Dataset Information

Towards understanding non-equivalence of α and β subunits within human hemoglobin in conformational relaxation and molecular oxygen rebinding.

ABSTRACT: Picosecond to millisecond laser time-resolved transient absorption spectroscopy was used to study molecular oxygen (O₂) rebinding and conformational relaxation following O₂ photodissociation in the α and β subunits within human hemoglobin in the quaternary R-like structure. Oxy-cyanomet valency hybrids, α₂(Fe²⁺-O₂)β₂(Fe³⁺-CN) and α₂(Fe³⁺-CN)β₂(Fe²⁺-O₂), were used as models for oxygenated R-state hemoglobin. An extended kinetic model for geminate O₂ rebinding in the ferrous hemoglobin subunits, ligand migration between the primary and secondary docking site(s), and nonexponential tertiary relaxation within the R quaternary structure, was introduced and discussed. Significant functional non-equivalence of the α and β subunits in both the geminate O₂ rebinding and concomitant structural relaxation was revealed. For the β subunits, the rate constant for the geminate O₂ rebinding to the unrelaxed tertiary structure and the tertiary transition rate were found to be greater than the corresponding values for the α subunits. The conformational relaxation following the O₂ photodissociation in the α and β subunits was found to decrease the rate constant for the geminate O₂ rebinding, this effect being more than one order of magnitude greater for the β subunits than for the α subunits. Evidence was provided for the modulation of the O₂ rebinding to the individual α and β subunits within human hemoglobin in the R-state structure by the intrinsic heme reactivity through a change in proximal constraints upon the relaxation of the tertiary structure on a picosecond to microsecond time scale. Our results demonstrate that, for native R-state oxyhemoglobin, O₂ rebinding properties and spectral changes following the O₂ photodissociation can be adequately described as the sum of those for the α and β subunits within the valency hybrids. The isolated β chains (hemoglobin H) show similar behavior to the β subunits within the valency hybrids and can be used as a model for the β subunits within the R-state oxyhemoglobin. At the same time, the isolated α chains behave differently to the α subunits within the valency hybrids.

SUBMITTER: Lepeshkevich SV

PROVIDER: S-EPMC8153241 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Hemoglobin (Hb) based oxygen carriers (HBOCs) are designed to minimize the toxicity of extracellular Hb, while preserving its high oxygen-carrying capacity for oxygen delivery to cells. Polymerized human Hb (PolyHb) is a novel type of nanosized HBOC synthesized via glutaraldehyde-mediated crosslinking of free Hb, and which preserves the predominant quaternary state during the crosslinking reaction (low oxygen affinity tense (T) quaternary state PolyHb is synthesized at 0% Hb oxygen saturation, and high oxygen affinity relaxed (R) quaternary state PolyHb is synthesized at 100% Hb oxygen saturation). Major potential applications for PolyHbs, and HBOCs in general, include oxygenation of bioreactor systems containing large liver cell masses, and ex-vivo perfusion preservation of explanted liver grafts. The toxicity of these compounds toward liver cells must be evaluated before testing their use in these complex systems for oxygen delivery. Herein, we characterized the effect of PolyHbs on the hepatoma cell line HepG2/C3A, used as a model hepatocyte and as a cell line used in some investigational bioartificial liver support devices. HepG2/C3A cells were incubated in cell culture media containing PolyHbs or unmodified Hb at concentrations up to 50 mg/mL and for up to 6 days. PolyHbs were well tolerated at a dose of 10 mg/mL, with no significant decrease in cell viability; however, proliferation was inhibited as much as 10-fold after 6 days of exposure at 50 mg/mL. Secretion of albumin, and urea, as well as glucose and ammonia removal were measured in presence of 10 mg/mL of PolyHbs or unmodified Hb. In addition, methoxy- and ethoxy-resorufin deacetylase (MROD and EROD) activities, which reflect cytochrome P450 metabolism, were measured. R-state PolyHb displayed improved or intact activity in 3 out of 7 functions compared to unmodified Hb. T-state PolyHb displayed improved or intact activity in 4 out of 7 functions compared to unmodified Hb. Thus, PolyHbs, both in the R-state and T-state, are safer to use at a concentration of 10 mg/mL as compared to unmodified Hb in static culture liver-related applications.

Project description:Purpose To generate magnetic resonance (MR) imaging-derived, oxygen-hemoglobin dissociation curves and to map fetal-placental oxygen-hemoglobin affinity in pregnant mice noninvasively by combining blood oxygen level-dependent (BOLD) T2* and oxygen-weighted T1 contrast mechanisms under different respiration challenges. Materials and Methods All procedures were approved by the Weizmann Institutional Animal Care and Use Committee. Pregnant mice were analyzed with MR imaging at 9.4 T on embryonic days 14.5 (eight dams and 58 fetuses; imprinting control region ICR strain) and 17.5 (21 dams and 158 fetuses) under respiration challenges ranging from hyperoxia to hypoxia (10 levels of oxygenation, 100%-10%; total imaging time, 100 minutes). A shorter protocol with normoxia to hyperoxia was also performed (five levels of oxygenation, 20%-100%; total imaging time, 60 minutes). Fast spin-echo anatomic images were obtained, followed by sequential acquisition of three-dimensional gradient-echo T2*- and T1-weighted images. Automated registration was applied to align regions of interest of the entire placenta, fetal liver, and maternal liver. Results were compared by using a two-tailed unpaired Student t test. R1 and R2* values were derived for each tissue. MR imaging-based oxygen-hemoglobin dissociation curves were constructed by nonlinear least square fitting of 1 minus the change in R2*divided by R2*at baseline as a function of R1 to a sigmoid-shaped curve. The apparent P50 (oxygen tension at which hemoglobin is 50% saturated) value was derived from the curves, calculated as the R1 scaled value (x) at which the change in R2* divided by R2*at baseline scaled (y) equals 0.5. Results The apparent P50 values were significantly lower in fetal liver than in maternal liver for both gestation stages (day 14.5: 21% ± 5 [P = .04] and day 17.5: 41% ± 7 [P < .0001]). The placenta showed a reduction of 18% ± 4 in mean apparent P50 values from day 14.5 to day 17.5 (P = .003). Reproduction of the MR imaging-based oxygen-hemoglobin dissociation curves with a shorter protocol that excluded the hypoxic periods was demonstrated. Conclusion MR imaging-based oxygen-hemoglobin dissociation curves and oxygen-hemoglobin affinity information were derived for pregnant mice by using 9.4-T MR imaging, which suggests a potential to overcome the need for direct sampling of fetal or maternal blood. Online supplemental material is available for this article.

Dataset Information

Towards understanding non-equivalence of α and β subunits within human hemoglobin in conformational relaxation and molecular oxygen rebinding.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets