STUDIES ON INVERTEBRATE HEMOGLOBINS (ERYTHROCRUORINS)

1. Two high molecular invertebrate hemoglobins (the erythrocruorins of Lumbricus terrestris and of Nereis virens) as well as the low molecular erythrocruorin of Glycera dibranchiata Ehlers were studied. Their physical chemical properties were compared with those of vertebrate hemoglobin. 2. The hemin of the blood pigment of Glycera dibranchiata Ehlers was shown to be identical with that of vertebrate hemoglobin. 3. The dissociation rates of Glycera and human oxyhemoglobin were measured in the reaction meter of DuBois and t ₅₀ (half time of the reaction) was found to be identical (0.027 second) for the two pigments. The t ₅₀ value for the high molecular Lumbricus erythrocruorin was 0.070 second. 4. The chemical constitution and physical chemical properties of erythrocruorins were compared with those of vertebrate hemoglobin and of hemocyanin.     

THE RATE OF ESCAPE OF HEMOGLOBIN FROM THE HEMOLYZED RED CORPUSCLE

A theoretical treatment is given of the rate of escape of hemoglobin from the hemolyzed red corpuscle. For complete permeability of the surface, as may perhaps be produced by strong lysins, the time taken for the hemoglobin to decrease to 10 per cent of its original concentration is calculated to be 0.16 seconds (for the human cell). For dilute saponin, giving complete lysis of human cells in 3 minutes, Ponder found a time of escape of 4 seconds, from which the permeability of the membrane to the pigment is calculated to be µ_H = 5 x 10^–5 cm./sec.     

Time-course of the polycythemic response in normoxic and hypoxic mice with high blood oxygen affinity induced by cyanate administration

The Hb-O₂ affinity and the erythropoietic response as a function of time were studied in mice treated with sodium cyanate for up to 2 months. Cyanate increased the Hb-O₂ affinity in normoxic mice more than in chronically hypoxic mice. The hemoglobin concentration rose as a function of time both in normoxic and hypoxic conditions but reached higher levels in hypoxia. After 42 days of study (21 days of hypoxia) hemoglobin reached maximum levels and thereafter showed a plateau in both cyanate and control animals. It is concluded that a chronic left-shifted oxygen dissociation curve does not avoid the development of hypoxic polycythemia in mice. Moreover, prolonged cyanate administration potentiates the crythropoietic response to chronic hypoxia. Since polycythemia is an index of tissue hypoxia, the results show that the high hemoglobin affinity did not prevent tissue hypoxia in low PO₂ conditions. Results showing beneficial effects of high hemoglobin oxygen affinity induced by cyanate based on acute hypoxic expositions should be cautiously interpreted with regard to their adaptive value in animals chronically exposed to natural or simulated hypoxia.Abbreviations Hb hemoglobin - NaOCN sodium cyanate - ODC oxygen dissociation curve - P ₅₀ PO₂ at which hemoglobin is half saturated with O₂     

Differential control of the synthesis of two hemoglobin beta chains in normal mice

A fetal-to-adult switch in the proportion of the mouse minor hemoglobin is described. Although mice have no fetal hemoglobin per se, the timing of this switch in the mouse suggests that the mechanism of its control may directly parallel that of the human switch from fetal to adult hemoglobin expression. The mouse minor hemoglobin is expressed only in strains with the "diffuse" allele for the beta chain complex locus. Fetal liver cells of these mice synthesize a much greater proportion of the betaminor globin chain that do adult hematopoietic cells. Consequently, circulating fetal erythrocytes carry a high level of the minor hemoglobin containing it. By the time of birth, a lowered proportion of betaminor is synthesized in the liver. This low proportion continues to be expressed during early erythroid maturation in the adult. The fetal-to-adult switch is the first indication that in normal mice, the two beta chain loci can be expressed noncoordinately. The similarity between the patterns of the decline of the minor hemoglobin in mice and of the disappearance of fetal hemoglobin in humans suggests that the minor hemoglobin in the "diffuse" mouse may function to some degree as a fetal hemoglobin in the period between the disappearance of the embryonic hemoglobins and the time of birth.     

Oxidation of hemoglobin by arenediazonium salts. The influence of dioxygen

The reactions of human hemoglobin with p-nitro- and p-chlorobenzenediazonium tetrafluoroborates in the presence and absence of molecular oxygen have been investigated in kinetic detail. The oxidation of iron(II) occurs with first order rate dependence on both the hemoglobin and diazonium salt concentrations, but inverse first order dependence on the concentration of molecular oxygen characterizes reactions performed in the presence of O₂. In the absence of O₂, nitrobenzene is the only product observed from hemoglobin oxidation by p-NO₂C₆H₄N₂⁺BF₄^?, and a 1:1 stoichiometry exists between nitrobenzene produced and Fe(II) oxidized. In the presence of O₂, p-nitrophenol is the dominant product, but product yield is dependent on the ratio of reactants. Electron transfer to the diazonium salt rather than its corresponding diazohydroxide or diazoate is inferred from the relative absence of pH dependence on the rate of oxidation. The composite results are consistent with a mechanism for hemoglobin oxidation that requires molecular oxygen dissociation from oxyhemoglobin prior to oxidation by the diazonium salt. Implications of this investigation for the mechanism of arylhydrazine reactions with hemoglobin are discussed.     

Inhibitory effect of deoxyhemoglobin A2 on the rate of deoxyhemoglobin S polymerization.

From a consideration of the primary sequence of hemoglobin A₂ and the reported 5 å molecular contacts between deoxyhemoglobin S molecules in a crystal, it is predicted that hemoglobin A₂ might act as an inhibitor of the polymerization of deoxyhemoglobin S in a manner similar to hemoglobin. F. This has been tested experimentally by measuring the rate of change of the transverse water proton relaxation times (T₂) in equimolar mixtures of hemoglobin S and one of the non-gelling hemoglobins A, F or A₂. Hemoglobins A₂ and F have far more pronounced inhibitory effects on the rate of polymerization than does hemoglobin A. These molecules contain several amino acid differences from hemoglobin A beta chains which are located in the 5 Å molecular crystal contacts and these altered crystal contacts result in a much stronger inhibition of the rate of polymerization. Since hemoglobin A₂ is a normal hemoglobin found in small amounts in all adult red cells, increased delta chain synthesis may have potential importance in therapy for sickle cell disease.     

Simplification of the intravenous glucose tolerance test (ivGTT) in rats

A simplified technique was established for the intravenous glucose tolerance test (ivGTT) in unanesthetized rats. In order to evaluate the relation between insulin secretion, glucose load and glucose disappearance rate, precatheterized rats were given glucose ranging in dose from 0.25 to 2.0 g/kg bw by intravenous injection. A highly linear correlation was observed in glucose disappearance rate during a period of 4-32 min. A glucose load greater than 0.5 g/kg ow induced a maximum response in insulin secretion. Small blood samples were collected using the orbital bleeding technique at 4, 16 and 28 minutes after a glucose load of 1.0 g/kg bw had been given and then T1/2, the time taken for the glucose level to fall by one half, was calculated. The mean T1/2 was significantly longer in alloxan- or cyproheptadin-diabetic rats than that in the intacts. These data indicate that a glucose load of 1.0 g/kg administered by intravenous injection with the T1/2 calculated between 4 and 32 minutes would provide an accurate means of assessing pancreatic endocrine function.     

Temperature Sensitivity of the Latent Phase in Ethylene-induced Elongation

The temperature sensitivity is reported for the latent period preceding ethylene-induced elongation in the adaxial half of the leaf petiole of Helianthus annuus. When intact plants were exposed to 10 μl of ethylene/l of air over the temperature range 18 to 35 C, the minimum latent time was 62 minutes at 28 C and the maximum was 132 minutes at 18 C. The temperature coefficient, Q₁₀, changed from 2.1 below 28 C, to 0.7 above. In 100 μl of ethylene/l of air, the latent time was reduced by 14% at 18 C, but was significantly increased at 28 and 38 C. These results show that the latent period in the elongation response of the petiole to ethylene cannot be reduced below about 60 minutes by raising either the leaf temperature or the atmospheric ethylene concentration.     

Tendency for oxidation of annelid hemoglobin at alkaline pH and dissociated states probed by redox titration

The redox titration of extracellular hemoglobin of Glossoscolex paulistus (Annelidea) was investigated in different pH conditions and after dissociation induced by pressure. Oxidation increased with increasing pH, as shown by the reduced amount of ferricyanide necessary for the oxidation of hemoglobin. This behavior was the opposite of that of vertebrate hemoglobins. The potential of half oxidation (E_{1 / 2}) changed from − 65.3 to + 146.8 mV when the pH increased from 4.50 to 8.75. The functional properties indicated a reduction in the log P₅₀ from 1.28 to 0.28 in this pH range. The dissociation at alkaline pH or induced by high pressure, confirmed by HPLC gel filtration, suggested that disassembly of the hemoglobin could be involved in the increased potential for oxidation. These results suggest that the high stability and prolonged lifetime common to invertebrate hemoglobins is related to their low tendency to oxidize at acidic pH, in contrast to vertebrate hemoglobins.     

LYTIC ACTIVITIES IN RENAL PROTEIN ABSORPTION DROPLETS : An Electron Microscopical Cytochemical Study

The digestive cycle following reabsorption of hemoglobin by cells of the proximal convoluted tubules in mouse kidney and the uptake of ferritin by glomerular mesangial cells in the kidney of normal and nephrotic rats were investigated by electron microscopical histochemical procedures. Mouse kidneys, sampled at closely spaced time points between 1 to 48 hours after intraperitoneal injection of hemoglobin, and rat (normal and nephrotic) kidneys, sampled at 30 minutes, 2 hours, and 48 hours after intravenous injection of ferritin, were fixed in glutaraldehyde, cut at 50 µ on a freezing microtome, incubated for acid phosphatase and thiolacetate-esterase, and postfixed in OsO₄. Satisfactory preservation of fine structure permitted the localization of the enzymatic reaction products on cell structures involved in uptake and digestion of exogenous proteins. The latter were identified either by their density (hemoglobin) or their molecular structure (ferritin). It was found that lysosomal enzymic activities and incorporated exogenous proteins occur together in the same membrane-bounded structures. In the cells of the proximal convolution, lytic activities become demonstrable within 1 hour after hemoglobin injection, appear first in apical vacuoles filled with hemoglobin, and persist in fully formed protein absorption droplets. At the end of the lytic cycle (~48 hours post injection), the cells have an increased population of polymorphic bodies which exhibit lytic activities. In smaller numbers, identical bodies occur in controls. It is concluded that they represent remnants of previous digestive events. The means by which the resorptive vacuoles acquire hydrolytic activities remain unknown. Fusion of newly formed vacuoles with residual bodies was not seen, and hemoglobin incorporation into such bodies was only occasionally encountered. Acid phosphatase activity was found sometimes in the Golgi complex, but enzyme transport from the complex to the resorbing vacuoles could not be established. Autolytic vacuoles containing mitochondria or mitochondrial remnants were frequently found during the early stages of hemoglobin resorption, but no definite conclusions about the mechanism involved in the segregation of endogenous material were obtained. In nephrotic rats ferritin was segregated in membrane-bounded bodies mainly in the mesangial cells and to a lesser extent in epithelial and endothelial cells. Most of these sites were marked by the reaction products of acid phosphatase and organophosphorus-resistant esterase and therefore identified as lysosomes connected with the digestion of incorporated exogenous proteins.     

Molecular dynamics simulations indicate that deoxyhemoglobin,oxyhemoglobin, carboxyhemoglobin,and glycated hemoglobin under compression and shear exhibit an anisotropic mechanical behavior

We developed a new mechanical model for determining the compression and shear mechanical behavior of four different hemoglobin structures. Previous studies on hemoglobin structures have focused primarily on overall mechanical behavior; however, this study investigates the mechanical behavior of hemoglobin, a major constituent of red blood cells, using steered molecular dynamics (SMD) simulations to obtain anisotropic mechanical behavior under compression and shear loading conditions. Four different configurations of hemoglobin molecules were considered: deoxyhemoglobin (deoxyHb), oxyhemoglobin (HbO₂), carboxyhemoglobin (HbCO), and glycated hemoglobin (HbA_1C). The SMD simulations were performed on the hemoglobin variants to estimate their unidirectional stiffness and shear stiffness. Although hemoglobin is structurally denoted as a globular protein due to its spherical shape and secondary structure, our simulation results show a significant variation in the mechanical strength in different directions (anisotropy) and also a strength variation among the four different hemoglobin configurations studied. The glycated hemoglobin molecule possesses an overall higher compressive mechanical stiffness and shear stiffness when compared to deoxyhemoglobin, oxyhemoglobin, and carboxyhemoglobin molecules. Further results from the models indicate that the hemoglobin structures studied possess a soft outer shell and a stiff core based on stiffness.     

Molecular oxygen binding with α and β subunits within the R quaternary state of human hemoglobin in solutions and porous sol–gel matrices

Nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O₂) rebinding to α and β subunits within oxygenated human adult hemoglobin in solutions and porous wet sol–gel matrices. Plasticity associated with the tertiary structure within R-state hemoglobin is explored through measurements that focus on the functional properties of hemoglobin under conditions designed to tune the tertiary structure without inducing the R to T transition. Inequivalence in the O₂ binding to the α and β hemes within the R quaternary structure is studied. The individual kinetic properties of the α and β subunits within the hemoglobin encapsulated in sol–gels and aged as the oxy derivative are shown to be independent of proton concentration over the pH range from 6.3 to 8.5. However, buffer effects on the subunits' properties are revealed in sol–gel-free mediums. Interestingly, the α and β subunits within the encapsulated hemoglobin possess the O₂ rebinding properties which fall within the range of the ones for oxygenated hemoglobin in the buffer solutions. The combined results show a pattern in which there is a progression of functional properties that are ascribed to a family of conformational substates of R-state hemoglobin. O₂ rebinding to the α and β subunits within the oxygenated R-state hemoglobin in both solutions and wet sol–gels is revealed to be modulated by tertiary structural changes in two quite different ways. The possible structural changes, which modify the O₂ rebinding properties, are discussed.     

Two-domain hemoglobin from the blood clam,Barbatia lima. The cDNA-derived amino acid sequence

The blood clam,Barbatia lima, from Kochi, Japan, expresses a tetrameric (α ₂ β ₂) and a polymeric hemoglobin in erythrocytes. The latter hemoglobin is composed of unusual 34-kDa hemoglobin with a two-domain structure, and its molecular mass (about 430 kDa) is exceptionally large for an intracellular hemoglobin. The 3′ and 5′ parts of the cDNA ofB. lima two-domain globin have been amplified separately by polymerase chain reaction and the complete nucleotide sequence of 1147 bp was determined. The open reading frame is 930 nucleotides in length and encodes a protein with 309 amino acid residues, of which 73 amino acids were identified directly by protein sequencing. The mature protein begins with the acetylated Ser, and thus the N-terminus Met is cleaved. The molecular mass for the protein was calculated to be 35,244 Da. The cDNA-derived amino acid sequence ofB. lima two-domain globin shows 89% homology with that of two-domain globin fromB. reeveana, a North American species. The sequence homology between the two domains is 75%, suggesting that the two-domain globin resulted from the gene duplication of an ancestral 17-kDa globin.     

Phloem unloading in soybean seed coats: dynamics and stability of efflux into attached ;empty ovules'

The time-course of sucrose efflux from attached seedcoats (having their embryos surgically removed) into aqueous traps placed in the `empty ovules' had three phases. The first phase lasted 10 minutes and probably was a period of apoplastic flushing. The second lasted 2 to 3 hours and is thought to be a phase of equilibration of seed coat symplast with the frequently refreshed liquid. The third phase of relatively steady efflux was postulated to reflect the continued import of sucrose from the plant, and hence to reflect the rate of sieve tube unloading. The average steady state efflux was equal under most conditions to the estimated rate of sucrose import. Efflux and import were unaffected by 150 millimolar osmoticum (mannitol or polyethylene glycol [molecular weight about 400]), by 0.5 millimolar CaCl<sub>2</sub>, or by pretreatments up to 20 minutes with p-chloromercuribenzenesulfonic acid (PCMBS); they were enhanced by 40 micromolar abscisic acid, 40 micromolar indoleacetic acid, 20 micromolar fusicoccin, and 1 millimolar dithiothreitol (DTT) and were inhibited by 100 micromolar KCN, by 0.03% H<sub>2</sub>O<sub>2</sub>, by 20 micromolar and 5 micromolar trifluoromethoxy (carbonyl cyamide) phenylhydrazone, by repeated 5 minutes per hour treatments with 5 millimolar PCMBS, and by 5 millimolar DTT. The `steady state' sucrose efflux was able to account for about half the rate of dry weight growth of the embryo, but stabilization of the system with <1 millimolar DTT taken together with other considerations is likely to give good correspondence between experimental unloading rates and in vivo growth rates.     

Ontogenetical studies on extracellular hemoglobins of Artemia salina

Ontogeny of the three extracellular hemoglobins of zygogenetic races of Artemia salina was studied. Hb-II was the first hemoglobin to appear in the swimming nauplius at about 2 hr posthatching, followed by Hb-III at about 8 hr posthatching. Hb-I was detectable by benzidine-H₂O₂ staining of cellulose acetate electrophoregrams only after the seventh to ninth day posthatching, when the functional gills were formed. Hb-III was always lower in quantity than Hb-II and disappeared completely from the hemolymph between the thirtieth and fourtieth day posthatching. This disappearance of Hb-III was earlier in the female than in the male. Both Hb-I and Hb-II were found to remain for most of the life period in the hemolymph of both the male and female, but Hb-II decreased gradually or sometimes disappeared in the old female, whereas this hemoglobin was continuously the major species in the old male. The general ontogetical pattern described in the present study appears to agree with the observations using different races of Artemia salina including several parthenogenetic ones. We conclude that ontogenesis is the factor most responsible for the observed hemoglobin polymorphism in Artemia salina.     

Water proton magnetic resonance studies of normal and sickle erythrocytes Temperature and volume dependence

The temperature and cell volume dependence of the NMR water proton linewidth, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T₂) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T₁) shows no significant change upon dexygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T₁ and T₂ as the cell hydration is changed indicate that these parameters only slightly by a 10–20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T₁ for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the “bound” water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at ?15°C to 500 Hz at ?36°C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T₁ data go through a minimum at ?35°C for measurements at 44.4 MHz and ?50°C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irratationally bound water, is altered during the sickling process.     

Magnetic circular dichroism studies of hemoglobin: The reduction of ferrihemoglobin by ferrocytochrome b5 and characterization of the high-spin hydroxy species of mixed-valence hemoglobin

The final step in the erythrocyte methemoglobin reduction pathway, the transfer of an electron from cytochrome b₅, to methemoglobin, has been studied using magnetic circular dichroism spectroscopy. Spectral analysis allowed us to determine accurately the concentration of each redox species in mixtures of the two heme-proteins and to follow simultaneously the kinetics of the appearance or disappearance of each of these species during reduction reactions. Our analysis detected a substantial increase in the high-spin hydroxymethemoglobin species in the partially reduced bovine hemoglobin tetramer. This species was sensitive to the degree of reduction and pH, and was spectrally similar to fluoride methemoglobin. At pH 7.8. 100% of the hydroxide component of methemoglobin was in the high-spin form when two or more subunits were in the ferrous form. Kinetic analysis of bovine methemoglobin reduction yielded values for the apparent first-order rates for the tetrameric species possessing four, three, two, and one ferric subunit. Further analysis showed that the reduction kinetics can also be described by an equilibrium state, pure competitive inhibition model for enzyme catalysis in which ferrous and ferric subunits of hemoglobin compete for cytochrome b₅ This analysis generated a K_D that depends on ionic strength and hemoglobin tetramer conformation, a V_max that was independent of these factors, and an inhibition constant that was equal to K_d. This model is consistent with the hypothesis that the reduction of methemoglobin can be separated into two steps, the ionic interaction between cytochrome b₅ and hemoglobin and the electron transfer.     

The effect of chlorine toxicity on certain blood parameters of adult rainbow trout (Salmo gairdneri)

Synopsis In an effort to assess the mode of chlorine action on rainbow trout (Salmo gairdneri), hematocrit percentage, and hemoglobin, methemoglobin, reduced glutathione, plasma protein, and plasma hemoglobin concentrations were determined in four tests in which duplicate groups of approximately 15 fish each were exposed to 3.86, 2.47, 2.75, and 1.09 mg 1^–1 TRC1₂ for 8, 19, 20, and 29 minutes, respectively. Blood from fish exposed to chlorine was darker and thicker than that of the control. Chlorine seemed to diffuse readily through the gills, oxidizing the hemoglobin to methemoglobin and disrupting the erythrocyte membranes, resulting in hemolysis. Stress polycythemia was also due to the substantial increase of the hematocrit values and hemoglobin concentration. Hemoconcentration led to a significant rise in the reduced gluthathione and plasma protein concentrations. The hemoconcentration seemed to interfere with the blood circulation and hinder the delivery of oxygen to tissues.     

Reverse Engineering the Cooperative Machinery of Human Hemoglobin

Hemoglobin transports molecular oxygen from the lungs to all human tissues for cellular respiration. Its α₂β₂ tetrameric assembly undergoes cooperative binding and releasing of oxygen for superior efficiency and responsiveness. Over past decades, hundreds of hemoglobin structures were determined under a wide range of conditions for investigation of molecular mechanism of cooperativity. Based on a joint analysis of hemoglobin structures in the Protein Data Bank (Ren, companion article), here I present a reverse engineering approach to elucidate how two subunits within each dimer reciprocate identical motions that achieves intradimer cooperativity, how ligand-induced structural signals from two subunits are integrated to drive quaternary rotation, and how the structural environment at the oxygen binding sites alter their binding affinity. This mechanical model reveals the intricate design that achieves the cooperative mechanism and has previously been masked by inconsistent structural fluctuations. A number of competing theories on hemoglobin cooperativity and broader protein allostery are reconciled and unified.     

Destruction of human hemoglobin in the presence of water and negative air ions generated by corona discharge

A buffered solution containing hemoglobin was exposed to negative air ions that were generated by corona discharge. In two hours, the optical absorbance at 405 nm decreased to ca., 40% of the value observed prior to the exposure to air ions. After 18 1/2 hours, the absorbance was further decreased to ca. 5% of the original values. The hemoglobin solution exposed to air for this duration did not show any appreciable change in absorbance at 405 nm. Concomitant with the decrease of absorbance at 405 nm, that at 205 nm increased several fold. The molecular weight of the specie(s) which absorbed strongly at 205 nm was ca. 400 daltons. Similar results were obtained when hemoglobin was exposed to ozone (O₃) instead of air ions. From these results and our earlier conclusion that O₃ is generated from negative air ions in the presence of water, it can be concluded that the destruction of hemoglobin was by O₃.