BBM 00710
Heinz-Jiirgen Steinhoff
lnstitut ]'fir Biophysik, Ruhr-Universitiit Bochum, 4630 Bochurn, F,R. G.
Summary
A method using nitroxide radical spin labels for determining both the isotropic rotational correlation
time ZR and the environmental polarity of the label is described. By means of a least square fitting
method, the values of an effective hyperfine tensor A' and of an effective g value tensor g ' of randomly
oriented spin labels are determined from X-band EPR spectra on the basis of an effective time-indepen-
dent Hamihordan. The traces of the tensors deliver the information about the environmental polarity of
the label and are not dependent on the rotational correlation time "rR. A new averaging parameter S
(~'R), calculated on the basis of the principal values of the tensor A', permits the evaluation of the
rotational correlation time z R in a very wide time range between 1 0 10 and 1 0 6 s. An application of
this method to spin-labeled methemoglobin over a large temperature range and in environments of
different polarity is discussed.
Introduction
The sensitivity of spin-label spectra to the rate of nitroxide rotational motion has
long been known [1,2]. Therefore, if a spin label can rigidly bind to a macromolc-
cule, its electronic paramagnetic resonance (EPR) spectrum should directly reflect
Correspondence address: H.-J. Steinhoff, Institut fi.ir Biophysik, Ruhr-Universit~it Bochum, 4630 Bochum.
F.R.G.
the motion of the macromolecule itself or some residual motion (librational motion)
of the label with respect to the whole macromolecule [3-5]. Rotational correlation
times ri~ for isotropic motions of slow-tumbling spin labels or spin-labeled biopoly-
mers have been determined by use of appro:dmate methods as we11 as simulation
methods [2]. These approaches utilize the property' that the positions of the outer
peaks in the X-band spin-label spectra (their separation is approximately 2Azz,)
shift inward from their rigid limit position, 2Azz, if the rotational motion increases.
On the other hand, the principal values of the hyperfine tensor __Aand of the g value
tensor g of a nitroxide radical depend significar~tly on the polarity of its environ-
ment [6,7]. Since a protein possesses hydrophobic and hydrophilic regions, it must
be considered that the environment of a protein-bound spin label may change its
polarity during a temperature-induced conformational change of the protein.
Another explanation of an observed temperature dependence of the peak separation
in the spectrum ma t' be the N O ' - H X hydrogen bound formation by, the spin label
[8,9~.
A special problem has been to separate the spectral effects of nitroxide libra-
tional motion from those due to hydrogen bonding interactions or polarity' change.
This problem may' bc partially overcome if one considers not only the hypcrfine
separation 2Azz but also line-width values of the absorption spectra [4,5,13]. In the
present work it is dcmonstra:cd, that this problem of" separation may be especially
overcome through determination of thc complete tensors g and A of the spin-labeled
protein using a fast least square fitting method. In the case of polarity change of the
spin-label environment, the change of the hyperfine separation 2Azz is correlated to
the change of the hyperfine tensor (TrAy trace. On the other hand. if an observed
change of the hyperfine separation is due to librational motion of the labcl, the trace
of the tensor _A must net alter, which follows directly from the rotational invarianee
of the trace of a tensor [111.
Therefore the starting point of the separation method presented here must be the
determination of the tensors g and A also in the slow motional region. However, a
least square fitting method in the slow tumbling region (~R -- 10 7 S) on the basis of
Freeds prograln [2] is very time consuming. A ~ypicat fit with seven parameters
including ten steps will last up to 2300 s (depending on rR) on a Cyber 855,
delivering the tensors g and A and the rotational correlation time ~'R- Instead of
this, the method presented here describes the EPR spectra of tumbling spin labels
using an effective time-independent Hamiltonian. This is a drastic simplification
and its relevance to experiment and theory has to be checked very carefully. Some
points of practical interest arc to be mentioned. To ensure the uniqueness of the fits
the number of parameters is reduced to a minimum: the six principal values of the g
and _A tensor. The line width function is determined from the spectrum in an
independent step. The fitting pr(rcedure is very fast so that an implementation of the
method using a personal computer is possible.
As an example of an application of this method, the tensors g and A of the spin
1abel .. :-(1-oxvl-_. 6.6-tetramethvl-4-piperidinvl) iodoacetami-de (JAA6) bound
within methemoglobin in different environments are determined in a temperature
range between 80 and 330 K.
239
Materials
Oxyhemoglobin was prepared from fresh horse blood samples by the method of
Benesch et al. [12]. Spin labeling (JAA6) followed the procedure of McConnell et al.
[13]. The oxidation of the labeled oxyhemoglobin to methemoglobin was achieved
by addition of a threefold amount of K 3 (Fe(CN)~,). The sample was desalted by
running it through a column of Sephadex G-25. Polycrystalline samples were
prepared by mixing the solutions of labeled methb with buffered anamonium sulfate
according to the method of Perutz [14].
EPR spectra were measured on home-made X-band and K-band (22 G H z )
spectrometers equipped with a modified Oxford ESR 9 variable temperature acces-
sory. The microwave power used was 0.1 mW, the modulation frequency was 50
kHz and the modulation amplitude 0.4 10 - 4 T. The magnetic field was measured
with an A E G Telefunken N M R instrumentation. 2,2-Diphenyl-l-picrylhydrazyl
(DPPH) powder served as a g value standard ( g = 2.0037). After analog-digital
conversion, the spectra were recorded in a personal computer (CBM 8296, Com-
modore) and then transmitted to a Cyber 855 (Control Data). There the tensors g
and _A were determined as described in the following section.
Methods
where H is the external field vector, /~ the Bohr magneton, S the electron spin
operator, I the nuclear spin operator, g the electron g value tensor and /t the
electron nuclear hyperfine interaction tensor. To a good approximation [15], the
eigen values of the Hamiltonian Eq. (1) are given by
E = g ( O , ~ ) B e H M s + A ( O , c ~ ) M s M I, (2)
with
g(O,O) = g x x l z x + g v v l z y + g z z l z z ,
COS~) 2, /ZY = (sin0, sin~) 2, lzz = (cos0) 2- Ms and M l are the eigen values of Sz
and lz, respectively. Considering the selection rules Eq. (2) gives the resonance
frequencies of every" spin-label orientation in the external magnetic field. A F o r t r a n
program was written to calculate the corresponding E P R spectrum. The E P R line
positions are calculated in steps of 3 for 0 and q~. The intensity of each EPR line
of a r a n d o m distribution of spin-label orientations in the laboratory fixed coordi-
nate system is proportional so the n u m b e r of molecules d N, for which Eq. (3) holds:
TABLE 1
VALUES OF THE TENSORS g' AND A', DETERMINED IN THE X- AND K-BAND AT T = 180 K
X-band K-band
(u = 9.00 GHz) (u = 22.00 GHz)
gxx 2.0089 + 2 x 10 -4 2.0089 + 5 x 10-4
gyy 2.0064+210 4 2.0060+510-4
gzz 2.0021 + 2 x 10-4 2.0021 + 5 x 10-4
Axx/MHz 17.4 +_1.2 18.2 +2.0
Ayy/MHz 21.3 *1.2 18.6 +2.0
Az~/MHz 104.5 _+0.1 104.0 + 2.0
1/3 TrA/MHz 47.7 _+0.4 46.9 +_2.0
241
(3
I I I
I I I I
0.810 0.815 0.820 0.825
B/Testa
Fig. 1. EPR spectra of polycryst~fine methemoglobin-JAA6 at T=180 K. (a) t,o=9.0 GHz: (b)
% = 22.0 GHz. The dotted lines show the least square fittings to the experimental spectra.
The corresponding values coincide. The greater standard deviation of the values
determined from the K-band compared to those determined from the X-band is due
to a lower signal to noise ratio. This result shows that the approximation in Eq. (2)
is valid in the K-band too.
No significant change in the values of g, _A and Q was observed when we
extracted the line shape function from the h~-gh field peak instead of the low field
peak. So the dependence of the line width on M~ and g is small and the assumption
of a single line shape function for the whole EPR spectrum is justified.
Sz = A z z - 1/3 TrA'
Azz - 1 / 3 YrA ' (5)
243
2
~'T. A.]0
/.
~.rg"10
-1 i i i i i i . i l i L , ,, i , ,L,~i[ i,.
Fig. 2. The de,Aation of TrA' and Trg' from the 'true' traces of the tensors, TrA and Trg, respectively, as
a function of the correlation time ~'R for Brownian rotational diffusion. The deviations o are defined as
OTrA = (TrA' - T r A ) / T r A (X) and ol-rg= (Trg' - T r g ) / T r g (C). The curves were determined from
fittings of spectra calculated on the basis of a time-independent effective Hamiltonian to spectral
simulations using the exact stochastic Liouville method of Freed [2]. The parameters for the calculated
spectra in the notation of [2] are: L_<I6, K_<12, A x x = 7 . 4 G, Avv=6.1 G, AT,z=37.5 G,
gxx = 2.0089, gvv = 2.0063, gzz = 2.0023 and the peak-peak first derivative line width is 3.0 G.
or, i n c l u d i n g all p r i n c i p a l v a l u e s
0-9
08
07
06
0.5
0.4
0.3
0"2f
ol
determining r R values from the effective hyperfine tensor ,4' in the whole time
range from 10 -6 to ]0 -1 s.
The values of the parameters S and TrA' shown in Figs. 2 and 3 were calculated
starting the fitting with varying initial sets of g ' and A'. The fitting converged in all
cases if the initial choice of g ' and A ' corres-ponds to S values, which deviate less
than 0.3 from the final (' true-~) value of S. The initial values of TrA' and T r g ' m a y
deviate from their true values more than 20% and 2 10 -3, respectively.
Azz/MHz
o
106
10/,
/',
102
,LA
100
98
96
80 ~oo ,20 ,,o 40 ,8o ~oo 220 2,0 2~o 28o ~/K
lira
_ _ _ "b , . : i
MHz
5O
~2
I I
80 100 120 lt,0 160 180 200 220 240 260 280 T/K
Fig. 4. (a) The parameter Azz as a function of temperature for methemoglobin-JAA6, which has been
immobilized by different methods: (A) frozen in aqueous solution, (z~) crystallized in a m m o n i u m sulfate
solution of high ionic strength, (O) lyophilized. (b) The trace of the hyperfine tensor, 1 / 3 TrA, as a
function of temperature for the same samples shown in (a).
linear correlation between 1 / 3 TrA and Azz and the values of the spin label bound
to the methemoglobin molecule fit the curve well. So we conclude that the
correlated change of Azz and 1 / 3 TrA by removing the water from the methe-
moglobin-JAA6 is due to a change of the polarity of the spin-label environment. On
the other hand, the temperature dependent change of Azz of all samples is not
correlated with a change of the trace of the tensor, the trace is even independent of
temperature in the whole temperature range investigated. Therefore a temperature-
induced change of the polarity of the spin-label environment in the protein can be
246
~-Tr A
MHz
50
48
z,8
44
42
~0 I I l l I t
Fig. 5. Correlation between the trace of the hyperfme tensor, 1/3 TrA, and Azz for the spin label JAA6
in systems of different polarity, determined at 120 K: ten) butanol, (11) ethanol, (o) lyophilizcd
methemoglobin-JAA6, (e) glycerol, (zx) methemoglobin-JAA6 in aqueous solution, (zx) crystallized
methemoglobin-JAA6.
e x c l u d e d a n d t h e o n l y e x p l a n a t i o n f o r t h e a b o v e r e s u l t s is t h e e x i s t e n c e o f a n i s o -
t r o p i c m o t i o n a l f l u c t u a t i o n s o f t h e l a b e l s e v e n in t h e f r o z e n s a m p l e s . A d e t a i l e d
a n a l y s i s o f t h e s e f l u c t u a t i o n s will b e g i v e n in a s u c c e s s i v e p a p e r .
Acknowledgements
S p e c i a l t h a n k s a r e d u e to P r o f e s s o r A. R e d h a r d t f o r t h e n u m e r o u s i n s p i r i n g
c o n v e r s a t i o n s . T h e a s s i s t a n c e o f C. B a s s a r i s a n d H. G r o t t h a u s m a n n i n t h e p r e p a r a -
t i o n o f t h e m a n u s c r i p t is g r a t e f u l l y a c k n o w l e d g e d .
247
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