Peroxidase activity of erythrocytes hemoglobin under action of low-frequency vibration

Background. Hemoglobin is a hemoprotein which in the presence of oxidative equivalents, such as H2O2, can act as peroxidase with a very high oxidative potential. Hemoglobin oxidation is accompanied by generation of highly oxidized forms of iron and globin radicals that have high oxidative activity and are toxic to cells. In addition, peroxidase activity may indicate structural changes that occur in the hemoglobin molecule as a result of chemical modification. Materials and Methods. Erythrocyte suspension was subjected to vibration for 3 h within the frequency range from 8 to 32 Hz with amplitudes of 0.5 ± 0.04 and 0.9 ± 0.08 mm. At certain intervals, hemoglobin peroxidase activity was determined together with the content of its ligand forms in the hemolysates of cells. Additionally, experiments were performed to investigate the mechanism and calculate the kinetic parameters of peroxidase reaction. Results and Discussion. Experimental data on low-frequency vibrations effect on erythrocyte hemoglobin peroxidase activity were analyzed. The kinetics of the oxidation reaction of p-phenylenediamine by hemoglobin in erythrocytes was studied. It was found that peroxidase oxidation has a ping-pong mechanism. The kinetic parameters of the peroxidase reaction involving hemoglobin were determined. The change of kinetic parameters after two-hour exposure to the incubation medium and low-frequency vibration was studied. A possible mechanism of action of hemoglobin in oxidation reactions involving H2O2 was proposed. Conclusion. Any effect that initiates the formation of methemoglobin leads to an increase in the peroxidase activity of hemoglobin due to the involvement of the latter in the pseudoperoxidase cycle and the formation of toxic reactive globin radicals. The high content of oxyhemoglobin in the cell, observed under vibrations within the frequency range of 16–32 Hz with an amplitude of 0.9 ± 0.08 mm, can prevent its oxidation and involvement in the pseudoperoxidase cycle.

Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit

In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules. We recently engineered a hemoglobin with a single novel, reactive cysteine residue on the surface of the alpha subunit creating a single PEGylation site (βCys93Ala/αAla19Cys). This enabled homogenous PEGylation by maleimide-PEG with >80% efficiency and no discernible effect on protein function. However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions and cross-conjugation to endogenous thiol species in vivo. We therefore compared our maleimide-PEG adduct with one created using a mono-sulfone-PEG less susceptible to deconjugation. Mono-sulfone-PEG underwent reaction at αAla19Cys hemoglobin with > 80% efficiency, although some side reactions were observed at higher PEG:hemoglobin ratios; the adduct bound oxygen with similar affinity and cooperativity as wild type hemoglobin. When directly compared to maleimide-PEG, the mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for seven days in the presence of 1 mM reduced glutathione. Hemoglobin treated with mono-sulfone-PEG retained > 90% of its conjugation, whereas for maleimide-PEG < 70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation is certainly stable enough for acute therapeutic use as an oxygen therapeutic, for pharmaceuticals intended for longer vascular retention (weeks-months), reagents such as mono-sulfone-PEG may be more appropriate.

SOME MATHEMATICAL ASPECTS OF ABIOGENESIS

In this paper, we try to modestly, but strictly, using a mathematical approach, show that life was created with a certain goal and that the possibility of accidental origin of life is equal to zero. To calculate the probability of accidental creation of only one protein molecule, the basic building block of living systems, we selected a hemoglobin molecule that, because it is well studied, has become the standard for protein research. For comparison, together with this calculation, we also made a calculation of the probability of the systematic stacking of chaotically scattered playing cards from a certain height on a certain surface, a probability that is generally accepted as - improbably!

COVID-19: hemoglobin, iron, and hypoxia beyond inflammation. A narrative review

Coronavirus disease-19 (COVID-19) has been regarded as an infective-inflammatory disease, which affects mainly lungs. More recently, a multi-organ involvement has been highlighted, with different pathways of injury. A hemoglobinopathy, hypoxia and cell iron overload might have a possible additional role. Scientific literature has pointed out two potential pathophysiological mechanisms: i) severe acute respiratory syndrome-coronavirus-2 (SARS-CoV- 2) interaction with hemoglobin molecule, through CD147, CD26 and other receptors located on erythrocyte and/or blood cell precursors; ii) hepcidin-mimetic action of a viral spike protein, inducing ferroportin blockage. In this translational medicinebased narrative review, the following pathologic metabolic pathways, deriving from hemoglobin denaturation and iron metabolism dysregulation, are highlighted: i) decrease of functioning hemoglobin quote; ii) iron overload in cell/tissue (hyperferritinemia); iii) release of free toxic circulating heme; iv) hypoxemia and systemic hypoxia; v) reduction of nitric oxide; vi) coagulation activation; vii) ferroptosis with oxidative stress and lipoperoxidation; viii) mitochondrial degeneration and apoptosis. A few clinical syndromes may follow, such as pulmonary edema based on arterial vasoconstriction and altered alveolo-capillary barrier, sideroblastic-like anemia, endotheliitis, vasospastic acrosyndrome, and arterio- venous thromboembolism. We speculated that in COVID-19, beyond the classical pulmonary immune-inflammation view, the occurrence of an oxygen-deprived blood disease, with iron metabolism dysregulation, should be taken in consideration. A more comprehensive diagnostic/therapeutic approach to COVID-19 is proposed, including potential adjuvant interventions aimed at improving hemoglobin dysfunction, iron over-deposit and generalized hypoxic state.

Direct observation of ligand migration within human hemoglobin at work

Hemoglobin is one of the best-characterized proteins with respect to structure and function, but the internal ligand diffusion pathways remain obscure and controversial. Here we captured the CO migration processes in the tense (T), relaxed (R), and second relaxed (R2) quaternary structures of human hemoglobin by crystallography using a high-repetition pulsed laser technique at cryogenic temperatures. We found that in each quaternary structure, the photodissociated CO molecules migrate along distinct pathways in the α and β subunits by hopping between the internal cavities with correlated side chain motions of large nonpolar residues, such as α14Trp(A12), α105Leu(G12), β15Trp(A12), and β71Phe(E15). We also observe electron density evidence for the distal histidine [α58/β63His(E7)] swing-out motion regardless of the quaternary structure, although less evident in α subunits than in β subunits, suggesting that some CO molecules have escaped directly through the E7 gate. Remarkably, in T-state Fe(II)-Ni(II) hybrid hemoglobins in which either the α or β subunits contain Ni(II) heme that cannot bind CO, the photodissociated CO molecules not only dock at the cavities in the original Fe(II) subunit, but also escape from the protein matrix and enter the cavities in the adjacent Ni(II) subunit even at 95 K, demonstrating the high gas permeability and porosity of the hemoglobin molecule. Our results provide a comprehensive picture of ligand movements in hemoglobin and highlight the relevance of cavities, nonpolar residues, and distal histidines in facilitating the ligand migration.

100 years of sickle cell disease research: etiology, pathophysiology and rational drug design (part 1)

Background Sickle cell disease (SCD) is a chronic hemolytic disease caused by an altered hemoglobin molecule (HbS) and was first termed as a molecular disease. Glutamic acid in the normal hemoglobin molecule (HbA), was replaced by valine in HbS at the sixth position of both β-chains. This alteration was proved to be due to a single point mutation GTG instead of GAG in the genetic code. Since the discovery of sickle cell disease in 1910, great efforts have been done to study this disease on a molecular level. These efforts aimed to identify the disease etiology, pathophysiology, and finally to discover efficient treatment. Despite the tremendous work of many research groups all over the world, the only approved drug up to this moment, for the treatment of SCD is the hydroxyurea. Main text In this review, the antisickling pharmaco-therapeutics will be classified into two major groups: hemoglobin site directed modifiers and ex-hemoglobin effectors. The first class will be discussed in details, here in, focusing on the most important figures in the way of the rational drug design for SCD treatment aiming to help scientists solve the mystery of this problem and to get clear vision toward possible required therapy for SCD. Conclusion Despite the large number of the antisickling candidates that have been reached clinical studies yet, none of them has been introduced to the market. This may be due to the fact that hemoglobin is a large molecule with different target sites, which requires highly potent therapeutic agent. With this potency, these drugs should be safe, with acceptable oral pharmacokinetic and pharmacodynamic properties. Such ideal drug candidate needs more efforts to be developed.

High-throughput assessment of hemoglobin polymer in single red blood cells from sickle cell patients under controlled oxygen tension

Sickle cell disease (SCD) is caused by a variant hemoglobin molecule that polymerizes inside red blood cells (RBCs) in reduced oxygen tension. Treatment development has been slow for this typically severe disease, but there is current optimism for curative gene transfer strategies to induce expression of fetal hemoglobin or other nonsickling hemoglobin isoforms. All SCD morbidity and mortality arise directly or indirectly from polymer formation in individual RBCs. Identifying patients at highest risk of complications and treatment candidates with the greatest curative potential therefore requires determining the amount of polymer in individual RBCs under controlled oxygen. Here, we report a semiquantitative measurement of hemoglobin polymer in single RBCs as a function of oxygen. The method takes advantage of the reduced oxygen affinity of hemoglobin polymer to infer polymer content for thousands of RBCs from their overall oxygen saturation. The method enables approaches for SCD treatment development and precision medicine.

Peroxide-induced oxidative modification of hemoglobin

The oxidative modification of human hemoglobin Hb treated with ydrogen peroxide was investigated. The method of mass spectrometry were detected oxidized amino acid residues of the hemoglobin molecule: αTrp14, αTyr24, αArg31, αMet32, αTyr42, αHis45, αHis72, αMet76, αPro77, αLys90, αCys104, αTyr140, βHis2, βTrp15, βTrp37, βMet55, βCys93, βCys112, βTyr130, βLys144, βHis146. The antioxidant potential of the Hb molecule in the intracellular space and when it enters the blood plasma is discussed.

Changes in erythrocytopoesis indices in dogs with babesiosis

Babesiosis is a common disease in dogs. Babesia canis (Piana & Galli-Valerio, 1895) (Sporozoa, Babesiidae) causes the destruction of erythrocytes, resulting in hypotensive shock and total tissue damage due to lack of oxygen. Because of babesiosis, anemia develops in dogs, and in the first hours of the disease it is normocytic, normochromic and nonregenerative, and on the 2–3rd day of the course, macrocytic, hypochromic anemia with reticulocytosis develops. Scientists have studied the most common indicators of evaluation of erythrocytopoesis during babesiosis (the number of red blood cells, hemoglobin, hematocrit index, indices of “red” blood MCH and MCV), but the age structure of erythrocytes, their acid resistance and the ferrum-transferrin complex for this parasitic pathology have not been sufficiently studied. We carried out research on dogs of service breeds, which were divided into two groups: the first (6–18 months old, n = 10) and the second (2–8 years, n = 15). According to the conducted studies, in dogs of different age groups with babesiosis revealed oligocythemia with anisocytosis and poikilocytosis, oligochromia, decreased hematocrit index and macrocytosis. Significant changes in the morpho-functional state of erythrocytes in dogs of both groups have been revealed, in particular, changes in the population (age) composition of red blood cells: the number of “old” erythrocytes increases (they are rapidly destroyed), the “young” forms of red blood cells decrease and the time for their hemolysis is reduced. As a result of the study of the ferrum-transferrin complex in dogs with babesiosis in both groups, an increase in the level of free ferrum (UIBC) and a decrease in the saturation of transferrin with the trace element was found, which makes it impossible to form a hemoglobin molecule in the bone marrow.