Proc. Natl. Acad. Sci. USA Vol. 74, No. 10, pp. 4271-4275, October 1977 Biochemistry 31P nuclear magnetic relaxation studies of phosphocreatine in intact muscle: Determination of intracellular free magnesium (nuclear Overhauser effect/13C nuclear magnetic resonance) SHEILA M. COHEN*t AND C. TYLER BURTt *Department of Chemistry, University of Illinois at Chicago Circle, Chicago, Illinois 60680; and *Department of Biological Chemistry, University of Illinois at the Medical Center, Chicago, Illinois 60612 Communicated by Irving M. Klotz, July 25, 1977 ABSTRACT 31P nuclear magnetic relaxation rates for nipulations were performed in a cold room at 40 and NMR phosphocreatine in intact frog gastrocnemius were compared accumulations in a probe maintained at 40 were begun im- with those observed in model solutions at 40, a temperature at which muscle maintains its physiological state for at least 5 hr. mediately. Both nuclear Overhauser effect and spin-lattice relaxation rate PCr was from Sigma Chemical Co.; Mg(NO3)2 (ultrapure) (1/Tj) experiments indicate that interactions form was from Alfa Products. Metal-free solutions of PCr were the dominant relaxation path fordirle-dipoleP in intact muscle and model prepared by passing constituents through a column of Chelex. solutions, independent of phosphocreatine and Mg concentra- The pH was adjusted to 7.4 (pD = meter reading + 0.4) at room tions. Spin-spin relaxation rates (1/T2) measured by modified temperature with HCO (or DCl) prepared by passing Carr-Purcell-Melboom-Gill spin-echo experiments suggest the HCl (DCl) importance of scalar coupling modulated by chemical exchange gas through H20 (D20). Solutions were 0.1 M in KCI. Solutions with free Mg. From these results, we estimate the free intra- in deuterated solvent were lyophilized three to four times in cellular Mg in intact muscle as 4.4 mM and demonstrate that 99.8% D20. The final concentration of PCr in solution was 31p T2 experiments can be used as a tool for studying free Mg determined by total P analysis (9). Analyses for Mg and Mn in levels with minimum disturbance of the intact cell. stock and final solutions were by Trace Elements Inc. (Park Ridge, IL); these analyses showed Mn in final solutions to be 31P nuclear magnetic resonance (NMR) studies of intact cells <0.1 ,uM. All solutions were degassed either by nitrogen bub- to date have concentrated on high-resolution spectra of me- bling or several freeze-pump-thaw cycles. tabolites in intact muscle (1, 2), erythrocytes (3), yeast (4), and High-resolution NMR spectra were obtained at 36.43 MHz Escherichia coli and Ehrlich ascites tumor cells (5, 6). We report on a hybrid Bruker HFX-90 spectrometer with 2H stabilization here on the feasibility of measuring the relaxation parameters operating in the Fourier transform mode as described (10). of phosphocreatine (PCr) in intact frog muscle at 4°. The Spinning 10-mm samples were used; the field was locked either spin-lattice relaxation time (TI) and the spin-spin relaxation to a capillary of D20 (muscles and PCr/H20 solutions) or D20 time (T2) of PCr in intact muscle are compared with those solvent. Typical conditions included a 900 pulse of ca 20 lisec, observed in model solutions at 4°. Both relaxation rates are 32-128 scans of 4000 data points each, sweep width of 1 kHz, valuable as dynamic probes of the internal environment in exponential multiplication introducing 0.6-Hz line broadening, muscle because T2 can be sensitive to processes that do not affect and 'H irradiation. T, at all. These relaxation studies also enable us to assign a value T, measurements were made with the following pulse se- for the free intracellular Mg concentration in intact gastroc- quence: 900 pulse, spoil field homogeneity, wait delay time r, nemius via an intrinsically nonintrusive method. 90° pulse, sample free-induction decay, and then spoil field It is now clear that metal ions are of central importance in homogeneity. This sequence (11), which was repeated n times, many biological reactions and knowledge of free Mg levels has afforded a considerable saving of time over the inversion-re- particular significance. For example, Stephenson and Podolsky covery method in the T, regimen of 31P in PCr, thus making (7) showed that in skinned muscle fibers, Ca transport by intact it possible to carry out T, measurements on muscle during the sarcoplasmic reticulum is strongly influenced by Mg concen- interval in which there was no more than a 10% change in tration. The present nonperturbing technique for free Mg is of PCr. special interest; of the few alternate methods, the one of choice The Carr-Purcell-Meiboom-Gill (CPMG) (12) T2 mea- is probably a differential absorption procedure involving the surements were performed on a Bruker B-KR pulsed spec- microinjection of a Mg-binding dye to measure ionized Mg in trometer operating in conjunction with the same Nicolet data the volume surrounding a dialysis capillary (8). system and a 21.14 kG magnet used for high-resolution spectra. Two home-built modifications to the probe of this pulsed MATERIALS AND METHODS spectrometer were made so that satisfactory results could be Adult frogs, 5-12.5 cm in length, from Lake Champlain Frog obtained on biological samples in which phosphate metabolites Farm (Alberg, VT) were stored at 4°. Frogs appearing to be in are in the mM range-namely: (i) because field-frequency drift good condition were decerebrated and the rear legs were iso- is known to have a deleterious effect on the results of the CPMG lated. The gastrocnemius was dissected out and placed in a method, we installed an external deuterium lock system in the 10-mm NMR tube with standard frog Ringer's solution (113 pulse probe, which was capable of holding the 31p field-fre- mM NaCI/2.5 mM KCI/1.8 mM CaCl, pH 7.2). These ma- quency stability within 1-2 Hz over several hours, and (ii) in Abbreviations: NMR, nuclear magnetic resonance; PCr, phospho- The costs of publication of this article were defrayed in part by the creatine; T1, spin-lattice relaxation time; T2, spin-spin relaxation time; payment of page charges. This article must therefore be hereby marked CPMG, Carr-Purcell-Meiboom-Gill; NOE, nuclear Overhauser effect; "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate DD, dipole-dipole. this fact. t Present address: Bell Laboratories, Murray Hill, NJ 07974. 4271 Downloaded by guest on September 26, 2021 4272 Biochemistry: Cohen and Burt Proc. Natl. Acad. Sci. USA 74 (1977) _ 100 0 0 ~0 80 .0 0 0~ "F 60- 40 (a) (b) %4- 0 T= 277 K 20 A B A B 0 _ I w0 50 100I- 150 200 250 300 Minutes FIG. 1. Change in concentration (expressed as % of the original value) of whole-muscle phosphate metabolite as a function of time. There was less than a 20% change in PCr concentration over 5 hr at 40. This demonstrates the feasibility of long-term relaxation experiments at this temperature. The insets show two representative 31p spectra ofthe time course for change in PCr concentration (peak B) in whole muscle relative to a sealed reference.capillary of trimethyl phosphate (peak A). Experimental conditions were maintained constant throughout. (Inset a) The 0 min spectrum; the ratio of the integrated peak areas (B/A) is 0.73. (Inset b) A spectrum taken 290 min later in which this ratio is 0.64. order to use the significant gain in sensitivity offered by the of muscle were maintained within normal limits longer than large nuclear Overhauser effect (NOE) of 31P of PCr in muscle at room temperature (15). Typical 31P NMR spectra of the time (see below), we added proton irradiation capability to the 31P course for change in PCr concentration in whole muscle at 40 pulse probe. Whereas heteronuclear spin decoupling has a are shown in Fig. 1 inset. Long-term experiments requiring 4-5 complex effect on echo formation, the use of the NOE for signal hr were practical at this temperature; the data in Fig. 1 dem- enhancement does not contain this complication (13). Repro- onstrate that our muscle preparation maintained at least 80% ducible T2 values were obtained with the pulse spacings, 2r, of of this high-energy phosphate at the end of our longest exper- 2-20 msec used here. So that echo amplitudes could be mea- iments. sured relative to the proper steady state, the radio frequency Observed T1 values for several intact muscle samples and pulses were not turned off until the spin-echo decays had model solutions are listed in Table 1. Differences observed proceeded to about 6T2. Time averaging with a recycle time between stretched and unstretched muscle cannot be consid- of 6T, was used (14). Prior to averaging, signals were passed ered significant. However, it is significant that the average T, through a B-KR pulse-gated integrator whose output gave the of 4.8 sec for muscle and the average T1 of 4.9 sec for PCr in envelope of the echoes directly. CPMG measurements were simple aqueous solutions were the same within the stated error made on nonspinning samples in 10-mm tubes with the sample limits. This suggests that the system of 31P nuclear spins in PCr constrained within the radio frequency coil for greater H1 is relaxing by the same mechanisms in both cases. Comparison homogeneity. With muscle samples, this was accomplished by of T1 for PCr in H20 solution with T1 in D20 indicated that the choosing frogs of appropriate size so that a single gastrocnemius 'H-31P dipole-dipole (DD) interaction was the dominant re- almost filled the radio frequency coil space.