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The interaction of cobalt with glycerophosphoryl choline and phosphatidyl choline bilayer membranes
JOURNAL
OF MAGNETIC
31,283-293
RESONANCE
(1978)
The Interaction of Cobalt with Glycerophosphoryl Choline and Phosphatidyl Choline Bilayer Membranes A.C. MCLAUGHLIN Biology
Department,
Brookhaven
National
Laboratory,
Upton,
Long
Island,
New
York
11973
AND
R.E. RICHARDS
~.GRATHWOHLAND Biochemistry Received
Department, August
Oxford
University,
23, 1977; revised
November
Oxford,
England
22, 1977
The interaction of cobalt with glycerophosphoryl choline (a model for the polar headgroup region of phosphatidyl choline membranes) has been investigated. Because of the long TzM the effects of cobalt on the ,‘P NMR spectrum are predominantly due to the hyperfine interaction, which considerably simplifies the analysis. The frequency of the bound species, dw,, is calculated to be -1442 ppm (at 7O”C), while tM, the lifetime of the complex, is calculated to be 2.95 x IO-’ seconds (at 70°C) with an activation energy of 10.0 kcal/mole. The observed effects of the 3’P NMR spectrum are used to calculate the amount of bound cobalt-glycerophosphoryl choline complex, and a binding constant for the complex (5.1 M). It is shown that 3’P NMR is very useful for studying the binding of cobalt (as a model divalent cation) to phospholipid bilayer membranes. In particular, the technique can distinguish direct binding from electrostatic screening.
INTRODUCTION
Because of physiological ramifications, the interaction of divalent cations with model phosoholipid bilayer membranes has been extensively investigated (1). Two effects may contribute to this interaction-direct binding (which involves the insertion of some part the phospholipid molecules into the first coordination sphere of the divalent cation’) and electrostatic screening (which involves the sequestering of divalent cations into the ionic “double layer” near the surface of charged membranes (1)). Most experimental techniques do not distinguish between these two effects. However, the large hyperfine interaction of paramagnetic divalent cations with the phosphorus nucleus in phosphate groups is extremely sensitive to the direct binding of these cations. The effects of paramagnetic divalent cations on the 31P NMR spectrum of phospholipid membranes may thus be used to quantitatively study the binding of paramagnetic divalent cations to membranes. r We ignore first hydration
an “outer-sphere” complex in which sphere of the divalent cation.
no part of the phospholipid 283
molecule
is inserted
into the
OOZZ-2364178103 12-0283$02,00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
284
MCLAUGHLIN,
GRATHWOHL,
AND
RICHARDS
Because of the extremely short electron spin relaxation time, the effects of cobalt are particularly amenable to analysis. However, the quantitation of the direct binding of cobalt to phospholipids requires independent knowledge of either the frequency shift of the bound species, dohI, or the lifetime of the complex, rM. Because of experimental limitations it is difficult to determine either of these parameters for phospholipid bilayer membranes directly. We have therefore determined these parameters for the model phosphodiester compound, glycerophosphoryl choline. The applicability of these parameters to phospholipids in bilayer membranes is discussed.
MATERIALS
AND
METHODS
Glycerophosphoryl choline was obtained from Sigma Chemical Company as the cadmium adduct and was converted to the sodium salt by passage through a Chelex100 ion exchange column prepared in the sodium form. Egg-yolk lecithin (Grade I) was obtained from Lipid Products, South Nutfield, England, and was used without further purification. All other reagents were Analar grade. Two NMR spectrometers were employed in the experiments: a Bruker WH-90 and an instrument constructed in the Biochemistry Department at Oxford operating at 129 MHz on phosphorus (3). Both instruments were interfaced with Nieolet 1080 computers and were operated in the Fourier transform mode. Both spectrometers were equipped with variable-temperature accessories which maintained the temperature to & 1OC. The accuracy of the temperature calibration was k l°C. Both spectrometers employed a deuterium “lock.” Unless otherwise noted, solutions contained 0.1 M NaCl, 10 mm of Tris(hydroxymethyl)aminomethane, pH 7.5. Solutions were prepared in 99.97% deuterium oxide (Ryvan), which was used without further purification. The sonication was performed at O°C under a stream of nitrogen gas with a probetype sonicator (Dawes). Typically, a 4- to 6-min total sonication time was employed (-30% duty cycle), during which the solution became clear. The samples were sonicated in the presence of cobalt. The sonicated vesicles were centrifuged to remove titanium and the sample was divided into two aliquots for use in the two spectrometers. Since the size of the vesicles did not substantially affect the observed linewidth in the presence of cobalt, the sonicated vesicle preparation was not fractionated. The temperature variations were performed by raising the temperature by 10°C steps and, after offsetting the temperature by 5OC, lowering the temperature in 10°C steps. This procedure would have given an indication of any irreversible phenomena, such as aggregation’ or precipitation. No time-dependent effects on the linewidths nor precipitation was observed over the period of the experiments (-8 hr).
THEORY
General Considerations The effects of cobalt on groups has been treated in where +’ is the fraction of cobalt on the width of the
the 31P NMR spectrum of compounds containing phosphate detail by Shulman et al. (4). In the “dilute” case (i.e., /Q 1, phosphate groups bound to cobalt) the observed effects of phosphorus resonance, l/T,,, and the frequency shift of the
COBALT-GLYCEROPHOSPHORYL
CHOLINE
INTERACTION
285
resonance, Au,, are given by the equations (5) 1/T2,(1/T2,
‘=p T ZP
+ l/r,)
rMM(l/TzM + l/Q2
Au,=
+ Aoh
+ rMAdo; ’
A%
f (G&4
+ l)* + Au&r&’
111 El
where Aw, is the frequency shift of the 31P NMR resonance of the bound phosphate group (i.e., the phosphate group in the first coordination sphere of the cobalt), T2M is the transverse relaxation time of the 31P NMR signal of the bound phosphate group, and rM is the lifetime of the phosphate group in the first coordination sphere of the cobalt ion.
Determination of Au, The frequency shift, AoM, is usually calculated from a measurement of the observed shift, do, under conditions of fast exchange, and +’ is determined either from a knowledge of the equilibrium constant for the complex, or by using a ligand which forms a stoichiometric complex. Neither of these methods is feasible for the glycerophosphoryl choline system since the dissociation constant is unknown, but it can be assumed to be too large to form a stoichiometric complex under feasible experimental conditions. There is, however, a straightforward procedure for determining do, without independent knowledge off. Under conditions of fast exchange the equations for 1/T,, and Aw, which are valid for arbitrary /are (6) 1/T,, =flT2M
+ f (1 -f)
Au, = fdo,
h&r,,
[31 [41
The data in Fig. 2 show that the 36.4-MHz linewidth data are in fast exchange at the high temperatures. Since l/T,, is expected to be rather insensitive to frequency and, if anything, to decrease as the frequency is increased (7,8), the contribution from the two terms in Eq. [31 may be separated by the frequency dependence of the linewidth. If the second term in Eq. [3] dominates the linewidth, the observed linewidth will increase by a factor of 11.3 for an increase in frequency of a factor of 3.54 (since f < 1). The observed change is a factor of 10.0 at 6O“C. Since the high-frequency data are not completely in fast exchange (see Fig. 1) this observed ratio implies that, at 60°C, the R-CH, R--H H,i-0
-P-0-CH,--N%H,I, v
FIG. 1. The relationship of glycerophosphoryl choline to the polar headgroup region of phosphatidyl choline. When R = OH the structure is glycerophosphoryl choline; when R = long-chain fatty acid (usually about 12 to 18 carbons) the structure is phosphatidyl choline.
286
MCLAUGHLIN,
GRATHWOHL,
AND
RICHARDS
exchange term completely dominates the linewidth, i.e., that l/T,,
HI
Q AC&Z,
at both frequencies. Equations 131 and 141 then become l/T,,
=
[61
(1 -+‘) do&z,,
do, = f’AwM.
171
The ratio of the observed linewidth to the observed shift is given by the expression T;;/Aw,
= (1 -4)
do,
z,.
LB1
If a titration with cobalt is performed under these conditions, (i.e., when Eq. 181is valid) the extrapolation of the ratio T&?/Au, to zero cobalt concentration (i.e., to f = 0) gives the value of Aw, Z~ directly. Using this value of do, r,, one may calculate the value of / at any arbitrary cobalt concentration from the ratio of the observed width to the observed shift using Eq. 181. A plot of the observed shift against the calculated values of /gives do, (from Eq. 171). Simplified Equations for do, and I/T,, With the method outlined above, the value of Aw, is calculated to be 330,000 rad/sec (at 36 MHz, 70°C) and the value of rM is calculated to be 2.95 x lo-’ set at 70°C (see Results). Using these values, Eq. [51 gives l/TzM 4 33,000 set-’ at 7O’C. The temperature dependence of T,, will be governed by the temperature dependence of rs (the cobalt electronic relaxation time) (4, 9). Since the activation energy for ts, determined from the temperature dependence of the water proton relaxation time in aqueous cobalt solutions, is 1.45 kcal/mole (IO), the maximum increase in T,, through the temperature range employed in these experiments (i.e., from 0 to 7O“C) will be factor of 2. Using the apparent activation energy for r, obtained from the low-temperature region of the linewidth data, one then calculates that
throughout the entire temperature simplify to
range. Under these conditions
Eqs. [Xl and 121
[91 do,= Equations 4, AwM, and temperature determination simultaneous of/and rH.
f
AWM 1 + do2 MMz2
’
1101
191 and 1101 are two simultaneous equations in three unknown parameters, zM. Thus, the simultaneous measurement of 1/T2P and Aw, at a single and a single magnetic field strength will not, in general, allow a of all three parameters. However, if do, is independently determined, the measurement of l/T,, and Aw, does allow an unequivocal determination
COBALT-GLYCEROPHOSPHORYL
CHOLINE
287
INTERACTION
The functional form of Eqs. 191 and [lOI may be examined by two experimental means-variation of the temperature and variation of the magnetic field strength. The lifetime tM would be expected to obey a typical Arrhenius rate law
tM = FMexp (AHJRT). When +’ < 1, / would be expected to be directly proportional to the association constant for the cobalt-phosphate complex. Thus
f =f” exp (AHdR T), where AH, is the enthalpy for the binding process. Also, Aw, will be directly proportional to the magnetic field strength and inversely proportional to temperature (II). Equations [9l and [lo] should explain the observed l/T,, and do, values over a wide range of temperature at different magnetic field strengths. However, it is still impossible to use the simultaneous fit of the temperature and magnetic field dependence of l/T,, and Aw, to Eqs. [91 and [ 101 to calculate unique values of do,, rM, and ,k This is because the two equations may be considered to be functions only of the two products, rM do, and fAwM. Fitting the two equations to the experimental data gives only the values for these two products, but within these constrains, the three parameters are undetermined. However, using the independently determined value of do, (see above), one may determine unequivocal values for /and rhl. RESULTS
Cobalt Binding to Glycerophosphoryl Choline Glycerophosphoryl choline (see Fig. 1) is a simple phosphodiester compound which should be a good model for the polar headgroup region of lecithins (the predominant phospholipid in most biological membranes). Figure 2 shows the effect of cobalt on the linewidth, l/T,,, and the line shift, AwP, of the 31P NMR resonance of
0250125t
, 30
,
,
3.2
,
,
,
34
,
3-6 IO’/T
FIG. cobalt. function 0.1 M; [91 and
2. ,iP NMR linewidth, l/T,,, and line shift, Aw,, of glycerophosphoryl choline in the presence of The data were taken at two different magnetic field strengths (0, 36.4 MHz; 0, 129 MHz) as a of temperature. Glycerophosphoryl choline, 53 mM; cobalt chloride, 13.7 m&4, sodium chloride, Tris chloride, 20 mM, pH 7.5. The solid lines are calculated from the simultaneous best fit to Eqs. IlOl.
288
MCLAUGHLIN,
GRATHWOHL,
AND
RICHARDS
glycerophosphoryl choline. The data are taken at two different frequencies, 129 and 36.4 MHz, through a temperature range 5 to 65°C. Several qualitative features of the linewidth data are readily apparent. (a) At low temperatures l/T,, has a positive temperature dependence; i.e., the linewidth increases as the temperature increases. Also, there is little frequency dependence of the linewidth. Both of their properties are consistent with the classical case of slow exchange, where the linewidth may be given as
(b) At high temperatures the linewidth has a negative temperature dependence. In this region the linewidth is a very sensitive function of frequency, increasing by an order of magnitude from 36.4 to 129 MHz. Both of these properties are consistent with the classical case of fast exchange, where the linewidth is given as 1/T2r N Acolt, ‘cM. (c) The maximum value of the linewidth occurs at a substantially higher temperature for the higher frequency. This is consistent with the maximum value of l/T,, occurring at the transition from fast to slow exchange, i.e., where
The qualitative features of the shift data are also consistent with the interpretation from the linewidth data. In the low-temperature region the shift is very small at both frequencies and does not begin to increase substantially until the onset of the transition to fast exchange. Since the transition to fast exchange occurs at a lower temperature for the 36.4-MHz data, the shifts for the 36.4-MHz data are higher than the shifts for the 129-MHz data in the low-temperature region. In the high-temperature region, where the data at both frequencies are approaching fast exchange, the 129-MHz shift is substantially larger than the 36.4-MHz shift. The data in Fig. 2 can therefore be qualitatively explained as indicating a transition TABLE
1
CALCULATED PARAMETERS FOR THE COBALT-GLYCEROPHOSCHOLINE COMPLEX~ PHORYL 1.0 x lo-l3 10 kcal/mole
e, AH, PO
AH,
2.49' -4.0
A%
-1442
T Zt.4
>3
set
k&/mole
ppm (70°C) x 10-5sec
n Parameters are defined under Theory in the text. b+O is calculated for the experimental conditions given for Fig. 1. c The shift is downfield, and is measured
in
parts
per
million.
COBALT-GLYCEROPHOSPHORYL
CHOLINE
INTERACTION
289
from slow to fast exchange through the available temperature range. The data may quantitatively fit using Eqs. 191 and 1101. Using the independently determined value of dw, (see below), one may determine the values of / and rM. These values are tabulated in Table 1. A deviation of either 4 or rM by more than about 20% from the best-fit values produces a significantly poorer fit to the data. If a 1 : 1 stoichiometry for the cobalt-glycerophosphoryl choline complex is assumed, the dissociation constant for the complex is calculated to be 5.1 M at 20” C. There is a moderate activation energy for the dissociation constant (-4 kcal/mole) with the binding becoming tighter as the temperature is raised. rM has a large activation energy of 10 kcal/mole.
The Determination
of do, For a determination of Aw, from a cobalt titration using Eq. 181 (see Theory), the fast-exchange conditions must be valid. Since these conditions are most closely
FIG. 3. The observed a’P NMR linewidth, l/T,,, and line shift, dw,, for glycerophosphoryl choline as a function of cobalt chloride concentration. Glycerophosphoryl choline, 100 mM, T= 70°C, 36.4 MHz. 0, pH = 7.0 (20 mM Pipes); 0, pH = 4.6 (no buffer). The dashed lines through the data are not theoretical fits.
approached at the lowest available frequency and the highest available temperature, the cobalt titration was performed at 36.4 MHz and 70°C. That fast exchange is achieved under these conditions is shown by the negative temperature dependence and the large frequency dependence of the linewidth. From the frequency dependence of the linewidth it may be determined that l/T,, 6 Aa& r, (see Theory) and thus that Eqs. [61,[71, and 181 are valid. The l/Tzp and the Au, data for the cobalt titration are shown in Fig. 3. The ratio T&!/Aw, is plotted as a function of cobalt concentration in Fig. 4. From Eq. 181, the extrapolation of this ratio to zero cobalt concentration gives a value of Aw, 5, = 0.097 at 70°C. Using this value of do, tM, one may calculate the value of +’ at each cobalt concentration. The observed shift, AC+., is plotted against the calculated values of /‘in Fig. 5. From Eq. 171, the slope of this plot gives do, = 330,000 rads/sec (+ 10%) at 36.4 MHz, 70°C. The cobalt titration was performed at pH 4.5 and 7.5 with no significant difference between the observed data (see Fig. 5).
290
MCLAUGHLIN.
GRATHWOHL.
AND RICHARDS
(T*,.Aw,)-’
IO
41 0
'
' ' ' ' ' ' ' J ' O-2 0.4 0.6 0.8 I.0 M Cobalt
FIG. 4. The ratio of the observed linewidth, i’$, to the observed line shift, dw,, for glycerophosphoryl choline as a function of cobalt concentration. Glycerophosphoryl choline, 100 mM; T = 70°C 36.4 MHz. l , pH = 7.0 (Pipes); 0, pH = 4.6 (no buffer).
FIG. 5. The observed line shift, do+, for glycerophosphoryl choline as a function of the calculated value of { (see text). Glycerophosphoryl choline, 100 mM; T= 70°C; 36.4 MHz. 0, pH = 7.0 (Pipes);0, pH = 4.6 (no buffer).
Cobalt Binding to Phosphatidyl Choline Bilayers Figure 6 shows the effect of cobalt on the width, l/T,,, and the shift do of the 31P NMR resonance of sonicated phosphatidyl choline bilayers. The data are t!ken at two different frequencies, 36.4 and 129 MHz, over the temperature range 5 to 65OC. Qualitatively the data are remarkably similar to the glycerophosphoryl choline data
COBALT-GLYCEROPHOSPHORYL
CHOLINE
INTERACTION
291
shown in Fig. 2. In particular, the maximum values of l/7’,, come at the same temperatures for both systems. It is difficult to measure do, directly for the phosphatidyl choline bilayer, since the high cobalt concentration necessary to signitlcantly saturate the phosphate groups in the membrane (> 1 M) may disrupt the bilayer structure. For this reason we will assume that the calculated value of do, for glycerophosphoryl choline may be used as a reasonable estimate for the value of do, in the bilayer membranes. This assumption is justified for a number of different reasons. First, glycerophosphoryl choline is chemically identical to the polar headgroup region of phospholipid membranes (see Fig. l), which is the region where divalent cation binding takes place. Second, the position of the maximum in the plot of l/T*, against inverse temperature falls at the same
I”
I
0-251
, 3.0
,
, 32
,
,
,
3.4
, 3.6
IO'/T
FIG. 6. The observed linewidth, l/T,,, and line shift, dw,, for phosphatidyl choline bilayer membranes in the presence of cobalt. The data were taken at two magnetic field strengths (0,36.4 MHz; 0, 129 MHz) as a function of temperature. Phosphatidyl choline, 44 mM; cobalt chloride, 6 mM; sodium chloride, 100 mM, Tris chloride, 20 mM; pH 7.5. The solid lines were calculated from the simultaneous best fit to E&s. 191 and I 101.
temperature (30 + 5OC at 36.4 MHz) for glycerophosphoryl choline, the neutral phosphatidyl choline bilayers, and the charged phosphatidyl glycerol bilayers (data not shown). This implies that the products Ao,z, are identical (to within &40%) for all three systems. Although this could be due to a fortuitous cancellation, it is unlikely that the cancellations would be the same in all three systems. Rather, it suggests that both do, and rM are very similar for all three systems. Third, this conclusion is further strengthened by the good agreement (to within 20%) for the Aw, calculated here for the diester, glycerophosphoryl choline, and the Au, values reported for the monoester, adenosine monophosphate (4). Also, the do, values for the three phosphate groups of adenosine triphosphate differ by only 40%, with the a-phosphate Aw, very similar to that in adenosine monophosphate, and the y-phosphate Aw, insensitive to protonation (12). Thus, Aw, appears to be an intrinsic molecular parameter for the cobaltphosphate complex which is rather insensitive to the nature of the remaining ligands on the phosphate group.
292
MCLAUGHLIN,
GRATHWOHL.
AND
RICHARDS
DISCUSSION
The effects of paramagnetic divalent cations on the 31P NMR spectrum of phospholipids provide a useful method for studying the binding of these divalent cations to phospholipid bilayer membranes. Of the four common paramagnetic divalent cations, cobalt is the preferred choice. Nickel is unsatisfactory because of its very slow exchange rate (12). The choice among manganese, copper, and cobalt is determined by the different electron spin relaxation time of each cation. The electron spin relaxation times for manganese and copper are rather long (Z2), giving a i’$ which is the same order as LIw~, while the very short cobalt electron spin relaxation time gives a negligible c&. Manganese and copper thus give larger broadening effects in the 31PNMR spectra, with no apparent shift in either the fast- or the slow-exchange region. Cobalt, however, gives well-defined 31P NMR shifts in the fast-exchange region, which enables a direct measurement of the hyperfine interaction. The negligible T$, also means that the data may be fitted by the considerably simplified Eqs. [91 and [lo], rather than the cumbersome Eqs. [ 11 and I21. Cobalt binds extremely weakly to the model diester glycerophosphoryl choline (K,, = 5.1 A4 at 2O’C). As expected, no effect of pH on the binding constant was observed between 4.5 and 7.5. However, the dissociation constant decreased (i.e., the binding became tighter) as the temperature was increased. This is similar to the effect of temperature on the binding of manganese to adenosine diphosphate; but opposite to the effect of temperature on the binding of manganese to adenosine triphosphate (23). The value of 7M at 20°C (3 x 10m6 set) is an order of magnitude faster than that observed for cobalt-adenosine monophate and cobalt-adenosine triphosphate complexes (12), which is consistent with the tighter binding in the latter complexes. However, the difference of only one order of magnitude in the observed 7M for glycerophosphoryl choline and adenosine triphosphate does not account for the difference of nearly five orders of magnitude in observed dissociation constants for these two cobalt complexes (14). The most plausible explanation is that the observed 7, value for the a-, j?-, and yphosphates in the cobalt-adenosine triphosphate complex is not the lifetime of the entire cobalt-adenosine triphosphate complex. Rather, the cobalt is continually “hopping” on and off of the phosphate groups, while maintaining ligands to one or both of the remaining phosphate groups. Thus, each of the individual phosphate groups would have a short lifetime in the first coordination sphere of the cobalt (of the order of the lifetime of the cobalt-glycerophosphoryl choline complex), while the lifetime of the entire complex will be considerably longer, and the dissociation constant considerably smaller. With reasonable estimates of 7, for cobalt (-lo-r2 set (12, 8), TzM will be very similar to r,, Thus the upper limit of qh of 33,000 set-’ is consistent with the value of CA of about lo3 set-’ for the cobalt-adenosine monophate complex (4). The observed value of dw, for the phosphate diester glycerophosphoryl choline is very similar to the reported values of Aw, for cobalt phosphate crystals (4,16). This implies that glycerophosphoryl choline forms an “inner-sphere” complex with cobalt, i.e., that the phosphodiester group directly displaces water ligands from the first hydration sphere of the cobalt. Thus, although it cannot be determined if the observed hyperfine interaction is due to a contact interaction or a pseudo-contact interaction, it is clear that it arises from an interaction within the first coordination sphere complex. The effects of
COBALT-GLYCEROPHOSPHORYL
CHOLINE
INTERACTION
293
cobalt on the 31PNMR spectrum of phospholipids in bilayer membranes thus provide a good method for distinguishing differences between binding (which involves a first coordination sphere complex’) and electrostatic screening (2). The results of binding studies to bilayer membranes formed from neutral and charged phospholipids and mixtures of neutral and charged phospholipids will be reported elsewhere. ACKNOWLEDGMENT We would
like to thank
Mrs.
E. Richards
for use of the Bruker
WH-90
spectrometer.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
A. S. D. R. T. A. A. N. L. R.
II. 12. 13. II. IS.
N. H. J. R. J.
G. LEE, Progr. Biophys. Mol. Biol. 29,3 (1975). G. A. MCLAUGHLIN, G. SZABO, AND G. EISENMAN, .I. Gen. Phys. 58,667 (1971). I. HOULT AND R. E. RICHARDS, Proc. R. Sot. London A 344,311 (1975). G. SCHULMAN, H. STERNLICHT, AND B. WYLUDA, J. Chem. Phys. 43,3 116 (1965). J. SWIFT AND R. E. CONNICK,J. Chem. Phys. 37,307 (1962). C. MCLAUGHLIN AND J. S. LEIGH, J. Magn. Reson. 9,296 (1973). W. NOLLE AND L. 0. MORGAN, J. Chem. Phys. 26,642 (1957). BLOEMBERGEN AND L. 0. MORGAN, J. Chem. Phys. 34,842 (196 1). 0. MORGAN AND A. W. NOLLE, J. Chem. Phys. 31,365 (1959). A. BERNHEIM, T. H. BROWN, H. S. GUTOWSKY, AND D. E. WOESSNER, J. Chem. Phys. (1959). BLOEMBERGEN, J. Chem. Phys. 27,595 (1957). STERNLICHT, R. G. SCHULMAN, AND E. W. ANDERSON, J. Chem. Phys. 43,3 123 (1965). M. JALLON AND M. COHN, Biochim. Biophys. Acta 222,542 (1970). PHILLIPS, Chem. Rev. 66,501 (1966). M. MAYs,Phys. Rev. 108. 1090 (1957).
30,950
OF MAGNETIC
31,283-293
RESONANCE
(1978)
The Interaction of Cobalt with Glycerophosphoryl Choline and Phosphatidyl Choline Bilayer Membranes A.C. MCLAUGHLIN Biology
Department,
Brookhaven
National
Laboratory,
Upton,
Long
Island,
New
York
11973
AND
R.E. RICHARDS
~.GRATHWOHLAND Biochemistry Received
Department, August
Oxford
University,
23, 1977; revised
November
Oxford,
England
22, 1977
The interaction of cobalt with glycerophosphoryl choline (a model for the polar headgroup region of phosphatidyl choline membranes) has been investigated. Because of the long TzM the effects of cobalt on the ,‘P NMR spectrum are predominantly due to the hyperfine interaction, which considerably simplifies the analysis. The frequency of the bound species, dw,, is calculated to be -1442 ppm (at 7O”C), while tM, the lifetime of the complex, is calculated to be 2.95 x IO-’ seconds (at 70°C) with an activation energy of 10.0 kcal/mole. The observed effects of the 3’P NMR spectrum are used to calculate the amount of bound cobalt-glycerophosphoryl choline complex, and a binding constant for the complex (5.1 M). It is shown that 3’P NMR is very useful for studying the binding of cobalt (as a model divalent cation) to phospholipid bilayer membranes. In particular, the technique can distinguish direct binding from electrostatic screening.
INTRODUCTION
Because of physiological ramifications, the interaction of divalent cations with model phosoholipid bilayer membranes has been extensively investigated (1). Two effects may contribute to this interaction-direct binding (which involves the insertion of some part the phospholipid molecules into the first coordination sphere of the divalent cation’) and electrostatic screening (which involves the sequestering of divalent cations into the ionic “double layer” near the surface of charged membranes (1)). Most experimental techniques do not distinguish between these two effects. However, the large hyperfine interaction of paramagnetic divalent cations with the phosphorus nucleus in phosphate groups is extremely sensitive to the direct binding of these cations. The effects of paramagnetic divalent cations on the 31P NMR spectrum of phospholipid membranes may thus be used to quantitatively study the binding of paramagnetic divalent cations to membranes. r We ignore first hydration
an “outer-sphere” complex in which sphere of the divalent cation.
no part of the phospholipid 283
molecule
is inserted
into the
OOZZ-2364178103 12-0283$02,00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
284
MCLAUGHLIN,
GRATHWOHL,
AND
RICHARDS
Because of the extremely short electron spin relaxation time, the effects of cobalt are particularly amenable to analysis. However, the quantitation of the direct binding of cobalt to phospholipids requires independent knowledge of either the frequency shift of the bound species, dohI, or the lifetime of the complex, rM. Because of experimental limitations it is difficult to determine either of these parameters for phospholipid bilayer membranes directly. We have therefore determined these parameters for the model phosphodiester compound, glycerophosphoryl choline. The applicability of these parameters to phospholipids in bilayer membranes is discussed.
MATERIALS
AND
METHODS
Glycerophosphoryl choline was obtained from Sigma Chemical Company as the cadmium adduct and was converted to the sodium salt by passage through a Chelex100 ion exchange column prepared in the sodium form. Egg-yolk lecithin (Grade I) was obtained from Lipid Products, South Nutfield, England, and was used without further purification. All other reagents were Analar grade. Two NMR spectrometers were employed in the experiments: a Bruker WH-90 and an instrument constructed in the Biochemistry Department at Oxford operating at 129 MHz on phosphorus (3). Both instruments were interfaced with Nieolet 1080 computers and were operated in the Fourier transform mode. Both spectrometers were equipped with variable-temperature accessories which maintained the temperature to & 1OC. The accuracy of the temperature calibration was k l°C. Both spectrometers employed a deuterium “lock.” Unless otherwise noted, solutions contained 0.1 M NaCl, 10 mm of Tris(hydroxymethyl)aminomethane, pH 7.5. Solutions were prepared in 99.97% deuterium oxide (Ryvan), which was used without further purification. The sonication was performed at O°C under a stream of nitrogen gas with a probetype sonicator (Dawes). Typically, a 4- to 6-min total sonication time was employed (-30% duty cycle), during which the solution became clear. The samples were sonicated in the presence of cobalt. The sonicated vesicles were centrifuged to remove titanium and the sample was divided into two aliquots for use in the two spectrometers. Since the size of the vesicles did not substantially affect the observed linewidth in the presence of cobalt, the sonicated vesicle preparation was not fractionated. The temperature variations were performed by raising the temperature by 10°C steps and, after offsetting the temperature by 5OC, lowering the temperature in 10°C steps. This procedure would have given an indication of any irreversible phenomena, such as aggregation’ or precipitation. No time-dependent effects on the linewidths nor precipitation was observed over the period of the experiments (-8 hr).
THEORY
General Considerations The effects of cobalt on groups has been treated in where +’ is the fraction of cobalt on the width of the
the 31P NMR spectrum of compounds containing phosphate detail by Shulman et al. (4). In the “dilute” case (i.e., /Q 1, phosphate groups bound to cobalt) the observed effects of phosphorus resonance, l/T,,, and the frequency shift of the
COBALT-GLYCEROPHOSPHORYL
CHOLINE
INTERACTION
285
resonance, Au,, are given by the equations (5) 1/T2,(1/T2,
‘=p T ZP
+ l/r,)
rMM(l/TzM + l/Q2
Au,=
+ Aoh
+ rMAdo; ’
A%
f (G&4
+ l)* + Au&r&’
111 El
where Aw, is the frequency shift of the 31P NMR resonance of the bound phosphate group (i.e., the phosphate group in the first coordination sphere of the cobalt), T2M is the transverse relaxation time of the 31P NMR signal of the bound phosphate group, and rM is the lifetime of the phosphate group in the first coordination sphere of the cobalt ion.
Determination of Au, The frequency shift, AoM, is usually calculated from a measurement of the observed shift, do, under conditions of fast exchange, and +’ is determined either from a knowledge of the equilibrium constant for the complex, or by using a ligand which forms a stoichiometric complex. Neither of these methods is feasible for the glycerophosphoryl choline system since the dissociation constant is unknown, but it can be assumed to be too large to form a stoichiometric complex under feasible experimental conditions. There is, however, a straightforward procedure for determining do, without independent knowledge off. Under conditions of fast exchange the equations for 1/T,, and Aw, which are valid for arbitrary /are (6) 1/T,, =flT2M
+ f (1 -f)
Au, = fdo,
h&r,,
[31 [41
The data in Fig. 2 show that the 36.4-MHz linewidth data are in fast exchange at the high temperatures. Since l/T,, is expected to be rather insensitive to frequency and, if anything, to decrease as the frequency is increased (7,8), the contribution from the two terms in Eq. [31 may be separated by the frequency dependence of the linewidth. If the second term in Eq. [3] dominates the linewidth, the observed linewidth will increase by a factor of 11.3 for an increase in frequency of a factor of 3.54 (since f < 1). The observed change is a factor of 10.0 at 6O“C. Since the high-frequency data are not completely in fast exchange (see Fig. 1) this observed ratio implies that, at 60°C, the R-CH, R--H H,i-0
-P-0-CH,--N%H,I, v
FIG. 1. The relationship of glycerophosphoryl choline to the polar headgroup region of phosphatidyl choline. When R = OH the structure is glycerophosphoryl choline; when R = long-chain fatty acid (usually about 12 to 18 carbons) the structure is phosphatidyl choline.
286
MCLAUGHLIN,
GRATHWOHL,
AND
RICHARDS
exchange term completely dominates the linewidth, i.e., that l/T,,
HI
Q AC&Z,
at both frequencies. Equations 131 and 141 then become l/T,,
=
[61
(1 -+‘) do&z,,
do, = f’AwM.
171
The ratio of the observed linewidth to the observed shift is given by the expression T;;/Aw,
= (1 -4)
do,
z,.
LB1
If a titration with cobalt is performed under these conditions, (i.e., when Eq. 181is valid) the extrapolation of the ratio T&?/Au, to zero cobalt concentration (i.e., to f = 0) gives the value of Aw, Z~ directly. Using this value of do, r,, one may calculate the value of / at any arbitrary cobalt concentration from the ratio of the observed width to the observed shift using Eq. 181. A plot of the observed shift against the calculated values of /gives do, (from Eq. 171). Simplified Equations for do, and I/T,, With the method outlined above, the value of Aw, is calculated to be 330,000 rad/sec (at 36 MHz, 70°C) and the value of rM is calculated to be 2.95 x lo-’ set at 70°C (see Results). Using these values, Eq. [51 gives l/TzM 4 33,000 set-’ at 7O’C. The temperature dependence of T,, will be governed by the temperature dependence of rs (the cobalt electronic relaxation time) (4, 9). Since the activation energy for ts, determined from the temperature dependence of the water proton relaxation time in aqueous cobalt solutions, is 1.45 kcal/mole (IO), the maximum increase in T,, through the temperature range employed in these experiments (i.e., from 0 to 7O“C) will be factor of 2. Using the apparent activation energy for r, obtained from the low-temperature region of the linewidth data, one then calculates that
throughout the entire temperature simplify to
range. Under these conditions
Eqs. [Xl and 121
[91 do,= Equations 4, AwM, and temperature determination simultaneous of/and rH.
f
AWM 1 + do2 MMz2
’
1101
191 and 1101 are two simultaneous equations in three unknown parameters, zM. Thus, the simultaneous measurement of 1/T2P and Aw, at a single and a single magnetic field strength will not, in general, allow a of all three parameters. However, if do, is independently determined, the measurement of l/T,, and Aw, does allow an unequivocal determination
COBALT-GLYCEROPHOSPHORYL
CHOLINE
287
INTERACTION
The functional form of Eqs. 191 and [lOI may be examined by two experimental means-variation of the temperature and variation of the magnetic field strength. The lifetime tM would be expected to obey a typical Arrhenius rate law
tM = FMexp (AHJRT). When +’ < 1, / would be expected to be directly proportional to the association constant for the cobalt-phosphate complex. Thus
f =f” exp (AHdR T), where AH, is the enthalpy for the binding process. Also, Aw, will be directly proportional to the magnetic field strength and inversely proportional to temperature (II). Equations [9l and [lo] should explain the observed l/T,, and do, values over a wide range of temperature at different magnetic field strengths. However, it is still impossible to use the simultaneous fit of the temperature and magnetic field dependence of l/T,, and Aw, to Eqs. [91 and [ 101 to calculate unique values of do,, rM, and ,k This is because the two equations may be considered to be functions only of the two products, rM do, and fAwM. Fitting the two equations to the experimental data gives only the values for these two products, but within these constrains, the three parameters are undetermined. However, using the independently determined value of do, (see above), one may determine unequivocal values for /and rhl. RESULTS
Cobalt Binding to Glycerophosphoryl Choline Glycerophosphoryl choline (see Fig. 1) is a simple phosphodiester compound which should be a good model for the polar headgroup region of lecithins (the predominant phospholipid in most biological membranes). Figure 2 shows the effect of cobalt on the linewidth, l/T,,, and the line shift, AwP, of the 31P NMR resonance of
0250125t
, 30
,
,
3.2
,
,
,
34
,
3-6 IO’/T
FIG. cobalt. function 0.1 M; [91 and
2. ,iP NMR linewidth, l/T,,, and line shift, Aw,, of glycerophosphoryl choline in the presence of The data were taken at two different magnetic field strengths (0, 36.4 MHz; 0, 129 MHz) as a of temperature. Glycerophosphoryl choline, 53 mM; cobalt chloride, 13.7 m&4, sodium chloride, Tris chloride, 20 mM, pH 7.5. The solid lines are calculated from the simultaneous best fit to Eqs. IlOl.
288
MCLAUGHLIN,
GRATHWOHL,
AND
RICHARDS
glycerophosphoryl choline. The data are taken at two different frequencies, 129 and 36.4 MHz, through a temperature range 5 to 65°C. Several qualitative features of the linewidth data are readily apparent. (a) At low temperatures l/T,, has a positive temperature dependence; i.e., the linewidth increases as the temperature increases. Also, there is little frequency dependence of the linewidth. Both of their properties are consistent with the classical case of slow exchange, where the linewidth may be given as
(b) At high temperatures the linewidth has a negative temperature dependence. In this region the linewidth is a very sensitive function of frequency, increasing by an order of magnitude from 36.4 to 129 MHz. Both of these properties are consistent with the classical case of fast exchange, where the linewidth is given as 1/T2r N Acolt, ‘cM. (c) The maximum value of the linewidth occurs at a substantially higher temperature for the higher frequency. This is consistent with the maximum value of l/T,, occurring at the transition from fast to slow exchange, i.e., where
The qualitative features of the shift data are also consistent with the interpretation from the linewidth data. In the low-temperature region the shift is very small at both frequencies and does not begin to increase substantially until the onset of the transition to fast exchange. Since the transition to fast exchange occurs at a lower temperature for the 36.4-MHz data, the shifts for the 36.4-MHz data are higher than the shifts for the 129-MHz data in the low-temperature region. In the high-temperature region, where the data at both frequencies are approaching fast exchange, the 129-MHz shift is substantially larger than the 36.4-MHz shift. The data in Fig. 2 can therefore be qualitatively explained as indicating a transition TABLE
1
CALCULATED PARAMETERS FOR THE COBALT-GLYCEROPHOSCHOLINE COMPLEX~ PHORYL 1.0 x lo-l3 10 kcal/mole
e, AH, PO
AH,
2.49' -4.0
A%
-1442
T Zt.4
>3
set
k&/mole
ppm (70°C) x 10-5sec
n Parameters are defined under Theory in the text. b+O is calculated for the experimental conditions given for Fig. 1. c The shift is downfield, and is measured
in
parts
per
million.
COBALT-GLYCEROPHOSPHORYL
CHOLINE
INTERACTION
289
from slow to fast exchange through the available temperature range. The data may quantitatively fit using Eqs. 191 and 1101. Using the independently determined value of dw, (see below), one may determine the values of / and rM. These values are tabulated in Table 1. A deviation of either 4 or rM by more than about 20% from the best-fit values produces a significantly poorer fit to the data. If a 1 : 1 stoichiometry for the cobalt-glycerophosphoryl choline complex is assumed, the dissociation constant for the complex is calculated to be 5.1 M at 20” C. There is a moderate activation energy for the dissociation constant (-4 kcal/mole) with the binding becoming tighter as the temperature is raised. rM has a large activation energy of 10 kcal/mole.
The Determination
of do, For a determination of Aw, from a cobalt titration using Eq. 181 (see Theory), the fast-exchange conditions must be valid. Since these conditions are most closely
FIG. 3. The observed a’P NMR linewidth, l/T,,, and line shift, dw,, for glycerophosphoryl choline as a function of cobalt chloride concentration. Glycerophosphoryl choline, 100 mM, T= 70°C, 36.4 MHz. 0, pH = 7.0 (20 mM Pipes); 0, pH = 4.6 (no buffer). The dashed lines through the data are not theoretical fits.
approached at the lowest available frequency and the highest available temperature, the cobalt titration was performed at 36.4 MHz and 70°C. That fast exchange is achieved under these conditions is shown by the negative temperature dependence and the large frequency dependence of the linewidth. From the frequency dependence of the linewidth it may be determined that l/T,, 6 Aa& r, (see Theory) and thus that Eqs. [61,[71, and 181 are valid. The l/Tzp and the Au, data for the cobalt titration are shown in Fig. 3. The ratio T&!/Aw, is plotted as a function of cobalt concentration in Fig. 4. From Eq. 181, the extrapolation of this ratio to zero cobalt concentration gives a value of Aw, 5, = 0.097 at 70°C. Using this value of do, tM, one may calculate the value of +’ at each cobalt concentration. The observed shift, AC+., is plotted against the calculated values of /‘in Fig. 5. From Eq. 171, the slope of this plot gives do, = 330,000 rads/sec (+ 10%) at 36.4 MHz, 70°C. The cobalt titration was performed at pH 4.5 and 7.5 with no significant difference between the observed data (see Fig. 5).
290
MCLAUGHLIN.
GRATHWOHL.
AND RICHARDS
(T*,.Aw,)-’
IO
41 0
'
' ' ' ' ' ' ' J ' O-2 0.4 0.6 0.8 I.0 M Cobalt
FIG. 4. The ratio of the observed linewidth, i’$, to the observed line shift, dw,, for glycerophosphoryl choline as a function of cobalt concentration. Glycerophosphoryl choline, 100 mM; T = 70°C 36.4 MHz. l , pH = 7.0 (Pipes); 0, pH = 4.6 (no buffer).
FIG. 5. The observed line shift, do+, for glycerophosphoryl choline as a function of the calculated value of { (see text). Glycerophosphoryl choline, 100 mM; T= 70°C; 36.4 MHz. 0, pH = 7.0 (Pipes);0, pH = 4.6 (no buffer).
Cobalt Binding to Phosphatidyl Choline Bilayers Figure 6 shows the effect of cobalt on the width, l/T,,, and the shift do of the 31P NMR resonance of sonicated phosphatidyl choline bilayers. The data are t!ken at two different frequencies, 36.4 and 129 MHz, over the temperature range 5 to 65OC. Qualitatively the data are remarkably similar to the glycerophosphoryl choline data
COBALT-GLYCEROPHOSPHORYL
CHOLINE
INTERACTION
291
shown in Fig. 2. In particular, the maximum values of l/7’,, come at the same temperatures for both systems. It is difficult to measure do, directly for the phosphatidyl choline bilayer, since the high cobalt concentration necessary to signitlcantly saturate the phosphate groups in the membrane (> 1 M) may disrupt the bilayer structure. For this reason we will assume that the calculated value of do, for glycerophosphoryl choline may be used as a reasonable estimate for the value of do, in the bilayer membranes. This assumption is justified for a number of different reasons. First, glycerophosphoryl choline is chemically identical to the polar headgroup region of phospholipid membranes (see Fig. l), which is the region where divalent cation binding takes place. Second, the position of the maximum in the plot of l/T*, against inverse temperature falls at the same
I”
I
0-251
, 3.0
,
, 32
,
,
,
3.4
, 3.6
IO'/T
FIG. 6. The observed linewidth, l/T,,, and line shift, dw,, for phosphatidyl choline bilayer membranes in the presence of cobalt. The data were taken at two magnetic field strengths (0,36.4 MHz; 0, 129 MHz) as a function of temperature. Phosphatidyl choline, 44 mM; cobalt chloride, 6 mM; sodium chloride, 100 mM, Tris chloride, 20 mM; pH 7.5. The solid lines were calculated from the simultaneous best fit to E&s. 191 and I 101.
temperature (30 + 5OC at 36.4 MHz) for glycerophosphoryl choline, the neutral phosphatidyl choline bilayers, and the charged phosphatidyl glycerol bilayers (data not shown). This implies that the products Ao,z, are identical (to within &40%) for all three systems. Although this could be due to a fortuitous cancellation, it is unlikely that the cancellations would be the same in all three systems. Rather, it suggests that both do, and rM are very similar for all three systems. Third, this conclusion is further strengthened by the good agreement (to within 20%) for the Aw, calculated here for the diester, glycerophosphoryl choline, and the Au, values reported for the monoester, adenosine monophosphate (4). Also, the do, values for the three phosphate groups of adenosine triphosphate differ by only 40%, with the a-phosphate Aw, very similar to that in adenosine monophosphate, and the y-phosphate Aw, insensitive to protonation (12). Thus, Aw, appears to be an intrinsic molecular parameter for the cobaltphosphate complex which is rather insensitive to the nature of the remaining ligands on the phosphate group.
292
MCLAUGHLIN,
GRATHWOHL.
AND
RICHARDS
DISCUSSION
The effects of paramagnetic divalent cations on the 31P NMR spectrum of phospholipids provide a useful method for studying the binding of these divalent cations to phospholipid bilayer membranes. Of the four common paramagnetic divalent cations, cobalt is the preferred choice. Nickel is unsatisfactory because of its very slow exchange rate (12). The choice among manganese, copper, and cobalt is determined by the different electron spin relaxation time of each cation. The electron spin relaxation times for manganese and copper are rather long (Z2), giving a i’$ which is the same order as LIw~, while the very short cobalt electron spin relaxation time gives a negligible c&. Manganese and copper thus give larger broadening effects in the 31PNMR spectra, with no apparent shift in either the fast- or the slow-exchange region. Cobalt, however, gives well-defined 31P NMR shifts in the fast-exchange region, which enables a direct measurement of the hyperfine interaction. The negligible T$, also means that the data may be fitted by the considerably simplified Eqs. [91 and [lo], rather than the cumbersome Eqs. [ 11 and I21. Cobalt binds extremely weakly to the model diester glycerophosphoryl choline (K,, = 5.1 A4 at 2O’C). As expected, no effect of pH on the binding constant was observed between 4.5 and 7.5. However, the dissociation constant decreased (i.e., the binding became tighter) as the temperature was increased. This is similar to the effect of temperature on the binding of manganese to adenosine diphosphate; but opposite to the effect of temperature on the binding of manganese to adenosine triphosphate (23). The value of 7M at 20°C (3 x 10m6 set) is an order of magnitude faster than that observed for cobalt-adenosine monophate and cobalt-adenosine triphosphate complexes (12), which is consistent with the tighter binding in the latter complexes. However, the difference of only one order of magnitude in the observed 7M for glycerophosphoryl choline and adenosine triphosphate does not account for the difference of nearly five orders of magnitude in observed dissociation constants for these two cobalt complexes (14). The most plausible explanation is that the observed 7, value for the a-, j?-, and yphosphates in the cobalt-adenosine triphosphate complex is not the lifetime of the entire cobalt-adenosine triphosphate complex. Rather, the cobalt is continually “hopping” on and off of the phosphate groups, while maintaining ligands to one or both of the remaining phosphate groups. Thus, each of the individual phosphate groups would have a short lifetime in the first coordination sphere of the cobalt (of the order of the lifetime of the cobalt-glycerophosphoryl choline complex), while the lifetime of the entire complex will be considerably longer, and the dissociation constant considerably smaller. With reasonable estimates of 7, for cobalt (-lo-r2 set (12, 8), TzM will be very similar to r,, Thus the upper limit of qh of 33,000 set-’ is consistent with the value of CA of about lo3 set-’ for the cobalt-adenosine monophate complex (4). The observed value of dw, for the phosphate diester glycerophosphoryl choline is very similar to the reported values of Aw, for cobalt phosphate crystals (4,16). This implies that glycerophosphoryl choline forms an “inner-sphere” complex with cobalt, i.e., that the phosphodiester group directly displaces water ligands from the first hydration sphere of the cobalt. Thus, although it cannot be determined if the observed hyperfine interaction is due to a contact interaction or a pseudo-contact interaction, it is clear that it arises from an interaction within the first coordination sphere complex. The effects of
COBALT-GLYCEROPHOSPHORYL
CHOLINE
INTERACTION
293
cobalt on the 31PNMR spectrum of phospholipids in bilayer membranes thus provide a good method for distinguishing differences between binding (which involves a first coordination sphere complex’) and electrostatic screening (2). The results of binding studies to bilayer membranes formed from neutral and charged phospholipids and mixtures of neutral and charged phospholipids will be reported elsewhere. ACKNOWLEDGMENT We would
like to thank
Mrs.
E. Richards
for use of the Bruker
WH-90
spectrometer.
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30,950