Biopreservation and Reversal of Oxidative Injury During Blood Storage by a Novel Curcumin-based Gel Formulation.

Blood storage lesion induces cytosolic and membrane changes driven in part by hemoglobin (Hb) oxidation reactions within red blood cells (RBCs). A novel gel formulation containing the antioxidant curcuminoids in a biocompatible solvent system was used to deliver curcumin into RBCs. Incubation of peroxide treated RBCs stored in PBS with curcumin gel led to a reduction in prooxidant ferrylHb and recovery in ATP.

Changes in hemoglobin oxidation and band 3 during blood storage impact oxygen sensing and mitochondrial bioenergetic pathways in the human pulmonary arterial endothelial cell model.

Red blood cells (RBCs) undergo metabolic, oxidative, and physiological changes during storage, collectively described as the "storage lesion." The impact of storage on oxygen homeostasis, following transfusion, is not fully understood. We show that RBC storage induces changes in oxygen binding that were linked to changes in oxygen sensing (hypoxia-inducible factor, HIF-1α) mechanisms and mitochondrial respiration in human pulmonary arterial endothelial cells (HPAECs).

Effects of α subunit substitutions on the oxidation of βCys93 and the stability of sickle cell hemoglobin.

The β subunit substitutions, F41Y and K82D, in sickle cell hemoglobin (Hb) (βE6 V) provides significant resistance to oxidative stress by shielding βCys93 from the oxidizing ferryl heme. We evaluated the oxidative resistance of βCys93 to hydrogen peroxide (HO) in α subunit mutations in βE6 V (at both the putative and lateral contact regions) that included (1) αH20Q/βE6 V; (2) αH50Q/βE6 V; (3) αH20Q/H50Q/βE6 V; (4) αH20R/βE6 V; and (5) αH20R/H50Q/βE6 V. Estimation by mass spectrometry of irreversible oxidation of βCys93 to cysteic acid (CA) was unchanged or moderately increased in the single mutants harboring a H20Q or H50Q substitution when compared to control (βE6 V).

Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles.

The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes-sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent β-globin posttranslational modifications, including the irreversible oxidation of βCys93 and the ubiquitination of βLys96 and βLys145.

Sodium nitrite potentiates renal oxidative stress and injury in hemoglobin exposed guinea pigs.

Methemoglobin-forming drugs, such as sodium nitrite (NaNO2), may exacerbate oxidative toxicity under certain chronic or acute hemolytic settings. In this study, we evaluated markers of renal oxidative stress and injury in guinea pigs exposed to extracellular hemoglobin (Hb) followed by NaNO2 at doses sufficient to simulate clinically relevant acute methemoglobinemia. NaNO2 induced rapid and extensive oxidation of plasma Hb in this model.

Post-translational transformation of methionine to aspartate is catalyzed by heme iron and driven by peroxide: a novel subunit-specific mechanism in hemoglobin.

A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and β subunits of adult Hb (HbA) Bristol-Alesha (β67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins.

Isolated Hb Providence β82Asn and β82Asp fractions are more stable than native HbA(0) under oxidative stress conditions.

We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the β-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or β-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two β-subunit variants with single amino acid mutations at βLys82→Asp (βK82D) and at βLys82→Asn (βK82N) positions and binds oxygen at lower affinity than wild type HbA.

Simultaneous quantification and identification using 18O labeling with an ion trap mass spectrometer and the analysis software application "ZoomQuant".

Stable isotope labeling with (18)O is a promising technique for obtaining both qualitative and quantitative information from a single differential protein expression experiment. The small 4 Da mass shift produced by incorporation of two molecules of (18)O, and the lack of available methods for automated quantification of large data sets has limited the use of this approach with electrospray ionization-ion trap (ESI-IT) mass spectrometers. In this paper, we describe a method of acquiring ESI-IT mass spectrometric data that provides accurate calculation of relative ratios of peptides that have been differentially labeled using(18)O.

ZoomQuant: an application for the quantitation of stable isotope labeled peptides.

The main goal of comparative proteomics is the quantitation of the differences in abundance of many proteins between two different biological samples in a single experiment. By differentially labeling the peptides from the two samples and combining them in a single analysis, relative ratios of protein abundance can be accurately determined. Protease catalyzed (18)O exchange is a simple method to differentially label peptides, but the lack of robust software tools to analyze the data from mass spectra of (18)O labeled peptides generated by common ion trap mass spectrometers has been a limitation.