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in the spin-echo experiment. At a Τ2 of 0.060 s, the majority of the spec t r u m consists of resonances from pro tons having relatively long T^s, main ly protons of the smaller, more mobile components of the intracellular medi um as well as those of certain structur al units of the hemoglobin. For exam ple, most of the resonances in the 6.5-9 ppm region are due to imidazole protons of the histidyl residues of he moglobin. Our work on red blood cells follows from our interest in glutathione (GSH), a tripeptide of the sequence 7-L-glutamyl-L-cysteinyl-glycine, which is naturally present in h u m a n red blood cells at the 2-3-mM level. For some time I have been intrigued by the idea t h a t it might be possible to study GSH directly in red blood cells by proton N M R spectroscopy. As shown by spectrum A in Figure 4, however, the one-pulse spectrum is an envelope of overlapping resonances. Fortunately, G S H is among the small red cell molecules whose resonances can be resolved by the H a h n spin-echo sequence. Resonance gl in spectrum C is due to the methylene protons of the glycine residue of GSH, resonance g2 those of the cysteinyl residue, and resonances g3 and g4 are due to those of the glutamyl residue. With the Hahn spin-echo tech nique, it has been possible to study in tracellular processes involving G S H directly (7). For example, one function of the GSH in the red cell is to protect the cellular membrane and compo nents from oxidizing species such as hydroperoxides. In doing so, the sulfhydryl group of GSH is oxidized to the disulfide form (GSSG), with the oxi dation catalyzed by glutathione perox idase. When the GSH is oxidized, the chemical shift of resonance g2 changes. Using the specificity of the enzyme-catalyzed conversion of GSH to its oxidized form and the intensity of resonance g2, we have followed by N M R the selective titration of the in tracellular GSH with t -butyl hydro peroxide. Then after complete conver sion to GSSG, glucose was added, and the time course for the enzymatic con version back to GSH was obtained while simultaneously obtaining the time course for the metabolism of glu cose to lactic acid. T h e Hahn sequence is the simplest of the spin-echo sequences. Others (for example, the Carr-Purcell and the Carr-Purcell-Meiboom-Gill se quences) use a series of 180° refocusing pulses (1 ). These sequences must be used when the magnitude of τ·ι in the Hahn sequence becomes so large t h a t molecules have time to diffuse to different regions of HQ; thus, the ef fects of Ho inhomogeneity on T?2 are no longer reversible. Resolution enhancement with the
1268 A · ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
spin-echo sequence is based on selec tive loss of signals from resonances having the shorter T2S. T o some ex tent, similar effects can be achieved with the one-pulse sequence by using digital filtering (convolution) tech niques (8-10) or by introducing a delay time between the end of the pulse and the start of data collection (11). These techniques take advantage of the differential rates of decay of sig nals in the FID due to differences in T 2 's. Inversion-Recovery Sequence T h e inversion-recovery sequence is a standard sequence for measuring Tj values, but it also can be used to en hance resolution by taking advantage of any differences in the TVs of over lapping resonances. T h e inversion-re covery sequence (sequence C in Figure 1) consists of a 180° pulse followed by a time interval of length τχ and then a 90° pulse. T h e 180° pulse inverts the magnetization along the 2 axis (se quence C in Figure 2), so t h a t immedi ately following the 180° pulse, the magnetization is of magnitude Mo b u t in the negative 2 direction. As time passes, magnetization along the nega tive 2 axis decreases, passes through zero, and then grows toward its equi librium value by spin-lattice relaxa tion. Application of the 90° pulse when the magnetization is in these three states results in a negative reso nance, no resonance, and a positive resonance, respectively, in the fre quency domain spectrum. T h e magni tude of the magnetization along the 2 axis at time ττ following the 180° pulse is given by M 0 ( l — 2 e~T,/Ti), ac cording to which the magnetization for a given resonance passes through a null a t r\ = 0.69 Tv T h u s , by careful choice of the time at which the 90° sampling pulse is applied, one can se lectively null resonances in overlap ping patterns on the basis of differ ences in their Ti's. T h e selective elimination of the re sidual HDO peak in N M R spectra of D2O solutions by applying the 90° pulse at τ\ = 0.69 T ] , H D O was one of
the first applications of the inversionrecovery sequence in this way (12). This technique, termed the watereliminated-Fourier-transform (WEFT) technique, can be used to ob serve resonances under the HDO reso nance if the Ti's of the HDO reso nance and the resonance of interest are different, and to solve the dynamic range problem encountered in pulsed Fourier transform N M R of dilute so lutions in D2O. An inherent disadvan tage in the latter application is t h a t the magnetization for the resonances of interest is most likely less than M0 at the time the 90° pulse is applied due to incomplete relaxation. T h u s , the signal strength in the inversion-
