Eur. J. Biochem. 81, 507-514 (1977)

Determination of the Solution Conformation of Dephospho Coenzyme A by Nuclear-Magnetic-Resonance Spectroscopy with Lanthanide Probes A Method for Analysis When More Than One Complex Species Is Present G Victor FAZAKFRLFY, Peter W LINDER, and David G REID Department of Inorganic Chemistrj nnd Depdrtment of Phyw,dl Chemistry. University 01 Cape Town (Received July 26. 1977)

The aqueous solution conformation of the 1 : 1 complex of dephospho CoA bound to a lanthanide ion has been determined by examination of the dipolar shift and induced relaxation at pH 6.4. The experimental data are shown to arise from the presence of both 1 : 1 and 1 : 2 metal : ligand complexes and a graphical method is described to divide the experimental data into information corresponding to each of the two species. The formation constants are also derived. For the 1:1 complex the ribosc is found in a 2E conformation with the adenine base predominantly anti. A small contribution from a ,s.yn conformation is evident. The pantoinic acid fragment of the chain is folded back towards the pyrophosphate while the remainder of the chain is extended.

Few detailed structural studies have been reported on coenzyme A and none on dephospho coenzyme A. It has been suggested from 'H and 31PNMR coupling constant studies [1,2] that CoA and acetyl-CoA exist in a dynamic equilibrium of linear and hairpin conformations although the lack or continuity in coupling along the pantetheine chain must render conclusions about its conformation suspect. Another coupling constant study [3] on the pantetheine fragment suggests the presence of intramolecular hydrogen bonding in aqueous solution. Possible intramolecular association has also been proposed for benzoyl-CoA [4] in which stacking may take place between the benzene and adenine rings. More recently two reports [5,6] of the transcarboxylase-bound CoA structure propose a U-shape conformation for CoA. We have studied the conformation of dephospho coenzyme A using lanthanide ions as structural probes. CoA with two siles for coordination presents considerable difficulty in interprctiiig the data, a complication not present in dephospho CoA. EXPERIMENTAL PROCEDURE

NMR spectra were run on a Brukei- WH-90DS operating in the Fourier tranform mode. 'H spectra were recorded at 90 h4Hz and 31P spectra at 36.44 MH7. The internal references used were sodium 3-(triinethylsilyl)-propane sulphonate and triphenyl Ahbrewation NMR, nuclear magnetic rcsondnce

methyl phosphonium bromide respectively. Longitudinal relaxation times, T I , were determined using a 180"-~-90"pulse sequence. Dephospho coenzyme A was of the highest grade commercially available and used without further purification. Solutions of the required concentration were made up in 'Hz0 (99.8%): adjusted to pH 6.4 with NaO'H and lyophilized three times. Lanthanide nitrate solutions were prepared by dissolving the oxides (99.99 %) in nitric acid. recryslallizcd, dissolved in 2 H z 0 and adjusted to pH 6.4. Experiments were carried out at 28 "C. The calculated shifts and internuclear distances were obtained from careful model building using Dreiding models corresponding to known bond lengths from the available crystal structure data [7]. The ribose conformation was studied in all its predicted forins [S]. The metal-oxygen bond distance used was 0.23 nm found from previous studies [9] which gave the best fit of the data. The reported data are corrected for the small shifts obtained by titration with diamagnetic lanthanum nitrate. All straight lines were drawn according to a leastsquares best fit.

THEORY In thc presencc of more than one species formed between the lanthanide (M) and the ligand (L) the

Conformation of Dephoapho C o h by NMR Spectroscopy

508

observed shift (A&>)measured relative to that of free ligand is given by :

where A,, A2, A 3 etc. are the bound shifts of the ML. ML2 etc. species for a particular observed nucleus and [L], is the total ligand concentration. The concentrations of the various species can be written as. [MLJI = B,[Ml [LY

Here, we consider only systems for which @4

of the formation constants the concentrations of [ML] and [MLz] at each point on the shift graphs are determined by solution of the mass balance equations with the aid of a computer program ECCLES [12]. To produce the stripped shift curves corresponding to the separate species we need to find A I and Az. Rearranging Eqn (l),

(2)

where [MI and [L] are the free metal ion and ligand concentrations, respectively, /Yj is the formation constant of ML,. Rearranging Eqn (1) yields the general from. (3) The total metal ion concentration [MI, can be expressed as, (4)

If we define a parameter, A*,

then by combining Eqns ( 3 ) and (4),

Thus a graphical plot of the left-hand side of Eqn (8) against [ML]j[ML2] yields a slope of A l and intercept of 2 A2. All the information required to draw up thc shift curves for the separate species is available. The determination of dl and A 2 in this way also provides a useful test of the method. At high lanthanide to ligand concentrations the observed shifts will tend towards the bound shift of [ML], that is A I . The search for a conformation of the lanthanidc complexes follows the normal method. The magnitude of the pseudo-contact interaction in terms of Y, the metal-ion - observed-proton internuclear distance, 0. the angle between the magnetic anisotropy axis and the vector joining the metal ion and the observed nucleus has been given by La Mar [13],

Av -=D

N

V

1

+ ,,fPj[L]’ 3=l

83,

........ have negligible values. From the values

’

which is a function of [L]. The well known method of ‘corresponding solutions’ [lo] can be adapted as follows. A set of shift experiments is performed in which the metal ion is titrated against ligand, with the total ligand concentration [L], kept constant in each experiment but varied over a suitable range within the set. Corresponding solutions are defined as having different values of [MI, and [L], but common values of [L] and ii, where ii the ligand number is, (7) A* is plotted against [MI, generating a curve for each value of [L], examined. For common values of A * the corresponding values of [L], and [MI, are read off. A plot of the resulting values of [L], against [MI, yields a straight line of slope 6 and intercept [L]. A set of such data for each common A * is obtained. The pairs of (fi, [L]) data can now be used to determine graphically the fl values by the LedenFronaeus method [ Ill.

(3 cos20-- 1) ~~

Y3

(9)

In the case of axial symmetry where D is a constant during the experiment. Relaxation data derived from gadolinium(II1) titrations under conditions of fast chemical exchange are related by the Solomon [14] and Bloembergcn [15] equations to the metal-ion - observed-proton internuclear distance r. Under conditions which have been described previously [16] (TI is proportional to Y+. RESULTS AND DISCUSSION

Sh f t Studies The incremental addition of 1.21 M praseodymium nitrate to a 17.5 mM solution of dephospho coenzyme A (Fig. 1) at pH 6.4 shows (Fig.2) that the observed shift for some but not all of the proton resonances rises to a maximum and declines tending towards a limiting shift. This same pattern is followed in all the other lanthanide titrations. Because of line broadening and eventual precipitation at high lanthanide concentrations for most of the lanthanides employed it was usually not possible to reach thc limiting shifts. The praseodymium titration gave, for most of the observed protons, the closest approach

G. V. Fazakerley, P. W. Linder, and D. G. Reid

509 100 -

so 0 0

-

I N

Fig. 1, Structure of dephospho coenzyme A

I?

-so-

\

5

10

15

[Pr3+]( m M )

Fig. 2. Obsewedsklff of dephospho Con (17.5 m M )protoizs. (a)H-8; (A)H-2, (M)H-1' (adenowx). (v)11-5, 5" (adenosine)

Fig 3 Shlft of adenosine H-8proton at various total hgand concentrations (0) 1 1 m M , (+) 5 5 mM, (A) 8 3 m M , (m) 17 5 m M , (0)37 mM

to the limiting shifts. The most plausible explanation for the reversal in direction of the shifts lies in the presence of more than one lanthanide-ligand complex in solution and where the bound shifts for the species are significantly different. Before attempting to interpret these data the question of whether the shifts arise from complexes possessing axial or rhombic magnetic symmetry must be examined. Experiments were carried out with five lanthanides, Eu3+, Pr3', Dy3+, Ho3' and La3+, the last to correct the data for diamagnetic shifts. For the paramagnetic ions the shift pattern is essentially the same. Normalizing on H-8, the shift ratios at points on the titration curves remain constant to f 15%. This approach to prove axial symmetry can only be used if (a) the formation constants for the different lanthanides are fairly similar and (b) the relative magnitudes of the stepwise formation consLants ( K I

to K2 in this case) are also similar. The lanthanide series acetylacetonate complexes have been studied by potentiometry 117,181 and show (a> similar values of K, tending to higher values passing across the lanthanide series and (b) good agreement for the ratio Kl: Kz. Thus the offset of the shift maximum between, for example, P?+ and Eu3+ agrecs with the expected higher formation constant for Eu3' and the error in the shift ratios at each point on the titration curves, though higher than the normal k 10% observed, is adequately explained by the non-exact agreement in the ratio of Kl : Kz for the two metalion complexes. We thus conclude that the complexes exhibit axial magnetic symmetry. The following discussion is based upon the results obtained for praseodymium shifts. Experiments were carried out over a wide range of ligand concentration (1.1 - 37 mM), the upper limit being below that at

510

Conformatioil of Dephospho CoA by NMR Spectroscopy

\

h

\

08

100

06

_.

N

1

-e

b

50

N

04

I

0

02

0 5

0

10 I P r 3 + ](mM)

15

-50

Fig. 4. Plot of A* ngninst [Pr' ' 1 u t rnrious totcd ligand conccnrrntion t~ give 'corresponding solutions'. (A) 1.1 mM: (B) 3.7 mM; (C) 5.5 m M ; (D) 8.3 mM: (E) 21.2 m M ; (F) 17.5 mM

300

to calculate the formation constants. This yields a value o f K l = 3 . 1 x 1031.n101~iand j32=6.5x10612.mol-2 ( K z = 2.1 x lo3 1 inol-') and shows that K3 is negligible. The formation constants allow calculation, using ECCLES [12], of the concentration of each of the species present at each point of the titration curve for a particular total ligand concentration. Knowing 4 " b h of each of these points for each observed proton plotting the left-hand side of Eqn (8) against [ML]/ [ML2] (Fig.5) yields lhe values of 41 and 4 2 for each proton. These values are shown in Table 1. The calculatcd values of d l and the observed limiting shifts in the praseodymium titrations are in excellent agreement. The titration curves can now be redrawn in terms of the shift of each of the species (Fig. 6). 8

0

0

Rehxution Sludies Fig.5. Cdciritrricwi of rlw h o t r i d .slii/fs. The slope is ALII and the intercept ~ A M L , (A) . H-8; (+I H-2; (W)Ii-l'(adenosine): (r) CH3 (2)

which intermolecular base stacking occurs. At the highest concentration of ligand it is not possible to titrale lo the region where [ML] predominates (Fig. 3) before precipitation of the hydroxide occurs. The observed shifts are replotted in the form of A* (Fig. 4). For a set of A* values the corresponding values of [MI, and [L], are read off. For each A* chosen the six pairs of data ([MI,, [L],) are plotted and give straight lines of slope ii and intercept free ligand concentration [L]. The resulting pairs ( ~[L]) , are manipulated [I I ]

I t is not possible to divide satisfactorily relaxation measurements which correspond to a weighted average of ML and ML2 species. Rather it is necessary to carry out the experiments under conditions where one of the two species is dominant. At very high lanthanide concentrations the concentration of ML2 declines. Calculations show thai for a total ligand concentration of 0.03 M in the presence of 0.5 M lanthanide more than 97 ?(,of the ligand is bound as ML. The relaxation experiment to providc information on the conformation of ML was thus carried out by titrating lhc ligand in the presence of0.5 M lanthanumnitrale wilh 0.001 M gadolinium nilrate. The observed values of are shown in Table 1. (7-1

51 1

G. V . FaLaherley. P. W-. Lmder. and D. G . Reid

Table 1. Bound shifts &rived from Iiruseor~~r~iiuw?iIII) tifru(ions ond ohserved reluxufion durci ,/ram girrk~li~iun7 / I I I ) tirr.utions ( T I ~ I )values -~ were those observed at 0.03 M dephospho coeiizyme A concentration in the prcsence of 0.50 M lantl~anuninitrate Molecule

Atom

n1

iTi M)- ’

A2

IIL Adenosine

Pdntetheme

KHz

H-2 H-8 H-I’ H-2’ H-3’ H-4’ H-5’ H-5”

6 39 - 95 - 95 95 - 95 - 400 - 400

70 140 56 - 17

H-1 H-I’ CHd1) CHd2) 11-3 11-6, h H-7, 7 H-10, 10’ H-11. 11’

- 400

- 240

-

- 400

- 240

-

~

~

-

~

15 66 29 78 20 ’0 19

~

-

17

- 240

~

-

11

- 240

-

5.4 12.1 6.3

18 36

~

~

5.9”

31

-

66 24 24 16

11 4

66 19 1 1

An average value for the two methyl resonances which were not sufficiently resolved to permit independent determination.

Unfortunately it is not possible to find a region where the concentration of ML is very small. As with the shift studies the data at very low lanthanide concentrations correspond to a dominance of ML2 species. Titration of the ligand in the absence of any buffering lanthanum nitrate requires a final concentration of 0.1 mM gadolinium nitrate to change the proton relaxation times significantly. However, even at this low concentration ofmetal ion, at the end ofthe titration a significant proportion of the ligand is bound as ML. Thus we are unable to obtain relaxation data which can be interpreted wholly in terms of the ML2 species.

The Corlfor-inationof the M L Species The bound shifts, 01, for the two methylene groups adjacent to the pyrophosphate are identical within experimental error. This might suggest, but does not confirm, that the magnetic anisotropy axis lies symmetrically between the two groups. As an additional test the 31Pspectra were recorded in the presence of 0.5 M lanthanum nitrate. Praseodymium shifted the two resonances equally downfield. While these shifts contain a large contribution from a contact interaction it may reasonably be assumed that the contact and pseudo-contact interaction on each 31P nucleus is the same and therefore that the magnetic anisotropy axis lies symmetrically with respect to the two nuclei. It has been shown that for praseodymium

removal of the contact contribution does not change the direction of the shift [19] and thus both ”P nuclei and their adjacent methylene groups lie within the cone of 54‘ 44’ (955.4 mrad) about the magnetic anisotropy axis. The conformational search was carried out in two parls : (a) the fragment adenosinc-pyrophosphatemethylene (of pantetheine) and (b) pantetheine-pyrophosphate-methylcne (of adenosine). The former will be considered first. As the magnetic axis lies syininetrically with respect to the pyrophosphate the lanthanide must bind to either one or two oxygen donors on each phosphate. Attempts to fit the data with bidentate coordination rapidly run into difficulties as soon as the ribose is considered. Tetradentate coordination is in any case more plausible and has been found in the lanthanide-ATP complex [20]. We find that the best fit is observed with the magnetic axis down the axis of the pyramid formed by the four lanthanideoxygen bonds and passing through the bridging oxygen between the two phosphorus nuclei. The observed near equivalent shift of the two protons in each of the methylene groups adjacent to the pyrophosphate is found to arise not as might be expected from common r and 8 values but from a compensation effect between these two parameters that nevertheless gives nearly equivalent calculated shifts. The data are shown in Table 2. The differences of approximately 8:; in the calculated shifts are in accord with the observed spectra. Both methylenes show substantial broadening on titration with praseodymium. much greater than for any other set of protons shified to the same extent. Unfortunately we were unable to shift them far enough to resolve the individual protons. At high praseodymium concentrations the ribose protons initially obscured by residual 2HOH move downfield and are well resolved with respect to each other. The separation between them remains constant on further titration. This strongly suggests that they retain the same order as in the free ligand, H-2’, H-3’, H-4’. Attempts to fit this by considering a varicty of ribose conformations reveals a very predominant 2E conformation. The agreement is well within experimental error as rather few data points are available during the titration. Any contribution from, for example, a 3E conformation must be very small. The calculated shift of H-2’ is approximately 50 ‘:.b of that observed. 2E ribose conformations have been observed in the rubidium salt of ADP [7] and one of the two molecules in the asymmetric unit of ATP 1211. The observed shifts of the base protons are in excellent agreement with a completely urzti conformation with respect to the ribose at a glycosyl torsion angle ~ C = N -50’ (-872 mrad). Limited relaxation data are available on the adenosine moiety but the relative observed distances to H-8 and H-2 do not fit at all

512

Conforination of Uephospho C a A by N M R Spectroscopy

Table 2. Observed and culcultried shiJis und metal-ion-observed- roto on internuclear distances normalized on udmosine H-I' for the I :1 metal :ligund species Calculated data are based o n a 'E ribose conformation Molecule

Adenosine

Atom

Observed shift ratio

H-2. anti stn 93 anti o hsd H-8 an t I wn 93 anti obsd H-I ' H-2' H-3' 11-4' H-5' H-5"

-

H-3 Ir-1' CHdI) CH3(2) H-3 H-6. 6' H-7, 7 ' €1-10, 10' H-11, 11'

r

deg(mrad)

A(nni)

58 (1012) 55 (954)

12.5 (1.25) 5.8 (0.58) 8.84 (0.884)

2.31 1 .00 2.45 2.45 2.45 10.3 10.3 10.26 10.26 0.35 1.70 0.74 - 2.00 0.50 0.50 - 0.49 ~

~~

-

r",h

1'

ohsd

0.07 0.05 0.07

calcd

1.03 1.02

21 (366) 53 (924)

~

Calcd shirt ratio

0.15

'x

Pantetheinc

0

38 39 45 35 28 I2

8.0 (0 80) 10.2 (1.02) 8.07 (0.807)

2.38 0.06 2.27

(663) (680) (785) (610) (488) (209)

8.6 (0.86) 6.4 (0.64) 5.2 (0.52) 7.0 (0.70) 4.7 (0.47) 5.3 (0.53)

1.00 2.38 2.76 2.24 9.76 9.52

25 (436) 35 (262) 52 (907) 36 (628) 44 (767) 75 (1308) 70 (1221) 93 ( 1622) 90 (1 570)

4.8 (0.48) 5.3 (0.53) 6.5 (0.65) 7.5 (0.75) 8.0 (0.80) 6.6 (0.66) 8.8 (0.88) 10.8 (1.08) 12.4 (1.24)

10.00 9.14 0.38 1.76 0.83 - 2.14 - 0.69 0.62 - 0.38 ~~

0.94 0.89 1 .00

1 .oo

0.85"

0.76 0.87

0.90 1.02 1.25 1.35

0.76 1.02 1.25 1.44

An average value for the two methyl resonances which were not suffientlj well resolved to permit independent determitiation

well a wholly unri conformation. While it is possible to adopt a unique conformation. To perform a that long distances appear somewhat shorter through thorough Fearch over the entire chain would bc inadequate compensation for outer sphere complexextremely difficult but fortunately there are two severe formation this is unlikely to provide an adequate restraints imposed by the shift and relaxation data. explanation in these experiments. Rathcr some conFirstly, the cone of 54' 44' (955.4 mrad) passes tribution from a syn conformation is necessary as between pantetheine H-3 and H-6, 6' and secondly found, for example, with the purine 3': 5'-nucleotides H-6, ti' and H-7, 7' lie fairly close to the metal ion. [22]. The relaxation and shift data might appear to be The reversal in sign of the H-6,6' protons and the in disagreement on this point but examination of a large shift observed indicates that the chain from model in syn conformation shows that the angle 0 the phosphate to this methylene must be folded back for both the base protons lies in the region 53 - 55" on itself towards the metal ion. We find that in order (924- 959 mrad). We would expect a near zero shift for to fit the data, the chain must be folded back the both of the protons. The observed shift data is merely maximum amount allowed by the intervening bonds. reduced in magnitude proportional to the time the Thereafter the shifts and calculated distances attenuate molecule spends in the syn form. Calculating the time rapidly and the molecule is extended. The observed spent in each conformation as described previously and calculated data are shown in Tables 1 and 2. shows that an excellent fit of the relaxation data arises Two views of the overall conformation of the when the molecule spends 7 %, of the time, in the s y ~ molecule are shown in Fig.7 for the adenosine in its form. This does appear to be a real contribution not anti form. arising through errors in the data as longer distances The metal : ligand 1 : 2 complex was also studied are observed to protons on the pantetheine chain. in the hope of finding a conformation for this species. Considering the pantetheine-pyrophosphate fragAs noted earlier we were unable to derive relaxation ment it might appear that, with a large number of data corresponding to this complex. The interpretation bonds about which rotation can occur, it is unlikely of the shift data is potentially extremely complex

513

G. V. Fazakerley, P. W. Linder, and 1). ti. Reid

L Fig 7 Two views of the overall conformation of the dephospko CoA molecule for the adenosine in its anti form

and probably impossible in the absence of further assumptions. Two attempts were made to fit the data. The first assumed the conformation of the two ligands to be identical. N o satisfactory fit could be achieved for the adenosine fragment. The second assumed that one ligand retained the conformation found for the 1 : 1 species and thus A 2 was separated into the two shifts corresponding to each ligand. This method would only be valid if D in Eqn (9) were the same for both 1 : 2 and 1 : 1 complexes and no evidence is available that this is so. An attempt made along these lines failed to produce an acceptable conformation for the adenosine fragment. In general there is probably not enough information to determine the conformation of 1 : 2 species unless the ligands are fairly rigid.

CONCLUSION

The solution conformation of the lanthanide complex of dephospho coenzyme A has been determined. The metal ion is found coordinated to four oxygen donors of the pyrophosphate. The ribose is found in a 2E conforination with the base in predominantly mzfi form. The pantoinic acid part of the chain is folded back towards the pyrophosphate while the remainder of the chain is extended.

This work was supported by grants from the University of Cape Town and the Council for Scientific and Industrial Research.

REFERENCES 1. Lee; C.-H. & Sarma, R. €1.(1974) FEBS Lett. 43, 271 -276. 2. Lee. C.-H. & Sarma. R. H. (1975) J . Am. C'hem. Soc. 97, 1225-1236. 3 . Wilson, Ci. E., Jr, Bazzone. T. J., Kuo, C. H. & Kinaldi, P. L. (1975) J . Am. Chem. SOC.97. 2907-2908. 4. Mieyal, J. J.. Webster. L. T.. Jr & Siddiqui, U. A. (1974) .J. B i d . C'hetn. 249, 2633 - 2640. 5. Fung, C. H., Feldman, R. J. & Mildvan, A . S. (1976) Biochcmi.s/ry, 15, 75-84. 6. Fung, C. H., Gupta, R. J. & Mildvan, A. S. (1976) Biorhernistry, 15, 85-92. 7. Viswamitra, M. A., Hosur, M. V., Shakked, 2. & Kennard, 0. (1976) Nature (Land., 262, 234-236. 8. Altona. C. & Sundaralingam, M . (1972) J . An?. Chem. SOC.94. 8205 -8212. 9. Barry, C. D., Glasel. J. A., Williams. R. J. P. & Xavier, A. V. (1974) J. Mol. B i d . 84, 471 -490. 10. Rossotti, F. J . C. & Rossotti, H. (1961) The Derermination of' Stability Constunts. pp. 282-284. McGraw Hill Book Co., New York. 11. Rossotti, F. J. C. & Rossotti, €1. (1961) The Determination of Stability Constanrs, pp. 108 - 110. McGraw Hill Book Co.. Ncw York. 12. May, P. M., Linder, P. W. & Williams, D. R. (1977) J . Chem. SOC.Dalton, 588- 595. 13. La Mar. C;. N., Horrocks, de W. H. & Allen, L. C. (1964) J . Chem. Phys. 41. 2126-2134. 14. Solomon. I. (1955) P h y ~Rev. . 99. 559 -565. 15. Bloembergen, N. (1957) J . Chem. Phys. 34. 842-850.

514

G. V . Fazakcrlcy, P. W . Linder, and D. G. Reid: Conformation of Dephospho CoA by N M K Spectroscopy

16. Fazakcrlcy. G. V. & Jackson, G. E. (1975) .I. Chctw. Suc. Pcvkiti 11.567 571. 17. Yoneda, 11.. Choppin. G. R., Bear, J . L. & Quagliano. J. V. (1964) fTl(7rg. c'hcnl. 3. 1642- 1644. 18. Dutt, N. K . & Bandyopadhyay, P. (1964) .I. lwnoig. Nwcl. Chem. 26, 729 - 736. 19. Dobson, C. M., Williams. R. .I.P. & Xavier. A. V. (1973) J . Ciaeni. Soc. Dnltoti, 2662- 2664. ~~

20. Tanswell, P., Thornton. J. M., Korda, A. V. & Williams, R. J . P. (1975) E w .J . Biochem. 57, 135 145. 21. Kennard, 0.. Isaacs, h'.W., Motherwell, W. D. S . . Coppola, J. C., Wampler, D. L., Larson. A. C. & Watson, D. G. (1971) Proc. R . Soc. Lonil. R. Rioi. Sci. 325, 401 -436. 22. Fazakerley, G. V., Russell. J. C. & Wolfc. M. A. (1975) J . Cheni. Suc. Chetn. Comniurz., 527.

G. V. Fazakerley and D . G. Reid. Department of Inorganic Chemistry. IJniversity of Cape Town.

Private Bag, Rondebosch. Cape Town, South Africa 7700

P. W. Linder. Departmen1 of Physical Chemistry. University of Cape Town, Private Bag, Kondebosch. Cape Town, South Africa, 7700