1,721,480 research outputs found
Peroxynitrite Detoxification by Human Haptoglobin:Hemoglobin Complexes: A Comparative Study.
No full textHaptoglobin (Hp) reacts with dimeric hemoglobin (Hb), shifts the equilibrium in favor of the αβ dimer and displays heme-based catalysis. Here, kinetics of peroxynitrite scavenging by ferric human haptoglobin1-1:hemoglobin and haptoglobin2-2:hemoglobin complexes (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively) is reported between pH 6.2 and 8.3 at 20.0 °C. The reactivity of Hp1-1:Hb(III) and Hp2-2:Hb(III) against peroxynitrite is similar to that of tetrameric Hb(III), reflecting the R-like structure of the αβ dimers of Hb(III) bound to Hp. To investigate the protective role of Hp1-1:Hb(III) and Hp2-2:Hb(III) against peroxynitrite-mediated nitration, the relative yield of nitro-l-tyrosine formed by the reaction of peroxynitrite with free l-tyrosine was determined. Interestingly, both Hp1-1:Hb(III) and Hp2-2:Hb(III) impair peroxynitrite-mediated nitration of free l-tyrosine. Therefore, Hp:Hb complexes could participate to the detoxification of reactive nitrogen and oxygen species in vivo, contributing to prevent extra-erythrocytic Hb-induced damage during hemolytic crisis
Kinetic and thermodynamic analysis of leech-derived tryptase inhibitor interaction with bovine tryptase and bovine trypsin
Full text linkThe interaction of leech-derived tryptase inhibitor (LDTI) with bovine liver capsule tryptase (BLCT) and bovine trypsin has been studied using both thermodynamic and kinetic approaches. Several differences were detected: (i) the equilibrium affinity of LDTI for BLCT (K-a = 8.9 x 10(5) M-1) is about 600-fold lower than that for bovine trypsin (K-a = 5.1 x 10(8) M-1); (ii) LDTI behaves as a purely non-competitive inhibitor of BLCT, while it is a purely competitive inhibitor of bovine trypsin. These functional data are compared with those previously reported for the LDTI binding to human tryptase, where tight inhibition occurs at two of the four active sites of the tetramer (K-a = 7.1 x 10(8) M-1). Amino acid sequence alignment of BLCT, human beta II-tryptase and bovine trypsin allows us to infer some possible structural basis for the observed functional differences
Oxygen dissociation from ferrous oxygenated human hemoglobin:haptoglobin complexes confirms that in the R-state α and β chains are functionally heterogeneous.
Full text linkThe adverse effects of extra-erythrocytic hemoglobin (Hb) are counterbalanced by several plasma proteins devoted to facilitate the clearance of free heme and Hb. In particular, haptoglobin (Hp) traps the αβ dimers of Hb, which are delivered to the reticulo-endothelial system by CD163 receptor-mediated endocytosis. Since Hp:Hb complexes show heme-based reactivity, kinetics of O2 dissociation from the ferrous oxygenated human Hp1-1:Hb and Hp2-2:Hb complexes (Hp1-1:Hb(II)-O2 and Hp2-2:Hb(II)-O2, respectively) have been determined. O2 dissociation from Hp1-1:Hb(II)-O2 and Hp2-2:Hb(III)-O2 follows a biphasic process. The relative amplitude of the fast and slow phases ranges between 0.47 and 0.53 of the total amplitude, with values of koff1 (ranging between 25.6 ± 1.4 s-1 and 29.1 ± 1.3 s-1) being about twice faster than those of koff2 (ranging between 13.8 ± 1.6 s-1 and 16.1 ± 1.2 s-1). Values of koff1 and koff2 are essentially the same independently on whether O2 dissociation has been followed after addition of a dithionite solution or after O2 displacement by a CO solution in the presence of dithionite. They correspond to those reported for the dissociation of the first O2 molecule from tetrameric Hb(II)-O2, indicating that in the R-state α and β chains are functionally heterogeneous and the tetramer and the dimer behave identically. Accordingly, the structural conformation of the α and β chains of the Hb dimer bound to Hp corresponds to that of the subunits of the Hb tetramer in the R-state
Ferric microperoxidase-11 catalyzes peroxynitrite isomerization
No full textMicroperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c offering the possibility to study the reactivity of the heme group relatively unshielded by the protein. Here, the peroxynitrite isomerizadon to NO3- catalyzed by ferric MP11 (MP11-Fe(III)) is reported. Data were obtained between pH 3.6 and 8.1, at 20.0 degrees C. The value of the second-order rate constant (k(on)) for peroxynitrite isomerization to NO3- by MP11-Fe(III) decreases from (1.1 +/- 0.1) x 10(5) M-1 s(-1), at pH 3.6, to (6.1 +/- 0.6) x 10(3) M(-1)s(-1), at pH 8.1.The pH dependence of k(on) (pK(a) = 6.9) suggests that peroxynitrous acid reacts preferentially with MP11-Fe(III). The MP11-Fe(III)-catalyzed isomerization of peroxynitrite to NO3- has been ascribed to the reactive penta-coordinated heme-Fe atom of MP11-Fe(III). In fact, cyanide binding to the sixth coordination position of the heme-Fe atom inhibits the MP11-Fe(III)-catalyzed isomerization of peroxynitrite to NW. The values of the first-order rate constant (K-0) for isomerization of peroxynitrite to NO3- in the presence of the MP11-Fe(III)-CN complex are superimposable to those obtained in the absence of MP-Fe(III). Values of k(on) for peroxynitrite isomerization to NO3- by MP11-Fe(III) overlap those obtained for penta-coordinated cardiolipin-cytochrome c complex and for carboxymethylated cytochrome c in absence and presence of cardiolipin. Present results highlight the role of the heme-Fe(III) co-ordination state in the modulation of cytochrome c reactivity. (C) 2014 Elsevier Inc. All rights reserved
The nitrite reductase activity of ferrous human hemoglobin:haptoglobin 1-1 and 2-2 complexes
No full textStructural investigations on Zmp1 from Mycobacterium tuberculosis, a Zn-metalloprotease that prevents inflammasome activation and phagosome maturation
No full textPeroxynitrite-mediated oxidation of ferrous carbonylated myoglobin is limited by carbon monoxide dissociation
No full textCyanide binding to ferrous and ferric microperoxidase-11
No full textMicroperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c (cytc). MP11 is characterized by a covalently linked solvent-exposed heme group, the heme-Fe atom being axially coordinated by a histidyl residue. Here, the reactions of ferrous and ferric MP11 (MP11-Fe(II) and MP11-Fe(III), respectively) with cyanide have been investigated from the kinetic and thermodynamic viewpoints, at pH 7.0 and 20.0 A degrees C. Values of the second-order rate constant for cyanide binding to MP11-Fe(II) and MP11-Fe(III) are 4.5 M-1 s(-1) and 8.9 x 10(3) M-1 s(-1), respectively. Values of the first-order rate constant for cyanide dissociation from ligated MP11-Fe(II) and MP11-Fe(III) are 1.8 x 10(-1) s(-1) and 1.5 x 10(-3) s(-1), respectively. Values of the dissociation equilibrium constant for cyanide binding to MP11-Fe(II) and MP11-Fe(III) are 3.7 x 10(-2) and 1.7 x 10(-7) M, respectively, matching very well with those calculated from kinetic parameters so that no intermediate species seem to be involved in the ligand-binding process. The pH-dependence of cyanide binding to MP11-Fe(III) indicates that CN- is the only binding species. Present results have been analyzed in parallel with those of several heme-proteins, suggesting that (1) the ligand accessibility to the metal center and cyanide ionization may modulate the formation of heme-Fe-cyanide complexes, and (2) the general polarity of the heme pocket and/or hydrogen bonding of the heme-bound ligand may affect cyanide exit from the protein matrix
Insights into Proteasome Conformation Dynamics and Intersubunit Communication
No full textA recently published paper applies cryo-electron microscopy (EM) studies and biochemical/genetic approaches for the elucidation of the mechanisms linking nucleotide binding by ATPases, proteasome conformation dynamics, and gate opening of the 20S core particle. These insights potentially represent a milestone in our understanding of the structural dynamics of the 26S proteasome
Ferricyanide-mediated oxidation of ferrous nitrosylated sperm whale myoglobin involves the formation of the ferric nitrosylated intermediate
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