Spectral characterization of 15N spin labels

Chemistry and Physics of Lipids 13 (1974) 49-62 © NORTH-HOLLAND PUBLISHING COMPANY

SPECTRAL

CHARACTERIZATION

O F 15 N S P I N L A B E L S

Alec KEITH, David HORVAT and Wallace SNIPES Department of Biophysics, The Pennsylvania State University, University park, Pennsylvania 16802, USA Received November 19, 1973 Accepted February 18, 1974 Nitrocide spin labels containing 1s N have hyperfine lines at magnetic field positions different from those for 14N spin labels. This allows the use of an 14N and an l s N spin label for specific experiments where two events can be monitored at the same time. Before this technique can be used to full advantage, the spectral characteristics of 15N spin labels under various conditions must be known. This report describes, in a comparative way, the effects of restricted rotational motion and nitroxide-nitroxide interactions on 15N.Tempon ~ and the corresponding 14N spin label. An interesting feature is that 15N extends the useful range of motion of spin labels to slower tumbling rates than those conveniently handled with 1 4 ,N. A general description of line shape changes due to spin label concentration effects is made without resorting to formalism.

I. Introduction Studies using spin labels can potentially yield information directly about molecular rotational velocity, magnetic dipole interactions, electron exchange interactions, and solvent interactions with the spin label. Indirect information can also be obtained about molecular translational velocity, such inferred parameters as Arrhenius activation energies, distances between spin labels or the spin label and some other paramagnetic species, and partitioning of the spin label into local environments having different properties. In the biological experiment these studies and the information from them may relate to non-random distribution o f molecules in specific cell localities, velocity o f diffusion of spin-labeled components, availability o f specific zones to spin-labeled molecules, and a variety o f other biological processes. In regard to the above parameters 15N spin labels can add an additional dimension to spin label studies: simultaneous observation of two different spin-labeled species in the same host matrix. This should allow considerably more flexibility in the design o f spin label experiments in multicomponent preparations. Before 15N spin labels can be generally useful for probing biological systems a description of 15 N spin label signals must be carried out. The present report describes, in comparative form with an equivalent 14N spin label, rotational motion in the fast tumbling range and concentration-dependent effects on spin label line shape.

50

A. Keith et al., Spectral characterization o f 1s N spin labels

II. Experimental The piperidine synthesis of Francis [1 ] was scaled down and modified to accommodate feasible quantities of 15NH 3. About 0.5 ml of liquid 15NH3 was added to 5 ml of cold acetone containing 1.6 g of CaC12. After stirring for four days the resulting 15 N-Tempone was extracted into ether and purified by thin-layer chromatography using diethyl ether as a solvent on a Silica Gel-G stationary phase. Our yield was very low but more careful procedures and somewhat larger quantities of 14NH3 resulted in higher yields. The structure of Tempone is shown below. J

S i

N *

4 An X-band JEOL spectrometer Model JES-ME-1X equipped with a laboratoryconstructed variable temperature unit accurate to better than + 0.5°C was used.

Ill. Rotational motion Rotational motion, characterized quantitatively by a rotational correlation time, Zc, is a primary information parameter in the existing body of spin label literature. There is a range of rotational motion over which values of r c can be determined quite readily from measurements of the widths and relative heights of the first-derivative hyperfine spectral lines. The extent of this range depends on the spectrometer:frequency employed and on the values of the g-tensor and hyperfine tensor elements. Specifically, measurements of Tc made from line widths and heights become invalid when the individual hyperfine lines begin to lose their basic line shape. Two major and quite distinct causes of this line shape distortion are apparent when the rotational motion of a nitroxide is restricted. First, the hyperfine lines may broaden to such an extent that they overlap significantly, thereby introducing some ambiguity into the measurements of widths and heights. Second, and likely more important, the individual principal spectral positions for a given hyperfine line may be expressed independently when rotational motion is severely restricted. This reduces the total area under the portion of the line from which measurements are

A. Keith et al., Spectral characterization o f 1s N spin labels

51

-50"C

:4

V oo

'~'L.21" N

'' ,

2 5 " C ~

35~__

3 5 " C ~

Fig. 1. Spectral series for 14N-Temponeand 1SN.Tempone dissolvedin glycerol. The viscosity of the solution was varied by operating at different temperatures. Various notations on the spectra are referred to in the text. These and all other spectra were recorded at X-band microwavefrequencies with the magnetic field intensity increasingfrom left to right.

52

A. Keith et al., Spectral characterization o f 1sN spin labels

being made, and therefore gives incorrect values for widths and heights. In general, both these effects are expected to be quantitatively different for 14N and 15N spin labels. In what follows we make a comparison of these effects, both as expected and as observed, for the spin label Tempone containing 14N and 15N. The ESR spectra of fig. 1 will be used for illustrating various aspects of restricted molecular motion. A. Line overlap

As a foundation for discussing the spectral features of 14N and 15N spin labels we can begin with the basic equation describing the ESR resonance condition: (1)

h u = g/3H

where h is Planck's constant, u is the spectrometer microwave frequency, g is the: spectroscopic splitting factor,/3 is the Bohr magneton, and H is the magnetic ficld intensity at which resonance occurs. When hyperfine coupling to a single nucleus exists, the ESR absorption is split into 21 + 1 lines of equal intensity, I being the nuclear spin. From equation ( 1 ) w e can derive an empirical relationship which defines the magnetic field position, FiM, of a principal hyperfine tensor line as: hu F i M = ~i ~ - M A i

(2)

where M is the nuclear quantum spin state, i is either the X, Y, or Z principal axis,

inflection

x

/

/

I

t

I gauss

f

Fig. 2. Comparison between the Lorentzian line shape (dashed) and that of 14N-Tempone taken at 10 .3 M in water (light solid). The Tempone absorption line was calculated by numerical integration of the recorded first-derivative tracing (heavy solid). Widths were adjusted to be identical at the inflection points and heights were adjusted to be identical at the peak.

A. Keith et al., Spectral characterization o f 15N spin labels

53

and gi and A i are the corresponding principal g-value and hyperfine couplings constants, respectively.* The g tensor is expected to be identical for a 14N spin label and the corresponding 15N spin label, but the A i and the allowable values of M will be different. For 14N, I = 1 so that M = - 1 , 0 , or +1, whereas for 15NI=½ and M = - ~1 or +~. Furthermore rheA i for I5N are about 1.4 times those for 14N and opposite in sign. It is this difference in magnitude which results in a smaller degree of line overlap with 15N as compared to 14N when the individual lines are of similar width for the two spin labels. In estimatiog the extent of overlap for a given hyperfine coupling and set of line widths, the shape of the hyperfine line must be known in some detail. Fig. 2 shows that the Tempone line shape departs significantly from that of a true Lorentzian line in the regions outside the inflection points. There is less area in the wings than a Lorentzian line shape would predict. Consequently, we have taken the actual Tempone line shape as a basis for calculating the extent of overlap for 14N and 15N Tempone. When line broadening is due to restricted molecular rotation, the individual hyperfine lines broaden differentially, as discussed in the next section. We have (I)

(0)

Wl:Wo:W-i 5:5:3

hl/K /

(-I) -h

== =~====

4:3:5

,,

5:4:7~7

6:5:8.~ 16 gauss ~

~6gaUSS

W i/=:Wl/t (--112) h7 ~-hl/i0 3:3 - "/~

= tl/2)

4:5 5:7.27 6:8.9 22.5 gauss

Fig. 3. Illustration of comparative line overlap for 14N and I SN. N u m b e r s to the left give the peak-to-peak linewidths for each hyperfine line. Solid bars cover the field positions between the inflection points while open bars extend to the field position at which the first-derivative line height is 10% of that at the peak. * This assumes that the g tensor and the hyperfine tensor are both diagonal in the same coordinate system.

54

A. Keith et al., Spectral characterization of iSN spin labels

taken typical line widths from spectra recorded under conditions of restricted motion and determined the field position at which the intensity has dropped to one-tenth that at the peak. This, in effect, establishes the degree of overlap at the level of 10% or greater. Fig. 3 shows a comparison of line overlap at this level for 14N and 15N. The solid lines represent the first derivative peak-to-peak widths and the open lines represent the line width position at 10% peak height. The numbers separated by colons in the margin of fig. 3 give the linewidths, in gauss, for the three 14N lines and for the two 15N lines. For the sake of comparison, comparable linewidths for the low-field and high-field lines of the two isotopes were chosen. As progressive broadening occurs it is apparent that more extensive overlap occurs between the 14N lines. Furthermore, the double overlap of the low and high-field lines with the mid-line of 14N makes partially immobilized signals difficult to analyze accurately. The line height ratio ho/h 1 is used as a relative measurement of rotational motion for 14N spin labels, or as a component in a r c expression, where h 0, h 1 are the first derivative peak-to-peak heights of the mid-field and high-field lines, respectively. We find that, with 15N, the ratio h_}/h] is a useful motion parameter and, due to the lesser degree of line overlap, can be used for slower tumbling states, either directly or in a r c expression, than is possible with 14N. This point is illustrated in fig. 4, where the line height ratios just mentioned are plotted logarithmically vs. the reciprocal of absolute temperature, for Tempone in glycerol. For 14N, the parameter ho/h_ 1 departs from linearity at about 25°C due to the strong overlap of the three hyperfine lines. Since both the low-field and high-field lines contribute to the mid-field line below 25°C, whereas only the mid-field line contributes to the high-field line, h o / h 1 has an apparent value higher than the true value. For 15N, the parameterh~/h~ is linear to about 20°C, where it starts to curve downward. In this case both hyperfine lines have overlap contributions from only one other hyperfine line, and the dominant effect appears to be the differential relative overlap contributions. In the region of overlap the high-field line contributes a less significant fraction to the low-field line than that contributed by the low-field line to the high-field line. This interpretation seems reasonable, in that the high-field line height is much less than the low-field line height, and any contribution to it from overlap will be more significant than an equivalent contribution to the larger h } An interesting and quite useful feature for 15N is that the effects of line overlap can be largely circumvented in a limited rotational range by obtaining h l/h, from I 1 . . . o --~f ~ . measurements o f ~ h _ } and ~h~, as mdmated m fig. 1 (12 C spectrum). This extends the linear range for this motion parameter to at least 10°C, as is shown by the open triangles in fig. 4. To appreciate this extension of the useful range for the motion parameter, we note that the bulk viscosity of glycerol at 10°C is about four times that at 25°C and an experiment held within the limits of biological temperatures often does not exceed this viscosity range.

A. Keith et al., Spectral characterization o f 1sN spin labels

55

*C

50

50

I0

.o

2 7: h.~o 14 h- I

._1

e

h~l/2 hi/2

I.O

I 3.1

I 3.7'

I 3.3

I 3.4

3.5

I x 10 3 0-~

Fig. 4. Line height ratios for 14N'Tempone and 1SN.Tempone in glycerol plotted on an Arrhenius plot. For lSN, the triangles are for measurements of ~h_~_/}h~_ as shown in fig. 1. B. Independent expression o f tensor elements

For a spin label tumbling isotropically the rotational correlation time is frequently calculated for 14N from the differential broadening of the hyperfine lines that occurs when tumbling is restricted and motional averaging of the anisotropies in the g tensor and hyperfine tensor is incomplete. In principal, r c can be related to the width of any hyperfine line [2], but this requires a knowledge of what portion of the line width is due to incomplete motional averaging for every condition of interest. In practice, it is usually assumed that all other sources of line broadening contribute equally to each hyperfine line, and r c is calculated from an expression such as

r e = K A B ( WA - WB)

(3)

where the widths of any two hyperfine lines may be used. The appropriate value of KAB depends on the spectrometer frequency, the principal values of the g tensor, and the choice of hyperfine lines to be used. From equation (3) a more familiar expression for r c is readily obtained by assuming the line shapes to be Lorentzian, such that the integrated intensity (/) is proportional to W2h. Equation (3) then becomes

56

A. Keith et al., Spectral characterization o f i sN spin labels ~4N (M = I,O,-t)

I

I

I

M

.11i

F ,M

I I I

M

I

I

15N (M = -~- , ~)

I

I I I

14

I I

FZ ,M F'y, M

F

X,M

50 Gauss

Fig. 5. Field positions for the principal tensor elements of 14N-Tempone and 1SN.Tempone, calculated from the single crystal parameters reported by Snipes et al. [6] for laN-Tempone. For 1SN.Tempone, the principal g values are assumed to be the same as and the principal hyperfine values 1.4 times those of laN-Tempone. 1

7"c = KABWB L\hA ] - t

]

(4)

so that one line width and two line heights are required. Usually, A and B are taken as the high-field and mid-field lines, respectively. Under conditions of restricted tumbling, the degree to which averaging of the individual hyperfine lines is effective depends not only on the state of motion but also on the number of gauss (or hv) over which a given nuclear spin state must average. Thus, for 14N at X-band, the spread in field position for the elements composing the mid-field line (Fx, O, Fy,o, Fz, O) is less than the spread for the low-field line (Fx, 1 , Fy,1, Fz, 1 ), which in turn is less than that for the high-field line (kx,_ 1 , Fy,_ 1 ,Fz,_l) (see fig. 5). This gives the familiar pattern of W0 < W1 < W_ 1 , or h 0 > h 1 > h 1 for 14N spin labels undergoing isotropic tumbling. Turning our attention to 15N, we see that, by way of comparison, the spread in field position for the high-field line (F x ,, F., ,, Ff,t ~) is less that that for the high-field line of 14N, so that W,_ (15N)~< (g'-~l ( N ) . This has the important consequence that an 15N spin label ~an be taken to a slower tumbling state than an 14N spin label before a given amount of line broadening results. The spectra of fig. 1 taken at 23°C and 12°C illustrate this point. The importance of this result in extending the range of motion to slower tumbling rates can be seen by the following considerations.

A. Keith et aL, Spectral characterization of 15N spin labels

57

Values of ~'c calculated from equation (4) are valid only so long as the lines being used maintain their basic shape so that accurate values of width and height can be obtained. As tumbling gets slower, averaging of the individual tensor components becomes less complete, and independent expression of the absorption of spin label molecules at distinct orientations becomes more pronounced. In particular, for 14N, the high-field line with its large spread of field positions is the first to be affected, and the z-component Fz,_ 1 is expressed independently. In fig. 1 this effect is noted by the letter c on the spectra at 12°C and 7°C. When this occurs, and frequently before the component Fz,_ 1 becomes prominent, significant area is lost from the line being measured and incorrect values of r c are obtained. The spectra of 15NTempone, by comparison, show this effect appearing at lower temperatures (b on 7°C spectrum) because the spread in field position over which averaging must take place is lesser in this case. A convenient, quantitative appraisal of the independent expression of the z-component can be made by comparing the values of W2h for the individual hyperfine lines in a spectrum. Values of r c can be considered relatively accurate so long as W2h is about the same for all lines. In fig. 1 these values are indicated for some of the spectra, and it is clear that 15N can be used for calculating r c at lower temperatures than 14N. This extended range may be quite important for some biological experiments.

IV. Spin label concentration effects in dilute solution Intermolecular interactions between spin label molecules have been used to make estimations of intermolecular distances and collision rates [3-5], and a comparison between the spectral features of 14N and 15N under such conditions seems appropriate. In relatively dilute solutions where the hyperfine components are resolvable, the absorption lines are broadened by interactions that are generally attributed to concentration-dependent magnetic dipole interactions and electron exchange. The electron-electron dipole interaction broadens each hyperfine line identically, and this effect is expected to be the same for 14N and 15N spinlabels. The spectral modifications are subject, of course, to the same line overlap considerations discussed in the previous section, due to the difference in hyperfine coupling to Ihe two isotopes. The effects of electron exchange will be somewhat more complicated, however, because this phenomenon produces both a broadening of the absorption lines and a reduction of the observable hyperfine splitting. In the extreme case, as the concentration is increased, the hyperfine lines collapse into a single line Which is positioned, for 14N, at the original mid-field line but which, for 15N, falls equidistant between the original hyperfine lines. This will give rise to qualitatively different spectral characteristics for the two isotopes in solutions where concentration-dependent interactions become prominent. Fig. 6 shows a series of spectra taken at different spin-label concentrations under

58

A. Keith et al., Spectral characterization o f 15N spin labels

C=I

, ss A , ,

-~X~;

2A.-"IN-TEMPONE

\

Fig. 6. Effects of spin-label concentration on the spectral features of 14N-Tempone and l SNTempone. Bars show the isotropic hyperfine coupling in dilute solution and illustrate the reduction in observed hyperfine splitting as electron exchange interactions appear. conditions of low viscosity. The interesting aspect, for 15 N, concerns the appearance of a central line at high concentrations in a position where no absorption occurred previously. Due to the gradual broadening and concomitant reduction of hyperfine splitting, no distinctly unique and separate "exchange line" appears as is observed in the spectra of biradicals. It should be pointed out that, in heterogeneous or biological preparations, the effect of increased concentration on line shape may be somewhat different, as local pools or aggregates of various sizes may occur. This non-uniform dispersion can lead to an "exchange line" appearing in the middle of the spectrum for etther 14N o f 15N while the outer hyperfine components are still resolvable.

A. Keith et al., Spectral characterization o f 1sN spin labels

104

I

59

I

iO ~

Area 13N A/2 / ~

IO2

/ IO

/

I I0

Mx 10.4

I 102

103

Fig. 7. A plot of Area (W2h) vs. molarity for 14N-Tempone and lSN-Tempone in water. Data are for the low-field line of each isotope and correspond to -13and ½, respectively, of the total spectral absorption for 14N and 15N.

Some quantitative aspects of the spectral broadening were investigated. As described in the previous section, the quantity W2h can be used as a sensitive indicator for changes in line shape. In fig. 7 this quantity, designated "Area", is plotted vs. spin label concentration on a log-log plot. The linearity extending up to about 0.1 M indicates that no drastic alteration in line shape occurs over this concentration range in water. It may be noted in fig. 7 that the area under each 15N hyperfine line is about 1.5 times the corresponding area for each 14N hyperfine line, because the total absorption for 15N is divided between two components while that for 14N is divided among three components. Multiplication of the data for 14N and 15N by three and two, respectively, results in coincident lines. Worth noting also is the fact that this difference in number of lines alone gives 15N an inherent sensitivity advantage of 50% over that of 14N. The degree of broadening was also investigated quantitatively by measuring the hyperfine line width (W) at different spin-label concentrations. The degree of broadening, as expected for an electron-electron dipole interaction, is the same for both 14N and 15N (see fig. 8). The exact relationship between the concentration-dependent broadening and molarity can best be seen by first subtracting the "minimum line width" that is due to concentration-independent factors. When this is done, the concentration-dependent component, indicated Wc in fig. 8, is a linear function of molarity over the range of 5 × 10 -4 molar to about 10 -1 molar.

60

A. Keith et al., Spectral characterization o f I sN spin labels

rin~ IC

w Or

255

118

55

I

I

I

26 '

W

Wc

(gouss) 0.I-

Wc 0.01

i0-~

i0 -4

10-3 Molarity

iO-Z

i0 -~

Fig. 8. Line width measurements plotted vs molarity on a log-log scale for 14N-Tempone (closed) symbols) and 1SN_Tempone (open symbols). The concentration-dependent contribution to line width, Wc (circles), was calculated by subtracting the minimum linewidth at large dilution from each of the observed data points (squares).

In an isotropic, uniformly dispersed solution the average spin label separation (r) is related to molarity by the expression r = 11.84 M L3

(5)

where r is taken between spin label centers. From this expression and the linear relationship between molarity and concentration-dependent line width, it follows that Wc -'- K / r 3

(6)

where the constant K may depend on l:actors that vary from system to system, While a general discussion of the sources contributing to Wc is beyond the scope of the present treatment, it may be noted that the dipole-dipole interaction has just the distance-dependence of equation (6).

V. Double isotope spectra

As a final point of illustration, fig. 9 shows spectra taken with approximately equimolar concentrations of 14N-Tempone and lSN-Tempone in glycerol, and indicates

A. Keith et al., Spectral characterization of 1 SN ~pin labels 14N

14N

30"C

61

151~1

I

~ / ~

NI/z

r~l/2 14L.

!

I

15

/

|

14N

Fig. 9. Spectra of a mixture of 14N-Tempone and 15N.Tempone (5 X 10 -4 M each) in glycerol taken at two temperatures. that the limiting feature on simultaneous use of both labels will ultimately be the ability to distinguish the absorptions of each individual isotope. It is apparent that this is possible at 30°C but is subject to some ambiguity at 20°C. The problem may be either easier or more difficult in biological systems where the two isotopic labels would logically have different structures and different physical and chemical properties. Careful experimetal design will be essential to achieve the full potential of this new parameter in spin label studies.

Acknowledgements This research was supported by AEC contracts AT(11-1)-2223 and AT(11-1)-2311. A.K. is also grateful to NIH (1-K4-GM70593-01).

References

[1] F. Francis, L Chem. Soc. (t927) 2897 [2] D. Kivelson, J. Chem. Phys. 33 (1960) 1094

62

[3] [4] [5] [6]

A. Keith et al., Spectral characterization of" i sN spin labels

W. Plachy and D. Kivelson, J. Chem. Phys. 47 (1967) 3312 E. Sackmann and H. Trauble, J. Amer. Chem. Soc. 94 (1972) 4492 P. Devaux and H.M. McConnell, J. Amer. Chem. Soc. 94 (1972) 4475 W. Snipes, J. Cupp, G. Cohn and A. Keith, Biophys. J. 14 (1974) 20