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The 31P NMR spectrum of energy-related metabolites under strictly aerobic conditions in rapidly respiring tissues under physiological conditions has been approached by the study of the 31P NMR signals in vivoand in freeze-trapped organs. Freezing the head of the anesthetized animal by liquid N2, excision of the brain tissue (white and gray matter) at -196°, and transfer to the NMR tube occurs without alteration of the metabolite concentrations. The sample is warmed to the region -15° to -10°, at which temperatures there is sufficient mobility for recording 31P NMR at concentrations characteristic of brain tissues (∼5 mM) with an adequate signal to noise ratio in 10 min but insufficient mobility for significant enzymatic activity. A ∼0.4-sec acquisition time is adequate for nuclear relaxation and a 10-min scan gives an adequate signal to noise ratio. Metabolism of creatine phosphate, Pi, and sugar phosphates occurs by 1 hr at -10° and 2 hr at -12°. Extrapolation of the approximately zero order kinetics of disappearance of creatine phosphate and appearance of Pisuggests that <10% of these two metabolites has been altered in the time of the first measurement.A comparison of the freeze-trapped state and the in vivometabolite pattern is afforded in preliminary experiments on the head of the living mouse (brain and skeletal tissue) in aerobic and anaerobic states. Longer relaxation times and mild hypoxia due to the restricted diameter of the NMR tube gives significantly lower creatine phosphate/ATP values for this condition. Both direct in vivoand freeze-trapped assays of energy-related metabolites afford excellent approaches to the detection of anoxia and to the evaluation of metabolic control in hypoxic conditions.

With the aid of two kinds of spin-labeled protohemins, the nature of the heme-protein interaction of various hemoproteins was investigated. Di- and mono-spin-labeled protohemins were prepared from protohemin and 2,2,5,5-tetramethyl-3-aminopyrrolidine-1-oxyl. The spin-labeled hemins were recombined with apoproteins of hemoglobin (Hb), myoglobin (Mb), cytochrome cperoxidase (EC 1.11.1.5) and horseradish peroxidase (EC 1.11.1.7). Electron paramagnetic resonance spectra of the di- and mono-spin-labeled hemoglobin in 0.1 M potassium phosphate buffer, pH 7.0, at 20°C exhibited moderate immobilization of the labels, while that of cytochrome cperoxidase showed stronger immobilization. Di-spin-labeled horseradish peroxidase showed an EPR spectrum of a simple broad line with peak-to-peak line width of 35 G. This broadening is due to spin-spin interaction between the two labels attached at the 6- and 7-positions of the porphyrin ring. Ligand binding to the spin-labeled hemoproteins altered the EPR line shapes and amplitudes. The former is attributed to the changes in the mobility of the labels and the latter to the magnetic dipolar interaction between the heme iron and free radical. From the strength of this interaction the distance between the iron and the nitroxide radical may be calculated. In the hemoproteins examined, the distances are: Hb 12.5 Å, Mb 12.0 Å, cytochrome peroxidase ∼14 Å, and horseradish peroxidase 9.0 Å.