Studies on the influence of nitrite on methemoglobin formation in Amazonian fishes

Twenty one species of fishes, collected from the Rio Solim?es and a tributary lake in the Amazon Basin near Manaus, showed a wide range of methemoglobin formation 1 hr after a dose of 30 mg/kg of sodium nitrite i.p. Methemoglobin formation in two experimental fishes, Brycon cf. melanopterum and Semaprochilodus insignis, maintained in tanks in our INPA laboratory, was studied in detail. Both fishes survived a dose of 10 mg/kg of nitrite i.p. but usually died within 3 hr of a dose of 30 mg/kg with levels of blood methemoglobin in excess of 80%. Methemoglobin produced in vitro by addition of nitrite to fresh blood was slowly reduced back to hemoglobin over a period of several hours at room temperature. Hemoglobin in hemolysates was auto-oxidized to methemoglobin at pH 6.1 and below but not at 6.9 and above.     

Analytical chromatography of hemins on silica gel.

The analytical chromatography of proto, meso, hemato, deutero, and diacetyldeutero hemins, and of Mn^III, Co^III, and Fe^III mesoporphyrins by tlc on silica gel is described.     

Proton NMR study of the cyanide metmyoglobin reconstituted with meso-tetraalkylhemins. Dynamic free rotation of the synthetic hemins in the heme pocket

Incorporation of the three synthetic hemins, Fe(III) meso-tetraalkylporphyrins with the methyl, ethyl, or n-propyl groups, into apomyoglobin was followed by spectrophotometry, and the stoichiometric complex formation was confirmed. The reconstituted myoglobins bind with an equimolar amount of cyanide to exhibit visible absorption peaks at 419, 570, and 608 nm. The spectral feature was independent of the cyanide concentrations. Proton NMR spectra of the cyanide complexes resolved the pyrrole-proton signals of the hemins in a -5 to -15-ppm region, which is comparable with that of the corresponding signals of deuterohemin-containing low-spin methemoproteins. These spectral observations indicate the presence of the NC-Fe-N(His-F8) structure in the presently reconstituted cyanide metmyoglobins. The pyrrole-proton NMR signals of the hemins in cyanide metmyoglobins appeared as a singlet, doublet, or quartet for the methyl, ethyl, or n-propyl hemin complexes, respectively. The systematic NMR spectral changes suggest the dynamic free rotation of the alkylhemins about the Fe-N(His-F8) bond. Temperature-dependent NMR spectral transition of the meso-tetraethylhemin-reconstituted myoglobin was consistent with thermally regulated dynamic free rotation of the hemin in the myoglobin heme pocket.     

Studies on the mechanism of selective retention of porphyrins and metalloporphyrins by cancer cells

Comparative studies of the toxicity, stability, and retention of the water-soluble porphyrin, tetraphenylporphyrin sulfonate (TPPS), and its complex with Mn(III), have been made with the human breast cancer cell line MCF-7 wild type, and an adriamycin-resistant line derived from it, termed AdrR. Based on growth inhibition, we determined the maximum non-toxic concentration of MnTPPS tolerated by these cells. The integrity of MnTPPS in vitro was investigated by fluorescence microscopy, and we found that there is very little dissociation of MnTPPS within these cells within 4 days. We report novel proton magnetic resonance relaxation measurements of the bulk water of cells in a gel matrix undergoing perfusion. A slightly greater net uptake of MnTPPS in the wild-type cells was observed compared to AdrR; however, there was no significant difference in retention of MnTPPS. These results indicate that over a period of several hours the mechanism of selective retention of these compounds in tumour cells is not due to specific interaction with heme-binding protein, of which there is enhanced expression in the resistant cells. The fact that the net rate of washout of MnTPPS is approximately the same as the net rate of uptake also appears to eliminate compartmentalization or enzymatic modification of MnTPPS within these cells.     

Molecular and complex-chemical studies on methemoglobin from Chironomus thummi thummi and various isolated fractions]

1. Six different hemoglobin (Hb) fractions were isolated and characterized from the larvae of Chironomus thummi thummi using column chromatographic procedures. 2. Chromatographic and sedimentation-analytic studies (sedimentation coefficients of 2.0 +/- 0.2 (S)) have shown three Hb fractions to exist basically in a monomeric form. The molecular weight of component M-2 was determined by sedimentation equilibrium technique to be 15,470 +/- 400. The dimeric Hb was found to have sedimentation coefficients of 3.0 +/- 0.1 (S) in the weakly acidic pH region. In alkaline milieu, the reversible dissociation proceeds into the monomeric molecules (S20, W = 1.9 +/- 0.1 (S)). Molecular weights vary between pH 5.7 and 9.8 not only with hydrogen ion concentration, but also with protein concentration in correspondence with a dissociation-association equilibrium consisting of monomers and dimers. 3. For the Hb fraction M-2, a friction ratio of f/fo = 1.03 was calculated, suggesting an almost spherical shape of this protein. In contrast, the dimeric component appears to have a much more asymmetric structure (f/fo = 1.19). 4. The indivdual MetHb fractions bind the ligands: fluoride, imidazole and azide with different affinities.