Haemozoin (Hz) is a haem aggregate produced in some blood-feeding organisms. There is a general belief that Hz formation would be a protective mechanism against haem toxicity. Here we show that when aggregated into Hz, haem is less deleterious than its free form. When haem was added to phosphatidylcholine (PC) liposomes, there was an intense stimulation of oxygen consumption, which did not occur when Hz was incubated with the same preparation. Evaluation of oxygen radical attack to lipids, by measurement of thiobarbituric acid reactive substances (TBARS), showed significantly lower levels of lipid peroxidation in samples containing PC liposomes incubated with Hz than with haem. However, TBARS production induced by Hz was much higher when using 2-deoxyribose (2-DR) as substrate, than with PC liposomes. Spin-trapping analysis by electron paramagnetic resonance (EPR) of Hz and tert-butylhydroperoxide (tert-BuOOH) showed that production of methoxyl and tert-butoxyl radicals was only slightly reduced compared to what was observed with haem. Interestingly, when large Hz crystals were used in 2-DR TBARS assays and tertBuOOH EPR experiments, the pro-oxidant effects of Hz were strongly reduced. Moreover, increasing concentrations of Hz did not induce erythrocyte lysis, as occurred with haem. Thus, the reduced capacity of Hz to impose radical damage seems to result from steric hindrance of substrates to access the aggregated haem, that becomes less available to participate in redox reactions. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
Paramagnetic 3d9 [Ni(CN)4]3- complexes, with the unpaired electron in a d
x
2−
y
2
orbital, have been generated
from diamagnetic Ni(II) 3d8 cyanide complexes in KCl or NaCl host lattices. The magnetic and quadrupolar
hyperfine interactions with the four 14N, hidden in the CW-EPR (continuous wave electron paramagnetic
ressonance) line width, are revealed by pulsed EPR and ENDOR (electron nuclear double resonance) angular
variation studies. Ab initio embedded UMP2 cluster calculations, which take into account short- and long-range crystal interactions, confirm the unpaired electron orbital assignment and are in agreement with the
measured hyperfine values. The trend of 14N A
iso values (7.7 MHz for NaCl and 6.8 MHz for KCl) is given
by the Ni−CN distance, modified in each host lattice. Small asymmetry factors (about 0.04) for the 14N
quadrupolar tensor are obtained both in experiment and in theory. The experimental lines and the calculations
indicate spin density at the cations of both lattices. Experimental and theoretical data indicate that lattice
chlorine ions near the Ni atom, in axial positions, are not chemically coordinated to Ni. Spin density on these
ions arises only from spin polarization of their valence orbitals and of the valence orbitals of the complex.
Electron-nuclear double-resonance (ENDOR) spectra of protons coupled to molybdenum(V) in reduced xanthine oxidase samples have been recorded. Under appropriate conditions these protons may be studied without interference from protons coupled to reduced iron-sulfur centers. Spectra have been obtained for the molybdenum(V) species known as Rapid, Slow, Inhibited, and Desulfo Inhibited. Resonances corresponding to at least nine protons or sets of protons are observed for all four species, with coupling constants in the range 0.08-4 MHz. Most of these protons do not exchange when 2H2O is used as solvent. Additional protons giving couplings up to 40 MHz are also detected. These correspond to EPR-detectable protons studied in earlier work. The strongly coupled protons may be replaced by 2H, through appropriate use of 2H2O or of 2H-substituted substrates, with consequent disappearance of the 1H resonances. In most cases the corresponding 2H ENDOR features have also been observed. The nature of the various coupled protons is briefly discussed. Results permit specific conclusions to be drawn about the structures of the Inhibited and Desulfo Inhibited species. In particular, the data indicate that the aldehyde residue of the Inhibited species has been oxidized and that the four protons derived from the ethylene glycol molecule in the Desulfo Inhibited species are not all equivalent. Recent assignments [Edmondson, D.E., & D'Ardenne, S.C. (1989) Biochemistry 28, 5924-5930] of the weakly coupled protons in the latter species appear not to be soundly based. The possibility of obtaining more detailed structural information from the spectra is briefly considered.(ABSTRACT TRUNCATED AT 250 WORDS)
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