Volume 156, number 6 CHEMICAL PHYSICS LETTERS 2 I April 1989

PHOSPHORUS-31 MAGNETIC RELAXATION OF ADENOSINE 5’-MONOPHOSPHATE, ADENOSINE 5’-DIPHOSPHATE AND ADENOSINE 5’-TRIPHOSPHATE IN SOLUTION

R. GASPAR Jr. I, W.S. BREY Jr., A. QIU and E.R. ANDREW Departments of Physics, Radiology, Chemistry and Nuclear Engineering Sciences. University of Florida. Gainesville, FL 3261 I, USA

Received 17 January 1989; in final form 11 February 1989

Measurements have been made ofthe longitudinal phosphorus-3 1 magnetic relaxation of the individual phosphorus resonances of AMP, ADP and ATP at 12 1.5 MHz from 278 to 333 K and at 40.5 MHz at room temperature in H,O solutions of the chemicals.

The phosphorus-3 1 spin-lattice relaxation times of all compounds are dominated by dipolar interactions and influenced by chem- ical shift anisotropy interactions to a different extent at the two frequencies. Phosphate group motion superimposed on the tum- bling of the molecules is the main source of phosphorus-3 1 spin-lattice relaxation. Activation energies characterizing the com- bined motion range between 17.1 and 20.0 kJ/mol.

1. Introduction

In recent years 3’P NMR lines of naturally occur- ring metabolites AMP, ADP and ATP have fre- quently been utilized in in vivo NMR spectroscopy. Their observation provided useful information about the metabolic state of a wide variety of tissues and organs [ l-41. Much interest has also been devoted to the NMR study of the ion-ATP interactions be- cause of their relevance in enzymatic reactions [ 5,6 1. The pH dependence of the 3’P longitudinal relaxa- tion of AMP and ATP has been established and the effect of chelating agents such as EDTA and CDTA on the T, of these molecules has been studied [7- 10].

Compared to their importance rather little is known about the relaxation mechanisms of the spin systems of these molecules [ 1 l- 13 1. Recently we be- gan to address this problem by describing the proton relaxation and dynamics of AMP, ADP and ATP.in solution and in the solid state [ 14-161.

In this article we describe a 3’P relaxation study of AMP, ADP and ATP. We measured T, for the in-

dividual phosphorus atoms of the molecules in their

’ Permanent address: Department of Biophysics, Medical Uni- versity School, 40 12 Debrecen, Hungary.

solutions as a function of temperature and fre- quency. The results provide insight into the phos- phorus magnetic relaxation mechanisms and the un- derlying dynamical behaviour of the compounds.

2. Experimental

The 31P spin-lattice relaxation time measure- ments were carried out on a Nicolet NT-300 NMR spectrometer at 12 1.5 MHz and on a Varian XL- 100 NMR spectrometer with a Nicolet TT-100 l?T ac- cessory at 40.5 MHz. An inversion recovery pulse sequence with signal averaging was used to measure T, values of the individual phosphorus resonances between 278 and 333 K. The delay between succes- sive pulse sequences was at least five times the long- est T,. The recovery of nuclear magnetization was always exponential within the experimental error. The T, values and activation energies (EA ) were cal-

culated using least-squares methods from the mea-

sured intensities of the resonance lines. The sample temperature was constant to + 0.5 K. The accuracy of the T, values was typically & 5% at 121.5 MHz and + 7% at 40.5 MHz.

The AMP, ADP and ATP sodium salts and CDTA were purchased from Sigma Chemical Co. HPLC

0 009-26 14/89/$ 03.50 0 Elsevier Science Publishers B.V. ( North-Holland Physics Publishing Division )

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Volume 156, number 6 CHEMICAL PHYSICS LETTERS 2 1 April 1989

quality HzO, NaOD and DC1 were purchased from Aldrich Chemical Co. They were used without fur- ther purification.

Procedures used in the preparation of samples to minimize the introduction of paramagnetic impur- ities are described in ref. [ 151. The )‘P NMR sam- ples were prepared from 0.1 M solutions in pure H,O. The pH was adjusted by the application of concen- trated solutions of NaOD and DCl, earlier found ad- equate for the purpose of T, measurements [ 15,161. To quench the effect of the paramagnetic ions on the observed T, values, 5.5 mM CDTA was added to the samples. To remove any dissolved paramagnetic ox- ygen, pure N, gas was bubbled through the samples. The screw-cap NMR sample tubes were closed under Nz atmosphere.

3. Results and discussion

To demonstrate the results, experimental T, val- ues for the different 3’P lines of AMP, ADP and ATP at 3 10 K, 121.5 MHz and pH 7.4 are displayed in table 1. This temperature and pH are selected close to those of the normal human body to be relevant to in vivo MRS experiments. It is seen that T, varies with the compound and within the same molecule (for ADP and ATP) at a given temperature. The T, values vary with temperature as illustrated in figs. l-

3. This temperature dependence of T, suggests that

the dominant relaxation mechanism is through the dipole-dipole interaction with no indication of the spin-rotation interaction mechanism, which would have an opposite temperature dependence. The

Table 1 Comparison of -“P spin-lattice relaxation times (s) of AMP, ADP andATPat 121.5MHzand310K

Sample

AMP AMP+CDTA ADP ADP+CDTA ATP ATP + CDTA

Resonance lines

P, PU P,

1.77 5.73 2.21 2.22 3.91 6.30 1.93 0.99 I .05 3.03 4.38 5.97

620

15

10 - 9-

a 7 1 l ‘., 6- *-k

T,(s),5 _ 1.

‘1

3- '\..

2- 3.0 3.2 3.4 3.6

IO3 /T(K-'1

Fig. 1. Activation energy plot of the P, (0 ) resonance of AMP at 12 1.5 MHz, pH 7.4. The T, values display a linear activation energy plot throughout the whole temperature range. For further details of the experimental conditions we refer to the text.

12.6

7 6

5

d

TJsl- 3

2

1.5

I I I I I I I I

3.0 3.2 3.4 3.6

103/T (K-l1

Fig. 2. Activation energy plots of the P, ( l ) and Ps ( A ) reso- nances of ADP at 12 1.5 MHz, pH 7.4. For further experimental details see the text.

strong frequency dependence indicates a contribu- tion from the chemical shift anisotropy mechanism. A change in the resonance frequency from 121.5 to 40.5 MHz roughly doubled T, at room temperature. Bendel and James [ 121 showed that in the absence of proton decoupling 3’P spin-spin relaxation in AMP is dominated by scalar relaxation of the second kind via scalar coupling to the 5’ protons, which have short T,. However on the basis of the coupling con- stants [ 121 and our proton T, values [ 15,161 we es- timate that the scalar contribution to the 31P longi- tudinal relaxation is negligible. Relaxation due to paramagnetic impurities has been reduced to a min-

Volume 156, number 6 CHEMICAL PHYSICS LETTERS 21 April 1989

1 I I I I I I I

3.0 3.2 34 3.6

IO3 I T (K"1

Fig. 3. Activation energy plots oftbe P, ( l ), P, ( A ) and P, (w) resonances of ATP at 121.5 MHz, pH 7.4. For further experi- mental details see the text.

imum by careful sample preparation and the addi- tion of CDTA.

Molecular motions modulate the dipolar interac- tions and provide an effective mechanism of 3’P re- laxation, particularly through intramolecular 3’P-3’P, 3’P-‘H and intermolecular 3’P-‘H interactions. In- tramolecular dipolar interactions have been ob- served between the a and p phosphates and the I$, and I& protons of ATP [ 171. At concentrations close to the 0.1 M employed, intermolecular 3’P-3’P in- teractions can be ruled out, since no change of T, has been observed with dilution in the range 0.02-0.1 M. The dipolar and chemical shift anisotropy inter-

actions are both modulated by motion of the whole molecule in solution and the motion of different groups within the molecule.

From figs. l-3 it is clear that the longitudinal re- laxation in this temperature range is in the fast-mo- tion limit, 0.1~7, -K 1. For identical spin-l /2 nuclei undergoing rotational motion characterized by a cor- relation time r,,

(l/C 11, = #y:h27J-6 ) (1)

and for non-identical nuclei

(llT,),.~=y:y~Z1*7~,r-~, (2)

where r is the internucIear distance. For the chemical shift anisotropy mechanism

where b,, and oI are principal values of the shielding

tensor, assumed to be axially symmetric. The ex- plicit field and frequency dependence of (T, )CSA is clear from (3). The relaxation rates are assumed to be additive, giving an effective relaxation time which is measured,

(1/T,),,=(1/T,),,+(1/Tl),s+(1/~,),,*. (4)

Because only the ( 1 /T, )CSA term in (4) is fre- quency-dependent, the CSA contribution can readily be separated from the other contributions. In table 2 the fractional contributions to T, of AMP, ADP

and ATP are displayed. The temperature dependence of 5, may be de-

scribed by the Arrhenius activation expression

7= =7” exp(E,JRT) . (5)

When there are internal motions of groups within the molecules which itself undergoes random reorien- tation as a whole, the correlation time rc is an effec- tive correlation time combining the effects of the various motions.

Substitutionof (I), (2), (3) and (5) into (4) in the fast-motion limit leads to a linear plot of In T, versus 1 /T from which an activation energy EA is derived, which is a measure of the potential barrier hindering the motions. Values of EA derived in this way are given in table 3 for the 3’P resonances of AMP, ADP and ATP with a typical accuracy of k 7%.

In ADP and ATP the dipolar interactions contrib- uting to 3’P relaxation will be those between adja- cent P atoms in the same molecule and 3’P-‘H in- teractions with some adenosine protons, particularly H,,,.,, H,., as well as protons of the solvent water molecules close to the phosphate groups. In AMP

Table 2 Chemical shift anisotropy contributions (%) to the spin-lattice relaxation rates ( 1 /T, ) of AMP, ADP and ATP determined at two different frequencies at room temperature

Sample

AMPfCDTA

ADP + CDTA

ATP+CDTA

Frequency Resonance lines (MHz)

P, PO P,

121.5 51*7 40.5 llk2

121.5 53+7 2529 40.5 11+2 4fl

121.5 59?? 6Sk7 34+g 40.5 14k2 19+2 5+1

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Volume 156, number 6 CHEMICAL PHYSICS LETTERS 21 April 1989

Table 3

Activation energy values (kJ/mol) ofthe different 3’P resonance lines of AMP, ADP and ATP

Sample

AMP+CDTA ADP+CDTA

ATP + CDTA

Resonance lines

P, P, P,

17.1 20.0 17.1

19.6 19.2 17.7

only the “P-‘H interactions are important because intermolecular ‘rP-3rP interactions have been ruled out by lack of concentration dependence. The rela- tive contributions of these dipolar interactions can- not be differentiated further.

Table 2 shows that in ADP and ATP the relative CSA contributions vary within the molecule, the outermost phosphates displaying significantly lower CSA fractional contributions. From (3) these dif- ferences could arise from differences in shielding an- isotropy or from differences in rc. If we accept the similarity of the shielding anisotropies observed for ATP [ 111, the differences arise from differences in 7c, indicating a relatively less restricted motion of the p and y phosphate groups of ADP and ATP respectively.

We can obtain further insight by eliminating T= from eqs. ( 1 ), (2) and (3 ) to obtain a value of r6(a,,-uL)’ from the relative values of (l/

T, )di,mlar and (l/TIhA. Using a published value for ATP ofa,,-o,=260 ppm [ll], weobtain r=O.14 nm. Since this is an effective value for a single di- polar interaction representing all dipolar interac- tions, this somewhat short value of r is not unrea- sonable. These values may now be substituted back into(l)and(3)toobtainr,=4xlO-“sat310K which is a reasonable result.

The values of EA in table 3 fall in the close range 17.1-20.0 kJ/mol, and are similar to those found from analysis of proton T, measurements [ l&l6 1. This suggests that torsional motion around the Cd.- C5. bond, important in securing proton relaxation, is also important for 3’P relaxation. It is to be noticed that the outermost phosphorus atoms have slightly lower E, values indicating internal motions of the

di- and tri-phosphate groups of ADP and ATP in the 3’P relaxation of these compounds.

The 3’P spin-lattice relaxation times reported here for the metal-free state of AMP, ADP and ATP are higher than the corresponding values for cells and tissues. In cellular systems additional relaxation may arise from metal-ion binding and intermolecular in- teractions with other molecular constituents of the systems.

Acknowledgement

The authors gratefully acknowledge support from Johnson and Johnson, the NIH and the Hungarian Academy of Sciences under grants 1 P41 RR02278, CA 42283 and OTKA 112.

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