Hiroshi Taniuchi

UNLABELLED: An ideal hypoxia imaging agent should have high membrane permeability for easy access to intracellular mitochondria and low redox potential to confer stability in normal tissue, but it should be able to be reduced by mitochondria with abnormally high electron concentrations in hypoxic cells. In this context, nitroimidazole residues are not considered to be essential. In this study, Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM), a 62Cu-bisthiosemicarbazone complex, with high membrane permeability and low redox potential, was evaluated as a possible hypoxia imaging agent, using electron spin resonance spectrometry and the Langendorff isolated perfused rat heart model as well as rat heart left anterior descending occlusion model. METHODS: Nonradioactive Cu-ATSM was incubated with rat mitochondria, after which reduction of Cu(II) to Cu(I) was measured with electron spin resonance. As a model of hypoxic mitochondria, rotenone (Complex I inhibitor)-treated mitochondria were used. RESULTS: In this study, Cu-ATSM was reduced by hypoxic but not by normal mitochondria. CONCLUSION: Thus, retention of 62Cu-ATSM was studied serially in perfused rat hearts under conditions of normoxia (95% O2 + 5% CO2), hypoxia (95% N2 + 5% CO2) and reoxygenation (95% O2 + 5% CO2). In normoxia and reoxygenation, 62Cu-ATSM injected as a single bolus showed low retention (23.77% and 22.80%, respectively) 15 min after injection, but retention was increased markedly under hypoxic conditions (81.10%). Also, in the in vivo left anterior descending occluded rat heart model, 62Cu-ATSM retention was inversely correlated with accumulation of 201Tl, a relative myocardial blood flow marker.

In order to assess the effect of the covalently bonded heme on the stabilization of the conformation of horse heart cytochrome c, both the heme-free apo-cytochrome c and the ironfree, porphyrin-cytochrome c were prepared.The properties of these derivatives in solution were compared with those of native cytochrome c by the study of their viscosity, circular dichroism, and fluorescence.Porphyrin-cytochrome c appears to have a compact structure similar to native cytochrome c, although it is less stable to heat denaturation.It appears that the coordination of the iron atom of the heme with histidine-18 and methionine-80 results in an increase in the stability of this protein, but is not required for the folding of porphyrin-cytochrome c into a compact conformation.Apo-cytochrome c has the properties of a disordered structure.However, apo-cytochrome c can bind with the disordered heme-containing fragment of residues 1 to 65, prepared by the method of Corradin and Harbury ((1970) Biochim.Biophys.Acfa 221, 489), and forms a product with spectral properties closely resembling native cytochrome c in the same manner as does the COOH-terminal fragment of residues 66 to 104 (CORRADIN, G., AND HARBURY, H. A. (1971) Proc.Nat.Acad.Sci.U. S. A. 68, 3036).The observations indicate that the presence of the porphyrin ring covalently bonded with cysteines 14 and 17 is essential for cytochrome c to exist in the stable native conformation.

Two inactive fragments of staphylococcal nuclease (a protein consisting of 149 residues, devoid of sulfhydryl groups and disulfide bonds) have been prepared; one (nuclease-P(,126)) contains Residues 1 through 126, and the other (nuclease-P(127_ 49)) contains Residues 127 through 149. Studies of nuclease-P(I 1 26), employing circular dichroism, optical rotation, immunodiffusion, and solvent perturbation, reveal a loose and disorganized structure very different from that of nuclease. Nuclease-P( 127 .149) is also structureless, as determined by measurements of circular dichroism and the fluorescence spectrum of the tryptophan residue.

The conformational parameters of a nuclease produced by growing cultures of Staphylococcus aureus have been determined. This nuclease exhibits both ribonuclease and deoxyribonuclease activities. It has been shown to have a pH optimum of 9.2 and an absolute requirement for calcium ions. The experimental data are compatible with a compact globular protein of mol wt 17,000 and with a helical content of approximately 45%.

Pyrocatechasewas purified from extracts of benzoateinduced cells of Pseudomonas arvilta.The most purified preparation had a specific activity of 29.6, about 50% higher than that previously reported, and was homogeneous as judged by ultracentrifugation and by electrophoresis.The molecular weight was estimated to be approximately 90,000.The enzyme contained 2 g atoms of iron per mole of enzyme protein.Although the chemical determination of the valency state of iron has given inconclusive results, experiments with various chelating agents and the effects of oxidizing and reducing agents indicated that the iron in the native enzyme may be in the trivalent state.The concentrated solution of highly purified pyrocatechase had a pronounced red color with a broad absorption between 390 and 650 mp.The peak was at about 440 ml.c, and the molecular absorbance at 440 rnp was estimated to be 46'70.The trivalent iron bound to the enzyme appears to be responsible for the visible absorption band and functions as an integral part of the enzyme, since under a variety of conditions a loss of enzyme activity occurred in parallel with the disappearance of the absorbance in the visible range.By the addition of the substrate, catechol, under anaerobic conditions, the color of the enzyme solution changed to greyish blue with a concurrent increase of the absorbance