Changes in various biochemical and physico-chemical properties of hemoglobin during its aging

It is shown that hemoglobin senescence leads to both a decrease of amide groups in it and redistribution of the radioactive label (55Fe) in electrophoretic fractions. It is supposed that an increase of the radio-active label in highly mobile hemoglobin fractions of the "old" erythrocytes by 78 and 43% as compared to that of the "young" erythrocytes is related to the deamidation-mediate growth of the negative charge of this protein molecule or partial denaturation of its quaternary structure. The resistance of hemoglobin of the "old" erythrocytes is shown to decrease to acid tissue proteinases of the lysosomal fraction as against hemoglobin of the "young" erythrocytes.     

Combined effect of gamma-irradiation and sodium nitrite on the oxidative sensitivity of rat hemoglobin

gamma-Irradiation has been defined to increase in the rats blood the methemoglobin level providing for shortening the initiation phase and accelerates the autocatalytic phase initiation, reduces the period of half transforming hemoglobin into methemoglobin and increases the velocity of its oxidation. Alongside with the latter there is observed a violation of methemoglobin concentration growth dependence on the animals irradiation dose (in the range of 0.16-0.50 Gr). The hemoglobin oxygenation reaction kinetics with the initial level of hemoglobin unexceeding 3% has been determined as having a biexponential character. The reaction kinetics parameters don't depend on ionizing radiation and number of sodium nitrite oxidized subunits formed in the process of reaction in the case if their composition unexceeds 50% of the total level.     

Pancreatic ascites hemoglobin contributes to the systemic response in acute pancreatitis

Upon hemolysis extracellular hemoglobin causes oxidative stress and cytotoxicity due to its peroxidase activity. Extracellular hemoglobin may release free hemin, which increases vascular permeability, leukocyte recruitment, and adhesion molecule expression. Pancreatitis-associated ascitic fluid is reddish and may contain extracellular hemoglobin. Our aim has been to determine the role of extracellular hemoglobin in the local and systemic inflammatory response during severe acute pancreatitis in rats. To this end we studied taurocholate-induced necrotizing pancreatitis in rats. First, extracellular hemoglobin in ascites and plasma was quantified and the hemolytic action of ascitic fluid was tested. Second, we assessed whether peritoneal lavage prevented the increase in extracellular hemoglobin in plasma during pancreatitis. Third, hemoglobin was purified from rat erythrocytes and administered intraperitoneally to assess the local and systemic effects of ascitic-associated extracellular hemoglobin during acute pancreatitis. Extracellular hemoglobin and hemin levels markedly increased in ascitic fluid and plasma during necrotizing pancreatitis. Peroxidase activity was very high in ascites. The peritoneal lavage abrogated the increase in extracellular hemoglobin in plasma. The administration of extracellular hemoglobin enhanced ascites; dramatically increased abdominal fat necrosis; upregulated tumor necrosis factor-α, interleukin-1β, and interleukin-6 gene expression; and decreased expression of interleukin-10 in abdominal adipose tissue during pancreatitis. Extracellular hemoglobin enhanced the gene expression and protein levels of vascular endothelial growth factor (VEGF) and other hypoxia-inducible factor-related genes in the lung. Extracellular hemoglobin also increased myeloperoxidase activity in the lung. In conclusion, extracellular hemoglobin contributes to the inflammatory response in severe acute pancreatitis through abdominal fat necrosis and inflammation and by increasing VEGF and leukocyte infiltration into the lung.     

Genetic engineering of Serratia marcescens with bacterial hemoglobin gene: effects on growth, oxygen utilization, and cell size

The bacterial hemoglobin from Vitreoscilla has been shown to increase growth yield and yield of genetically engineered product in Escherichia coli. To test the generality of this phenomenon, the approximately 560-bp bacterial (Vitreoscilla) hemoglobin gene (vgb) (including the native promoter), cloned into the vector pUC8 in two constructs containing about 1650 and 850 bp, respectively, of Vitreoscilla DNA downstream of vgb, was transformed into Serratia marcescens. After several transfers of the transformants on selective media, both plasmids became stable in this host and the resulting strains produced hemoglobin. Both transformants were compared, regarding growth in liquid Luria-Bertani (LB) medium, with untransformed S. marcescens and S. marcescens transformed with pUC8. The vgb-bearing strains had about 5 times lower maximum viable cell numbers than the strains without hemoglobin, but the former also had late log or early stationary phase cells that were 5-10 times larger than those of the latter. Further, on a dry cell mass basis the presence of vgb inhibited cell growth in liquid media. In contrast, growth of the vgb-bearing strains on LB plates based on cell mass (determined from colony size) was markedly enhanced compared with that of the pUC8 transformant. Respiration of the vgb-bearing strains was lower than that of the strains without vgb on a cell mass basis. These results show that the presence of vgb can have idiosyncratic effects and is not always an aid to cell growth so that its use for genetic engineering must be tested on a case by case basis.     

The effects of Efaproxyn (efaproxiral) on subcutaneous RIF-1 tumor oxygenation and enhancement of radiotherapy-mediated inhibition of tumor growth in mice

Efaproxiral, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity, facilitating oxygen release from hemoglobin, which is likely to increase tissue pO(2). The purpose of this study was to determine the effect of efaproxiral on tumor oxygenation and growth inhibition of RIF-1 tumors that received X radiation (4 Gy) plus oxygen breathing compared to radiation plus oxygen plus efaproxiral daily for 5 days. Two lithium phthalocyanine (LiPc) deposits were implanted in RIF-1 tumors in C3H mice for tumor pO(2) measurements using EPR oximetry. Efaproxiral significantly increased tumor oxygenation by 8.4 to 43.4 mmHg within 5 days, with maximum increases at 22-31 min after treatment. Oxygen breathing alone did not affect tumor pO(2). Radiation plus oxygen plus efaproxiral produced tumor growth inhibition throughout the treatment duration, and inhibition was significantly different from radiation plus oxygen from day 3 to day 5. The results of this study provide unambiguous quantitative information on the effectiveness of efaproxiral to consistently and reproducibly increase tumor oxygenation over the course of 5 days of treatment, modeling the clinical use of efaproxiral. Also, based on the tumor growth inhibition, the study shows the efaproxiral-enhanced tumor oxygenation was radiobiologically significant. This is the first study to demonstrate the ability of efaproxiral to increase tumor oxygenation and to increase the tumor growth inhibition of radiotherapy over 5 days of treatment.     

Increased carbonic anhydrase activity in Friend erythroleukemia cells during DMSO-stimulated erythroid differentiation and its inhibition by BrdU.

Carbonic anhydrase activity is increased in Friend erythroleukemia (FL) cells during the enhancement of erythroid differentiation in the presence of dimethylsulfoxide (DMSO) or butyric acid. Untreated FL cells show an increase in enzyme activity associated with logarithmic growth. The increase in the specific activity of carbonic anhydrase in the differentiating treated cells, however, appears to be due to at least two additional general mechanisms: (1) an induction of carbonic anhydrase paralleling the stimulation of hemoglobin synthesis and (2) the stability and/or retention of active carbonic anhydrase as compared to most of the other cell proteins. The stimulation of carbonic anhydrase activity in the treated cells is inhibited by 5-bromo-2'-deoxyuridine (BrdU). This is the first demonstration of BrdU inhibition of a DMSO induced product not directly related to hemoglobin.     

Induction of hemoglobin synthesis in K562 cells by carbon dioxide deficiency

Proliferation and differentiation are inversely related in many cell culture systems. The study of inducible systems is facilitated by optimal growth conditions in order that whatever differentiation is observed may be attributed to a specific effect of the inducer, rather than to a nonspecific effect of adverse growth conditions. To investigate the role of CO2 supply in an inducible system, the K562 human leukemia cell line inducible for hemoglobin synthesis was studied at 10%, 5% and 1.5% CO2 concentrations. The lower the CO2 concentration, the higher the percentage of benzidine-positive cells but the slower the growth rate. This increase in benzidine positivity reflected hemoglobin synthesis as indicated by incorporation of 3H-leucine into globin chains. If, in addition to reducing CO2 concentration, the complete medium was replaced by a bicarbonate-free medium, the percentage of benzidine-positive cells was further increased and growth further slowed. However, if endogenously produced CO2 was retained by sealing the culture vessel, these effects were mitigated. Since addition of ribosides blocked these effects, the mechanism for these effects appears to be inhibition of riboside biosynthesis due to the depletion of CO2 as a substrate. The implication of this work is that, for reproducibility in studies of inducible systems in which reduction of proliferation may itself increase the probability of differentiation, the CO2 tension, the bicarbonate concentration in the medium and the rate of egress of endogenously produced CO2 must be kept constant.     

The process of hypoxic induction of Daphnia magna hemoglobin: subunit composition and functional properties

The process of oxygen-dependent hemoglobin induction in Daphnia magna was studied over an 11-day period of hypoxia (ambient oxygen partial pressure: 3 kPa). Along with the increase of hemoglobin concentration in the hemolymph, hemoglobin became the dominant protein fraction in gel filtration experiments using extracts of whole animals. The size of the native aggregates was constant. However, subunit composition depended on the duration of hypoxia: the pattern of predominantly expressed subunits under hypoxia deviated from that of normoxic individuals. The varying degree of hypoxic induction for different hemoglobin subunits was confirmed by autoradiography. Along with changes in hemoglobin subunit composition, oxygen affinity of the respiratory protein increased. The dynamics of the hemoglobin induction process was analysed. Newly synthesized hemoglobin can be detected within 18 h after the onset of hypoxia. A marked increase in hemoglobin concentration is evident from the third day of hypoxia, and a steady state of hemoglobin concentration is reached within 11 days. The changes of hemoglobin subunit expression in response to hypoxia form the structural basis for the observed adjustments of hemoglobin function leading to enhanced oxygen transport at low ambient oxygen concentrations.     

Role of ERK activation in growth and erythroid differentiation of K562 cells

Inhibition of signaling through Ras in BCR-ABL-positive pluripotent K562 cells leads to apoptosis and spontaneous differentiation. However, Ras-induced activation of the mitogen-activated protein kinase ERK has been suggested to play a critical role in either growth or differentiation in different model systems. We studied the role of ERK activation in the growth-promoting and anti-apoptotic effect of Ras and its involvement in hemin-induced nonterminal erythroid differentiation using the BCR-ABL-positive K562 cell line as a model. K562 cells were stably transfected with ERK1 or the dominant inhibitory mutant of ERK1 (ERK1-KR). Overexpression of ERK1-KR inhibited cell growth with an approximately fourfold increase in doubling time and induced apoptosis in K562 cells. Incubation with the MEK1 inhibitor UO126 inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner as well. In the presence of exogenously added hemin, K562 cells differentiate into erythroblasts, as indicated by the production of large amounts of fetal hemoglobin. We examined the activation of MAP kinases during hemin-induced differentiation. The ERK1 and 2 activity increased within 2 h post hemin treatment and remained elevated for 24-48 h. During this time, fetal hemoglobin synthesis also increases from 0.8 to 10 pg/cell. There was no activation of JNK or p38 protein kinases. The hemin-induced accumulation of hemoglobin was inhibited in ERK1-KR overexpressing cells and was enhanced in the wild-type ERK1 transfectants. Our results suggest that ERK activation is involved in both growth and hemin-induced erythroid differentiation in the BCR-ABL-positive K562 cell line.     

Thermodynamic analysis of the chemical inhibition of sickle-cell hemoglobin gelation.

When added to a solution of deoxygenated sickle-cell hemoglobin, a variety of compounds are known to increase the minimum gelling concentration. In the present analysis, this increase is attributed to a corresponding increase in the solubility of non-aggregated hemoglobin, which in turn is attributed to preferential interaction of additive with the isolated hemoglobin molecule in solution relative to that in the aggregate. An equilibrium thermodynamic model for chemical inhibition of gelation, based on these concepts, is presented. Using the model, experimental data in the literature are interpreted in terms of the hemoglobin-binding properties of the various additive species.     

Physicochemical and functional properties of hemoglobin of mice infected with Ehrlich's carcinoma]

Injection of white mice with Ehrlich's carcinoma triggers an increase in the mice blood erythrocyte fraction suspended in the 14% sucrose concentration zone. As established by acid erythrograms, a quantitative increase of this red blood cell population is due to an increased rate of erythroblast maturation and occurrence of immature cell forms in the blood stream. As shown by alkali denaturation of hemoglobin, tumour development causes an increase in alkali-resistant hemoglobin fraction in the erythrocytes. On the basis of the data on alkali denaturation of hemoglobin, it is suggested that in the infected mice increased rate of erythrocyte maturation go in line with selective binding of alkali resistant hemoglobin fraction to red cell membrane.     

Expression of vitreoscilla hemoglobin improves growth and levels of extracellular enzyme in Yarrowia lipolytica

Enhancement in oxygen uptake by high-cell-density cultivations has been achieved previously by expression of the bacterial hemoglobin gene from Vitreoscilla. The Vitreoscilla hemoglobin (VHb) gene was expressed in the yeast Yarrowia lipolytica to study the effect of expression in this commercially important yeast. The expression of VHb in this yeast was found to enhance growth, contrary to reported observations in wild-type Saccharomyces cerevisiae in which there was no significant growth enhancement. VHb-expressing Y. lipolytica exhibited higher specific growth rate, enhanced oxygen uptake rate, and higher respiratory activity. We report the beneficial effects of VHb expression on growth under microaerobic as well as under nonlimiting dissolved oxygen conditions. Earlier studies in Y. lipolytica have demonstrated inhibition of mycelia formation by respiratory inhibitors and poor nitrogen source, conditions poor for growth. VHb(+) Y. lipolytica cells were more efficient at forming mycelia, indicating better utilization of available oxygen as compared with the VHb(-) cells. Expression of VHb was also found to increase the levels of enzyme ribonuclease secreted into the medium, a property that may be beneficial for producing heterologous proteins in Y. lipolytica.     

Characterization of hemoglobin binding to Actinobacillus actinomycetemcomitans

In this study, binding of hemoglobin to Actinobacillus actinomycetemcomitans was characterized. The ability of A. actinomycetemcomitans to utilize hemoglobin as an iron source was examined by growth studies. Although the bacterial growth was limited almost completely by adding 400 microM 2, 2'-dipyridyl to culture medium, addition of hemoglobin recovered the growth in a dose-dependent manner, revealing that hemoglobin can be utilized effectively as an iron source by A. actinomycetemcomitans. Binding of A. actinomycetemcomitans to hemoglobin was examined by dot-blot assay. Optimal hemoglobin-binding activity occurred at pH 6 and activity under acidic conditions was found to be higher than that under alkaline conditions. Hemoglobin-binding activity was higher under anaerobic conditions than under aerobic conditions, and iron restriction in culture medium decreased the activity by 55%. Heat and trypsin treatments of the bacterial components reduced the activity by 28% and 60%, respectively. Globin inhibited the activity by 49%, while transferrin, lactoferrin and tested amino acids and sugars had little or no inhibitory effects. These results indicate that proteinaceous components of the bacterial cells may be involved in hemoglobin binding and that globin moiety of the hemoglobin molecule may be essential for the binding. In order to identify hemoglobin-binding proteins, the bacterial cell components extracted with n-octyl-beta-D-thioglucoside were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with hemoglobin and bound hemoglobin was detected with anti-hemoglobin antibodies. About 40- and 65-kDa proteins from A. actinomycetemcomitans reacted with hemoglobin. The 65-kDa protein was detected despite the iron concentration in culture medium, whereas expression of the 40-kDa protein was enhanced only when the organism was grown in iron-restricted culture medium. From these results, it is suggested that 40- and 65-kDa proteins of A. actinomycetemcomitans may be involved in hemoglobin binding.     

Degradation of Host Heme Proteins by Lysine- and Arginine-Specific Cysteine Proteinases (Gingipains) of Porphyromonas gingivalis

Porphyromonas gingivalis can use hemoglobin bound to haptoglobin and heme complexed to hemopexin as heme sources; however, the mechanism by which hemin is released from these proteins has not been defined. In the present study, using a variety of analytical methods, we demonstrate that lysine-specific cysteine proteinase of P. gingivalis (gingipain K, Kgp) can efficiently cleave hemoglobin, hemopexin, haptoglobin, and transferrin. Degradation of hemopexin and transferrin in human serum by Kgp was also detected; however, we did not observe extensive degradation of hemoglobin in serum by Kgp. Likewise the beta-chain of haptoglobin was partially protected from degradation by Kgp in a haptoglobin-hemoglobin complex. Arginine-specific gingipains (gingipains R) were also found to degrade hemopexin and transferrin in serum; however, this was observed only at relatively high concentrations of these enzymes. Growth of P. gingivalis strain A7436 in a minimal media with normal human serum as a source of heme correlated not only with the ability of the organism to degrade hemoglobin, haptoglobin, hemopexin, and transferrin but also with an increase in gingipain K and gingipain R activity. The ability of gingipain K to cleave hemoglobin, haptoglobin, and hemopexin may provide P. gingivalis with a usable source of heme for growth and may contribute to the proliferation of P. gingivalis within periodontal pockets in which erythrocytes are abundant.     

Hemoglobin and Escherichia coli, a Lethal Intraperitoneal Combination

Intraperitoneal injection into mice of approximately 8 x 10(6) washed cells of Escherichia coli suspended in a lysate of washed human red blood cells or an aqueous solution of crystalline hemoglobin was lethal. E. coli suspended in washed intact erythrocytes, whole blood, plasma, or saline was innocuous. Fractionation of non-hemoglobin proteins from hemoglobin in lysates showed that only hemoglobin promoted a lethal infection. Overwhelming intraperitoneal growth of E. coli was attained in about 12 hr in lethal infections. The polymorphonuclear leukocytic response was ineffective against this rapid growth. The lethal mechanism is hypothesized to center on a unique role for free hemoglobin in inhibiting peritoneal absorption and stimulating an intraperitoneal exudate which supports luxuriant bacterial growth. Death is attributed to a lethal intoxication from bacterial endotoxins. This role for hemoglobin involves neither enhanced bacterial virulence nor lowered host resistance, and it would be of importance not only in peritonitis but also in problems where hemolysis and infection coexist.     

Pathogenic neisseriae can use hemoglobin, transferrin, and lactoferrin independently of the tonB locus

Redundant TonB systems which function in iron transport from TonB-dependent ligands have recently been identified in several gram-negative bacteria. We demonstrate here that in addition to the previously described tonB locus, an alternative system exists for the utilization of iron from hemoglobin, transferrin, or lactoferrin in Neisseria meningitidis and Neisseria gonorrhoeae. Following incubation on media containing hemoglobin, N. meningitidis IR3436 (tonB exbB exbD deletion mutant) and N. gonorrhoeae PD3401 (tonB insertional mutant) give rise to colonies which can grow with hemoglobin. Transfer of Hb(+) variants (PD3437 or PD3402) to media containing hemoglobin, transferrin, and/or lactoferrin as sole iron sources resulted in growth comparable to that observed for the wild-type strains. Transformation of N. meningitidis IR3436 or N. gonorrhoeae PD3401 with chromosomal DNA from the Hb(+) variants yielded transformants capable of growth with hemoglobin. When we inactivated the TonB-dependent outer membrane hemoglobin receptors (HmbR or HpuB) in the Neisseria Hb(+) variants, these strains could not grow with hemoglobin; however, growth was observed with transferrin and/or lactoferrin. These results demonstrate that accumulation of iron from hemoglobin, transferrin, and lactoferrin in the pathogenic neisseriae can occur via a system that is independent of the previously described tonB locus.     

Nanobiotechnological modification of hemoglobin and enzymes from this laboratory

Polyhemoglobin is formed by the nanobiotechnological assembling of hemoglobin molecules into soluble nanodimension complex. A further step involves the nanobiotechnological assembly of hemoglobin, catalase and superoxide dismutase into a soluble nanodimension complex. This acts both as oxygen carrier and antioxidant to prevent the oxidative effects of hemoglobin. A further step is the preparation of nanodimension artificial red blood cells that contain hemoglobin and all the enzymes present in red blood cells. Other approaches include a polyhemoglobin-fibrinogen that acts as an oxygen carrier with platelet-like activity, and a polyhemoglobin-tyrosinase to retard the growth of a fatal skin cancer, melanoma.     

Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa are differently effected by the genetically engineered Vitreoscilla hemoglobin gene

Vitreoscilla hemoglobin is a good oxygen trapping agent and its presence in genetically engineered Escherichia coli helps this bacterium to grow better. Here, the potential use of this hemoglobin, for improving the growth and the oxygen transfer properties of Pseudomonas aeruginosa as well as Escherichia coli, was investigated. To stably maintain it in both bacteria, a broad-host range cosmid vector (pHG1), containing the entire coding sequence for Vitreoscilla hemoglobin gene and its native promoter on a 2.3 kb fragment, was constructed. Though at different levels, both bacteria produced hemoglobin and while the oxygen uptake rates of vgb-bearing strains were 2-3-fold greater than that of non-vgb-bearing strains in both bacteria, the growth advantage afforded by the presence of Vitreoscilla hemoglobin was somewhat varied. As an alternative to the traditional method of the improvement of oxygen transfer properties of the environment in which cells are grown, the genetic manipulation applied here improved the oxygen utilization properties of cells themselves.