P-450scc presents a more complicated picture when bound to substrate, postulated reaction intermediates, and product. P-450~c bound to cholesterol is high spin; bound to 20-ahydroxycholesterol or 22-R-hydroxycholesterol is low spin; bound to 20,22-dihydroxycholesterol is high spin; and bound to pregnenolone is low spin. Further it is possible to determine from the EPR spectra which of the steroids producing a low-spin complex is bound. 9 Similarly, high9 N. R. Orme-Johnson, Fed. Proc., Fed. Am. Soc. Exp. Biol. 33, 1246, Abstr. 124 (1974).
254
MICROSOMAL ELECTRON TRANSPORTAND CYT P-450
[26]
spin P-450 complexes do not all have the same g values. For example in rat adrenal mitochondria, the P-450~l,.deoxycorticosterone complex has gmnx = 7.9 while the P-450~e'cholesterol complex has gmax = 8.1, so that it is possible to monitor these two species separately in the intact organelles,l° though the spectra are overlapped and may require spectral simulation for satisfactory quantitation.
Low-Spin Heine---Detection and Quantitation EPR spectra of low-spin cytochrome P-450 are easily observed at temperatures <80°K. For any of the quantitative methods to be applied to an EPR spectrum, it is necessary that the spectrum not be saturated by the incident microwave power so that its full intensity may be observed. It is wise to check first for saturation before applying any of the rather laborious methods outlined below, l'z'n There are several methods for determining the concentration of low-spin heme (or indeed any paramagnetic species) in the EPR sample. 1. In the classical method, the double integral of the entire EPR spectrum of the low-spin heme is calculated, either manually or using a computer, and compared to the double integral of the EPR spectrum of an appropriate standard (e.g., 1 mM Cu(II) in 10 mM EDTA). L2,11 The double integral is used since EPR spectra are normally the first derivative of the microwave absorption and the spin concentration is proportional to the integrated microwave absorption. Corrections must be made for differences in temperature, incident microwave power, scan width, amplification, modulation amplitude, tube size (square of the inside diameter of the tube), and gave (transition probability). It is obviously an advantage to keep constant as many of these variables as possible; in particular it is desirable to keep the temperature and modulation amplitude the same for both samples. The concentration of low-spin heme in the sample may be calculated from Eq. (1). (a Csample -
-
Cstandard
1
1
A21 11) gave amplification ~pp ~ T M sample 1
1
-
(i)
(
A gave amplification
A2 l _ . ~ ¢ l
X/p
)
T
standar0
where C = concentration of paramagnetic species; A = value of double integral; gave [1/3(gx 2 + g 2 + gz2)]1/2; amplification = amplifier gain of ----
lOA. C. Brownie, J. Alfaho, C. R. Jefcoate, W. H. Orme-Johnson, and H. Beinert, Ann. N . Y. A c a d . Sci. 212, 344 (1973). 11M. L. Randolph, "Biological Applications of Electron Spin Resonance" (H. M. Swartz, J. R. Bolton, and D. C. Borg, eds.), p. 119. Wiley (Interscience), New York, 1972.
[26]
DETECTION AND QUANT1TATION OF CYTOCHROME P-450
255
instrument; A = width in gauss of interval used in calculating double integral; P = incident microwave power; ~b = cross-sectional area of EPR sample tube; T = sample temperature (°K); M = modulation amplitude. 2. Another primary method is that of Aasa and Viinngfird; tz this method does not require that you double-integrate the entire spectrum of the sample. It is necessary only to determine the area (i.e., the first integral) under an isolated absorption-like peak (g~ or gz not g~) and to know the g values for the species. This number is compared to the double integral of a standard via Eq. (2). Again, it is desirable to keep factors such as the temperature and modulation amplitude constant. I Tobs
,.
Csample
(gave)standard
Cstandard
(A Az
t'~-"~
1 1 1 1) A amplification X/P T ~b~ sample
1 1 1 amplification X/'ff ~ b
) standard
where Tobs = value of the single integral gave' = ~[~i(gx2 + gu2 + g 2)]1/2 + ~[~(gx + gu + gz)] T MI = f l
g Z + guZ
hv2[(l g'g21v2"~(1- g~2~1'/2 -
-~2)j
where TM~is the theoretical area under the absorption-like peak, v is the microwave frequency, /3 is the Bohr magneton, h is Planck's constant, and the other symbols are defined as for Eq. (1). 3. The third type of procedure consists simply of comparison of some feature of the low-spin P-450 spectrum (e.g., peak height of some peak, area under one peak) to the same feature of a low-spin P-450 spectrum of a sample of known low-spin heme concentration. The same sorts of corrections for differences in incident microwave power, amplification, etc., must be applied as for method 1. This procedure is particularly useful for comparing spectra of the same complex of the same P-450. One must take care when comparing spectra of different P,450 species that the gave and linewidth are closely similar. The following experiment suggests that such integrations may be rather accurate: We formed the low-spin complex of 20-ot-hydroxycholesterol with P-450~ee, and calculated the low-spin heme concentration of the sample by method 1. We also formed the reduced CO complex of the same sample and determined the absorbance at 450 nm. Comparison of these two numbers yielded a molar absorbancy of 90,000 for the reduced 12 R. Aasa and T. Viinng~rd, J. Magn. Reson. 19, 308 (1975).
256
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[26]
CO complex, in good agreement with results 13 based on other estimates of heme, for other cytochrome P-450 species.
High-Spin Heme--Detection and Quantitation EPR spectra of high-spin P-450 are not generally observed above 20°K. This species is not so easily saturable as the low-spin form, and powers in the mW region may often be used. Again it is necessary to be sure that the signal is not saturated by the microwave power used before the spectrum is quantitated. There is an additional problem, that of zerofield splitting, in the case of high-spin heme as opposed to low-spin heme. The existence of three energy levels in a S = 5/2 Kramers system, only one of which yields EPR at normal (i.e., 9 GHz) frequencies, leads to a temperature dependence in the recovery of integrated spins. This is because the energy separation between the levels (the zero-field splitting) is often comparable to the average thermal energy of the molecules at the low temperatures needed for observation of EPR. An example of the relationship between population and temperature, calculated 14 for the P-450cam.camphor complex, is given in Fig. 2. As the temperature approaches 0°K the occupation fraction of the EPR-active ground state approaches 1, while as the temperature increases, the occupation fraction approaches 1/3. At intermediate temperatures the fraction is between these limits, and comparison of a high-spin P-450 with a standard must take this into account. A satisfactory solution may be to use another cytochrome P-450 with a similar zero-field splitting, as a standard. More generally the zero-field splitting may be estimated from the temperature dependence of the signal intensity, and a plot similar to Fig. 2 constructed in a given case. Specifically, the method described for the quantitation of the low-spin heme species can be applied to high-spin hemes, if the correction for lack of recovery of spin because of zero-field splitting is made. (a) The method described by Eq. (1) is seldom used for high-spin hemes; these paramagnetic species yield EPR spectra with a large spread of g values. Such broad spectra are intrinsically more difficult to integrate accurately by method 1 than are narrower spectra. This is because much of the value of the integral comes from points that lie close to the base line, so that a large percentage of error is involved in their measurement. (b) The method of Aasa and V/innghrd given by Eq. (2) is very useful for high-spin P-450 complexes, since usually the absorption-like resonances near g = 2 and g = 8 are well isolated and thus the area is easy to evaluate. Aasa et al.15 discussed the integration 13 R. W. Estabrook and J. Werringloer, this volume [22]. 14 j. Peisach and W. Blumberg, personal communication. 15 R. Aasa, S. P. J. Albracht, K.-E. Falk, B. Lanne, and T. V~inngard, Biochirn. Biophys. Acta 422, 260 (1976).
[26]
DETECTION AND QUANTITATION OF CYTOCHROME P - 4 5 0
257
1.0 0.9
Populolion -+ I/2 level
0.8 07 E 0.6
E ~ 05 0.4
Population ± 3/z level
03 u.
0.2
0.1
I0
20 30 40 Temperature, =K
50
60
70
FIG, 2. Population of energy levels, and observed signal intensity, as a function of temperature of the EPR experiment (J. Peisach and W. Blumberg, personal communication). The populations are calculated from the Boltzmann equation for a three-level system, where the distance from the ground level (--+1/2) to the first excited state (-+3/2 level) is ll.7°K, while the distance to the second excited state (±5/2 level) is 33.3°K. The EPR signal intensity (integrated amplitude) is the population of the -+1/2 state corrected by a factor of I/T to account for the Curie law dependence of the EPR signal. These curves represent the behavior of the P-450eam system at low temperature [R. Tsai, C. A. Yu, I. C. Gunsalus, J. Peisach, W. Blumberg, W. H. Orme-Johnson, and H. Beinert, Proc. Natl. Acad. Sci. U.S.A. 66, 1157 (1970)]. The curve labeled signal intensity shows the rate of change of the EPR signal size, assuming that the linewidths do not change with temperature. Since linewidths commonly broaden with increasing temperature, the decline of the observed EPR signal with temperature will be if anything even sharper than indicated here.
o f less well r e s o l v e d high-spin h e m e spectra. T h e c o m p a r i s o n o f p e a k heights ( w h e n the linewidths are c l o s e l y similar) o r integrated p e a k areas is also useful w h e r e o n l y relative c o n c e n t r a t i o n s are n e e d e d . T h e p r o b l e m s d e s c r i b e d here m a k e it difficult to integrate high-spin h e m e signals with a n y t h i n g like the c o n f i d e n c e attainable with the low-spin cases, but relative m e a s u r e m e n t s are j u s t as a c c u r a t e in either c a s e , p r o v i d e d that g o o d t e m p e r a t u r e c o n t r o l is maintained. This is especially i m p o r t a n t with high-spin heroes, since the zero-field splitting and s t r o n g t e m p e r a t u r e d e p e n d e n c e o f relaxation m a k e the heights o f these signals v a r y c o n s i d e r a b l y m o r e s t r o n g l y t h a n the 1/T d e p e n d e n c e seen for lowspin h e m e s .
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[26]
[26] D e t e c t i o n a n d Q u a n t i t a t i o n o f F r e e C y t o c h r o m e P-450 and Cytochrome P-450 Complexes by EPR Spectroscopy By NANETTE R. ORME-JOHNSON and W. H. ORME-JOHNSON
Cytochromes P-450 in the oxidized (Fe 3÷) state exhibit electron paramagnetic resonance (EPR) spectra at low temperature, both in the free form and when bound to substrates, some intermediates and products, or certain inhibitors. The EPR 1"2 spectra fall into two categories, those arising from a high-spin heme species (S = 5/2) with absorption maxima at approximately g = 8, 4, and 1.8 and those arising from a low-spin heme species (S = 1/2) with absorption maxima at approximately g = 2.4, 2.25, and 1.9; which form is observed depends on the state of ligation of the cytochrome. Figure 1 shows high-spin and low-spin signals from P-450eam. 3 In general, free (uncomplexed) cytochrome P-450 is the low-spin state. 4 This is true for example of P-450cam from P s e u d o m o n a s p u t i d a , 3 the P-450s from liver microsomes, 5 the P450 in placental microsomes, e and the P-450 (P-45011~) from adrenal mitochondria involved in the conversion of deoxycorticosterone to corticosterone. 7 In general, the binding of amines or other nitrogen containing ligands to P-450's produces low-spin species while the binding of subtrates (or some inhibitors) produces high-spin species, s A 1 G. Palmer, this series, Vol. 10, p. 598. 2 j. A. Fee, this series, Vol. 49 [20]. 3 R. Tsai, C. A. Yu, I. C. Gunsalus, J. Peisach, W. Blumberg, W. H. Orme-Johnson, and H. Beinert, Prec. Natl. Acad. Sci. U.S,A. 66, 1157 (1970). * An apparent exception to this is the P-450 (P-450~c) isolated from adrenal mitochondria and involved in the conversion of cholesterol to pregnenolone. This cytochrome is predominantly high spin as isolated; however, the isolated preparations contain at least a stoichiometric amount of cholesterol so that this high-spin species may be the P-450~ccholesterol complex. 5 A. Y. H. Yu, K. W. Junk, and M. J. Coon, J. Biol. Chem. 44, 3714 (1969). e j. A. Canick, K. J. Ryan, N. R. Orme-Johnson, W. H. Orme-Johnson, and A. C. Brownie, Meeting of the Endocrine Society, 58th, 1976, San Francisco, Calif., p. 113. 7 C. R. Jefcoate, R. Hume, and G. S. Boyd, FEBS Lett. 9, 41 (1970). s Even when an excess of a ligand is present, there may be an equilibrium between the high-spin and low-spin forms so that measuring the amount of P-450 in the predominant spin form will not yield the total amount. However, in the cases for which this phenomenon has been quantitated, the amount in the alternate spin state is less than 10% of the total amount. Therefore, quantitation of the predominant form yields numbers that are very close indeed to the true value, and, quantitation of both spin forms gives the true value [S. G. Sligar, Biochemistry 15, 5399 (1976)].
[26]
253
DETECTION AND QUANTITATION OF CYTOCHROME P-450
79* K 245
15 ° K tge
I
2~15226 1~t911/~
450
PHE-
.IM ) y_)/ f~S----
P450 ÷ CAMPHOR W
FIG. 1. Electron paramagnetic spectra of Pseudomonas putida P-450 at 79 ° and 15°K. Top: 1.1 rnM P-450 untreated; center: after addition of 0.25 volume of a 6 mM solution of 1phenylimidazole; bottom: after addition of 0.25 volume of a 6 mM solution of D-(+)camphor. The conditions of EPR spectroscopy were at 79 ° and 15°K, respectively: microwave power, 27 and 0.27 mW; scanning rate 160 and 625 G/min; modulation amplitude 6 G and time constant 0.25 sec throughout. The amplification of the center and bottom curves, recorded at 79°K, is 1.25 times that of the top curve to correct for dilution and the amplification of the bottom curve recorded at 15°K is 3.1 times that of the others. From R. Tsai, C. A. Yu, I. C. Gunsalus, J. Peisach, W. Blumberg, W. H. Orme-Johnson, and H. Beinert, Pro('. Natl. Acad. Sci. U.S.A. 66, 1157 (1970).
few specific examples may hint at the enormous amount of information available from EPR spectra. P-450scc presents a more complicated picture when bound to substrate, postulated reaction intermediates, and product. P-450~c bound to cholesterol is high spin; bound to 20-ahydroxycholesterol or 22-R-hydroxycholesterol is low spin; bound to 20,22-dihydroxycholesterol is high spin; and bound to pregnenolone is low spin. Further it is possible to determine from the EPR spectra which of the steroids producing a low-spin complex is bound. 9 Similarly, high9 N. R. Orme-Johnson, Fed. Proc., Fed. Am. Soc. Exp. Biol. 33, 1246, Abstr. 124 (1974).
254
MICROSOMAL ELECTRON TRANSPORTAND CYT P-450
[26]
spin P-450 complexes do not all have the same g values. For example in rat adrenal mitochondria, the P-450~l,.deoxycorticosterone complex has gmnx = 7.9 while the P-450~e'cholesterol complex has gmax = 8.1, so that it is possible to monitor these two species separately in the intact organelles,l° though the spectra are overlapped and may require spectral simulation for satisfactory quantitation.
Low-Spin Heine---Detection and Quantitation EPR spectra of low-spin cytochrome P-450 are easily observed at temperatures <80°K. For any of the quantitative methods to be applied to an EPR spectrum, it is necessary that the spectrum not be saturated by the incident microwave power so that its full intensity may be observed. It is wise to check first for saturation before applying any of the rather laborious methods outlined below, l'z'n There are several methods for determining the concentration of low-spin heme (or indeed any paramagnetic species) in the EPR sample. 1. In the classical method, the double integral of the entire EPR spectrum of the low-spin heme is calculated, either manually or using a computer, and compared to the double integral of the EPR spectrum of an appropriate standard (e.g., 1 mM Cu(II) in 10 mM EDTA). L2,11 The double integral is used since EPR spectra are normally the first derivative of the microwave absorption and the spin concentration is proportional to the integrated microwave absorption. Corrections must be made for differences in temperature, incident microwave power, scan width, amplification, modulation amplitude, tube size (square of the inside diameter of the tube), and gave (transition probability). It is obviously an advantage to keep constant as many of these variables as possible; in particular it is desirable to keep the temperature and modulation amplitude the same for both samples. The concentration of low-spin heme in the sample may be calculated from Eq. (1). (a Csample -
-
Cstandard
1
1
A21 11) gave amplification ~pp ~ T M sample 1
1
-
(i)
(
A gave amplification
A2 l _ . ~ ¢ l
X/p
)
T
standar0
where C = concentration of paramagnetic species; A = value of double integral; gave [1/3(gx 2 + g 2 + gz2)]1/2; amplification = amplifier gain of ----
lOA. C. Brownie, J. Alfaho, C. R. Jefcoate, W. H. Orme-Johnson, and H. Beinert, Ann. N . Y. A c a d . Sci. 212, 344 (1973). 11M. L. Randolph, "Biological Applications of Electron Spin Resonance" (H. M. Swartz, J. R. Bolton, and D. C. Borg, eds.), p. 119. Wiley (Interscience), New York, 1972.
[26]
DETECTION AND QUANT1TATION OF CYTOCHROME P-450
255
instrument; A = width in gauss of interval used in calculating double integral; P = incident microwave power; ~b = cross-sectional area of EPR sample tube; T = sample temperature (°K); M = modulation amplitude. 2. Another primary method is that of Aasa and Viinngfird; tz this method does not require that you double-integrate the entire spectrum of the sample. It is necessary only to determine the area (i.e., the first integral) under an isolated absorption-like peak (g~ or gz not g~) and to know the g values for the species. This number is compared to the double integral of a standard via Eq. (2). Again, it is desirable to keep factors such as the temperature and modulation amplitude constant. I Tobs
,.
Csample
(gave)standard
Cstandard
(A Az
t'~-"~
1 1 1 1) A amplification X/P T ~b~ sample
1 1 1 amplification X/'ff ~ b
) standard
where Tobs = value of the single integral gave' = ~[~i(gx2 + gu2 + g 2)]1/2 + ~[~(gx + gu + gz)] T MI = f l
g Z + guZ
hv2[(l g'g21v2"~(1- g~2~1'/2 -
-~2)j
where TM~is the theoretical area under the absorption-like peak, v is the microwave frequency, /3 is the Bohr magneton, h is Planck's constant, and the other symbols are defined as for Eq. (1). 3. The third type of procedure consists simply of comparison of some feature of the low-spin P-450 spectrum (e.g., peak height of some peak, area under one peak) to the same feature of a low-spin P-450 spectrum of a sample of known low-spin heme concentration. The same sorts of corrections for differences in incident microwave power, amplification, etc., must be applied as for method 1. This procedure is particularly useful for comparing spectra of the same complex of the same P-450. One must take care when comparing spectra of different P,450 species that the gave and linewidth are closely similar. The following experiment suggests that such integrations may be rather accurate: We formed the low-spin complex of 20-ot-hydroxycholesterol with P-450~ee, and calculated the low-spin heme concentration of the sample by method 1. We also formed the reduced CO complex of the same sample and determined the absorbance at 450 nm. Comparison of these two numbers yielded a molar absorbancy of 90,000 for the reduced 12 R. Aasa and T. Viinng~rd, J. Magn. Reson. 19, 308 (1975).
256
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[26]
CO complex, in good agreement with results 13 based on other estimates of heme, for other cytochrome P-450 species.
High-Spin Heme--Detection and Quantitation EPR spectra of high-spin P-450 are not generally observed above 20°K. This species is not so easily saturable as the low-spin form, and powers in the mW region may often be used. Again it is necessary to be sure that the signal is not saturated by the microwave power used before the spectrum is quantitated. There is an additional problem, that of zerofield splitting, in the case of high-spin heme as opposed to low-spin heme. The existence of three energy levels in a S = 5/2 Kramers system, only one of which yields EPR at normal (i.e., 9 GHz) frequencies, leads to a temperature dependence in the recovery of integrated spins. This is because the energy separation between the levels (the zero-field splitting) is often comparable to the average thermal energy of the molecules at the low temperatures needed for observation of EPR. An example of the relationship between population and temperature, calculated 14 for the P-450cam.camphor complex, is given in Fig. 2. As the temperature approaches 0°K the occupation fraction of the EPR-active ground state approaches 1, while as the temperature increases, the occupation fraction approaches 1/3. At intermediate temperatures the fraction is between these limits, and comparison of a high-spin P-450 with a standard must take this into account. A satisfactory solution may be to use another cytochrome P-450 with a similar zero-field splitting, as a standard. More generally the zero-field splitting may be estimated from the temperature dependence of the signal intensity, and a plot similar to Fig. 2 constructed in a given case. Specifically, the method described for the quantitation of the low-spin heme species can be applied to high-spin hemes, if the correction for lack of recovery of spin because of zero-field splitting is made. (a) The method described by Eq. (1) is seldom used for high-spin hemes; these paramagnetic species yield EPR spectra with a large spread of g values. Such broad spectra are intrinsically more difficult to integrate accurately by method 1 than are narrower spectra. This is because much of the value of the integral comes from points that lie close to the base line, so that a large percentage of error is involved in their measurement. (b) The method of Aasa and V/innghrd given by Eq. (2) is very useful for high-spin P-450 complexes, since usually the absorption-like resonances near g = 2 and g = 8 are well isolated and thus the area is easy to evaluate. Aasa et al.15 discussed the integration 13 R. W. Estabrook and J. Werringloer, this volume [22]. 14 j. Peisach and W. Blumberg, personal communication. 15 R. Aasa, S. P. J. Albracht, K.-E. Falk, B. Lanne, and T. V~inngard, Biochirn. Biophys. Acta 422, 260 (1976).
[26]
DETECTION AND QUANTITATION OF CYTOCHROME P - 4 5 0
257
1.0 0.9
Populolion -+ I/2 level
0.8 07 E 0.6
E ~ 05 0.4
Population ± 3/z level
03 u.
0.2
0.1
I0
20 30 40 Temperature, =K
50
60
70
FIG, 2. Population of energy levels, and observed signal intensity, as a function of temperature of the EPR experiment (J. Peisach and W. Blumberg, personal communication). The populations are calculated from the Boltzmann equation for a three-level system, where the distance from the ground level (--+1/2) to the first excited state (-+3/2 level) is ll.7°K, while the distance to the second excited state (±5/2 level) is 33.3°K. The EPR signal intensity (integrated amplitude) is the population of the -+1/2 state corrected by a factor of I/T to account for the Curie law dependence of the EPR signal. These curves represent the behavior of the P-450eam system at low temperature [R. Tsai, C. A. Yu, I. C. Gunsalus, J. Peisach, W. Blumberg, W. H. Orme-Johnson, and H. Beinert, Proc. Natl. Acad. Sci. U.S.A. 66, 1157 (1970)]. The curve labeled signal intensity shows the rate of change of the EPR signal size, assuming that the linewidths do not change with temperature. Since linewidths commonly broaden with increasing temperature, the decline of the observed EPR signal with temperature will be if anything even sharper than indicated here.
o f less well r e s o l v e d high-spin h e m e spectra. T h e c o m p a r i s o n o f p e a k heights ( w h e n the linewidths are c l o s e l y similar) o r integrated p e a k areas is also useful w h e r e o n l y relative c o n c e n t r a t i o n s are n e e d e d . T h e p r o b l e m s d e s c r i b e d here m a k e it difficult to integrate high-spin h e m e signals with a n y t h i n g like the c o n f i d e n c e attainable with the low-spin cases, but relative m e a s u r e m e n t s are j u s t as a c c u r a t e in either c a s e , p r o v i d e d that g o o d t e m p e r a t u r e c o n t r o l is maintained. This is especially i m p o r t a n t with high-spin heroes, since the zero-field splitting and s t r o n g t e m p e r a t u r e d e p e n d e n c e o f relaxation m a k e the heights o f these signals v a r y c o n s i d e r a b l y m o r e s t r o n g l y t h a n the 1/T d e p e n d e n c e seen for lowspin h e m e s .