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1H NMR has detected both the deoxygenated proximal histidyl NδH signals of myoglobin (deoxyMb) and deoxygenated Hb (deoxyHb) from human gastrocnemius muscle. Exercising the muscle or pressure cuffing the leg to reduce blood flow elicits the appearance of the deoxyMb signal, which increases in intensity as cellular[Formula: see text] decreases. The deoxyMb signal is detected with a 45-s time resolution and reaches a steady-state level within 5 min of pressure cuffing. Its desaturation kinetics match those observed in the near-infrared spectroscopy (NIRS) experiments, implying that the NIRS signals are actually monitoring Mb desaturation. That interpretation is consistent with the signal intensity and desaturation of the deoxyHb proximal histidyl NδH signal from the β-subunit at 73 parts per million. The experimental results establish the feasibility and methodology to observe the deoxyMb and Hb signals in skeletal muscle, help clarify the origin of the NIRS signal, and set a stage for continuing study of O2regulation in skeletal muscle.

The detection of the 1H NMR signal of myoglobin (Mb) in tissue opens an opportunity to examine its cellular diffusion property, which is central to its purported role in facilitating oxygen transport. In perfused myocardium the field-dependent transverse relaxation analysis of the deoxy Mb proximal histidyl NdeltaH indicates that the Mb rotational correlation time in the cell is only approximately 1.4 times longer than it is in solution. Such a mobility is consistent with the theory that Mb facilitates oxygen diffusion from the sarcoplasm to the mitochondria. The microviscosities of the erythrocyte and myocyte environment are different. The hemoglobin (Hb) rotational correlation time is 2.2 longer in the cell than in solution. Because both the overlapping Hb and Mb signals are visible in vivo, a relaxation-based NMR strategy has been developed to discriminate between them.

Upon titration with palmitate, the 1 H NMR spectra of metmyoglobin cyanide (MbCN) reveal a selective perturbation of the 8 heme methyl, consistent with a specific interaction of myoglobin (Mb)

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The 'H NMR signal from oxymyoglobin, a low-concentration diamagnetic protein, is visible in myocardial tissue. The methyl group of the Val-Ell resonates in a dear spectral region at -2.76 ppm and responds to dynamic changes in cellular oxygenation. With CO, the signal shifts to -2.4 ppm. The Val-Ell peak assignment and its response to oxygen and CO agree perfectly with previous myoglobin solution studies. Intracellular oxygen level can now be determined in vivo with the signal intensity ratio of oxymyoglobin/deoxymyoglobin, reflected by the Val-Ell and His-F8 peaks in the 1H NMR spectra. Moreover, protein structure-function relationship in vivo can now be probed. perfused heart studies that the 1H NMR signal ofthe proximal histidine NH in deoxymyoglobin (deoxyMb) is visible and is sensitive to changes in cellular oxygenation (6). Its visibility and response are predicated on the heme iron, which in the paramagnetic deoxygenated state, hyperfine shifts the resonance to a clear spectral region 75 ppm downfield of the water line (7). Upon oxygenation, Mb becomes diamagnetic and the signal disappears.To obtain cellular oxygenation levels with only the deoxyMb signal poses a quantitation difficulty. Neither the total Mb nor the oxymyoglobin (oxyMb) concentration is known. A deoxyMb/oxyMb ratio would circumvent the obstacle and lead directly to a value of oxygen partial pressure in the cell. However, detecting the oxyMb, a diamagnetic protein, signal in vivo is a daunting proposition and appears to be untenable, especially in light of its low concentration and the interfering endogenous signals in the diamagnetic spectral region. To our knowledge, no NMR signal from a diamagnetic protein in intact tissue under physiological conditions has been reported.We have, however, focused on the ring current shifted signal from the Val-Ell methyl group in oxyMb and report that under optimal pulsing conditions, it is indeed observable and responsive to incremental changes in cellular oxygenation. The MATERIALS AND METHODS Perfused Heart Preparation. Male Sprague-Dawley rats (350-400 g) were anesthetized by an intraperitoneal injection of sodium pentobarbital (65 mg/kg) and heparinized (1000 units) by injection into the femoral vein. The heart was isolated and perfused using a modified Langendorff technique (6). Hearts were perfused at 23-250C at a constant perfusion rate of 11 ml/min, which was maintained by a peristaltic pump (Rabbit, Rainin, Woburn, MA). Heart rate and perfusion pressure were continuously monitored by means of a perfusate-filled cannula connecting the aortic cannula with a Statham P23XL strain gauge transducer and a Gould RS 3200 oscillographic recorder. Perfusion pressure under control conditions was 65-75 mmHg; hearts were beating spontaneously at a rate of60-80 min'. Perfusate flowing from the pulmonary artery bathed the heart. An overflow tubing above the heart withdrew the perfusate for recirculation by the peristaltic pump. The total recirculation volume was 200 ml. The perfusion medium was a mo...

Pulsed field gradient NMR methods have determined the temperature-dependent diffusion of myoglobin (Mb) in perfused rat myocardium. Mb diffuses with an averaged translational diffusion coefficient (DMb) of 4.24-8.37x10(-7)cm2/s from 22 degrees C to 40 degrees C and shows no orientation preference over a root mean-square displacement of 2.5-3.5 microm. The DMb agrees with the value predicted by rotational diffusion measurements. Based on the DMb, the equipoise diffusion PO2, the PO2 in which Mb-facilitated and free O2 diffusion contribute equally to the O2 flux, varies from 2.72 to 0.15 in myocardium and from 7.27 to 4.24 mmHg in skeletal muscle. Given the basal PO2 of approximately 10 mmHg, the Mb contribution to O2 transport appears insignificant in myocardium. In skeletal muscle, Mb-facilitated diffusion begins to contribute significantly only when the PO2 approaches the P50. In marine mammals, the high Mb concentration confers a predominant role for Mb in intracellular O2 transport under all physiological conditions. The Q10 of the DMb ranges from 1.3 to 1.6. The Mb diffusion data indicate that the postulated gel network in the cell must have a minimum percolation cutoff size exceeding 17.5 A and does not impose tortuosity within the diffusion root mean-square displacement. Moreover, the similar Q10 for the DMb of solution versus cell Mb suggests that any temperature-dependent alteration of the postulated cell matrix does not significantly affect protein mobility.

H NMR techniques can discriminate the deoxy-Mb and deoxy-Hb proximal histidyl N δ H signals in vivo. These signals reflect the change in tissue and vascular P O 2. As P O 2 falls, the proximal histidyl N δ H signal of deoxy-Mb and deoxy-Hb signal intensity increases (Kreutzer et al., 1992;Ponganis et al., 2002;Tran et al., 1999 , consistent with the value observed in rat myocardium. Equipoise P O 2 analysis revealed that Mb is the predominant intracellular O 2 transporter in elephant seals during eupnea and apnea.