Thermohemolysis of erythrocytes in the temperature range including physiologic (autohemolysis)]

The activation energy of thermohemolysis of erythrocytes changes from 36 +/- 5 kcal/mol (35-45 degrees C) to 97 +/- 5 kcal/mol (45-55 degrees C) at the temperature about 45 degrees C in isotonic buffer. The break on Arhenius' plot is preserved also when erythrocytes are placed into plasma. The character of Arhenius' plot is the same when erythrocyte hemoglobin is totally oxidated into methemoglobin by chemical way, though thermal stability of such erythrocytes is decreased. The scheme is presented in which thermohemolysis of erythrocytes occurs by two independent ways: thermodenaturation of hemoglobin (limiting stage of the process when t greater than 45 degrees C) and modification of membrane proteins by hemin, the last being a product of hemoglobin oxidation (limiting stage of the process when t less than 45 degrees C).     

Relationship between red blood cell uptake and methemoglobin production by nitrobenzene and dinitrobenzene in vitro

Nitrobenzene increases methemoglobin formation when incubated with native hemoglobin but not when incubated with red blood cell suspensions. These experiments were designed to determine if transport of nitrobenzene across the red blood cell membrane is a limiting factor for methemoglobin production by red blood cell suspensions. Incubation of [14C]-m-, o- or p-dinitrobenzene, but not mononitrobenzene, with red blood cell suspensions caused a time-dependent increase in methemoglobin. All three dinitrobenzenes and mononitrobenzene crossed the red blood cell membrane and accumulated in the erythrocytes after only 1 min of incubation. Incubation of mononitrobenzene with hemolysates did not result in methemoglobin production. Incubation of red blood cells with the dinitrobenzenes or mononitrobenzene for 1 and 10 min at 4 degrees C did not influence red blood cell uptake of the nitrobenzenes, suggesting that these compounds do not enter the red blood cell by an active process. Dinitrobenzene-induced methemoglobin production was markedly inhibited at 4 degrees C, and may be a result of decreased interaction with hemoglobin and/or decreased metabolism to reactive intermediates which mediate methemoglobin production. These data indicate that red blood cell transport of nitrobenzene is not the limiting factor in methemoglobin production in vitro.     

Effects of crosslinking on the thermal stability of hemoglobin. I. The use of bis(3,5-dibromosalicyl) fumarate

The double-headed aspirin, bis(3,5-dibromosalicyl) fumarate, has been used to crosslink hemoglobin A between Lys 82 beta 1 and Lys 82 beta 2 (J. A. Walder et al. (1979) Biochemistry 18,4265). Denaturation experiments were used to compare the stability of this crosslinked protein to that of hemoglobin A. Thermal denaturations, done in 0.01 M 4-morpholine-propanesulfonic acid, pH 7, containing 0.9 M guanidine to prevent precipitation at high temperatures, were monitored by changes in absorbance between 190 and 650 nm using a diode array spectrophotometer. The sample was heated from 25 to 70 degrees C at 0.3 degrees C/min. The data were analyzed by using both a two-state model and a novel first derivative method. As expected, methemoglobin A had a single, broad transition with a midpoint of 40.7 degrees C. The crosslinked methemoglobin showed a transition at 57.1 degrees C. Two minor transitions, one of which was apparently due to residual unmodified hemoglobin, were also observed in the crosslinked sample. Thus, a single crosslink between only two of the four subunits can lead to a significantly more stable molecule. These results can be explained by Le Chatelier's principle, since crosslinking prevents dissociation of the beta-subunits and, thereby, holds the entire tetramer together.     

Effect of cryoprotectors on binding of bromthymol blue by bovine methemoglobin

Spectrophotometric method was used for study the binding of bromthymol blue dye (BTB) with bovine methemoglobin in 15% solutions of ethanol, glycerol and polyethylene glycol with molecular mass of 1.5 kDa (PEG-1500). It was shown, that adsorption of BTB by methemoglobin decreased in the sequence: glycerol > ethanol > PEG-1500. It is supposed that adsorption of the alcohols on the BTS sites of binding on methemolglobin led to the decrease of the amount of binding sites accessible for the dye.     

Encapsulation of hemoglobin in phospholipid vesicles

Hemoglobin has been encapsulated in phospholipid vesicles by extrusion of hemoglobin/lipid mixtures through polycarbonate membranes. This technique avoids the use of organic solvents, sonication, and detergents which have proven deleterious to hemoglobin. The vesicles are homogeneous, with a mean size of 2400 A as determined by photon correlation spectroscopy. The encapsulated hemoglobin binds oxygen reversibly and the vesicles are impermeable to ionic compounds. Hemoglobin encapsulated in egg phosphatidylcholine vesicles converts to methemoglobin within 2 days at 4 degrees C. By contrast, when a mixture of dimyristoyl phosphatidylcholine, cholesterol and dicetyl phosphate is used there is no acceleration in methemoglobin formation, and the preparation is stable for at least 14 days at 4 degrees C.     

Temperature and domain size dependence of sickle cell hemoglobin polymer melting in high concentration phosphate buffer.

Deoxygenated sickle cell hemoglobin (Hb S) in 1.8 M phosphate buffer, and carbon monoxide (CO) saturated buffer were rapidly mixed using a stopped-flow apparatus. The binding of the CO to the Hb S polymers and the polymer melting was measured by time resolved optical spectroscopy. Polymer melting was associated with decreased turbidity, and CO binding to deoxy-Hb S was monitored by observation of changes in the absorption profile. The reaction temperature was varied from 20 degrees C to 35 degrees C. Polymer domain size at 20 degrees C was also varied. The data for mixtures involving normal adult hemoglobin (Hb A) fit well to a single exponential process whereas it was necessary to include a second process when fitting data involving Hb S. The overall Hb S-CO reaction rate decreased with increasing temperature from 20 degrees C to 35 degrees C, and increased with decreasing domain size. In comparison, Hb A-CO reaction rates increased uniformly with increasing temperature. Two competing reaction channels in the Hb S-CO reaction are proposed, one involving CO binding directly to the polymer and the other involving CO only binding to Hb molecules in the solution phase. The temperature dependence of the contribution of each pathway is discussed.     

Stoichiometry of the reaction of oxyhemoglobin with nitrite.

During the reaction of oxyhemoglobin (HbO2) with nitrite, the concentration of residual nitrite, nitrate, oxygen, and methemoglobin (Hb+) was determined successively. The results obtained at various pH values indicate the following stoichiometry for the overall reaction: 4HbO2 + 4NO2- 4H+ leads to 4Hb+ + 4NO3- + O2 + 2H2 O (Hb denotes hemoglobin monomer). NO2- binds with methemoglobin noncooperatively with a binding constant of 340 M-1 at pH 7.4 and 25 degrees C. Thus, the major part of Hb+ produced is aquomethemoglobin, not methemoglobin nitrite, when less than 2 equivalents of nitrite is used for the oxidation.     

Engineering temperature-sensitive hydrogel nanoparticles entrapping hemoglobin as a novel type of oxygen carrier

Temperature-sensitive oxygen carriers that are responsive to changes in temperature while maintaining colloidal stability would benefit physiological conditions characterized by tissue hypoxia due to decreased body temperature. These conditions are often accompanied with reduced blood flow and vasoconstriction. Temperature-sensitive oxygen carriers should ideally possess increased oxygen affinity when the body temperature is reduced, to selectively target tissues that are hypoxic as a result of temperature drops. This study expands on previous work, which introduced hydrogel based oxygen carriers as a new class of oxygen carrier that can be synthesized within liposomal reactors via photoinitiated free radical polymerization [Patton, J. N.; Palmer, A. F. Biomacromolecules 2005, 6, 414-24]. In addition to the ability of poly(N-isopropylacrylamide) hydrogel nanoparticles encapsulating bovine hemoglobin to swell and shrink in response to physiological changes in temperature, the effect of temperature changes on zeta potential, oxygen affinity, and cooperativity are also examined. The methemoglobin level and hemoglobin encapsulation efficiency of hydrogel-based oxygen carriers are also presented. It was observed that nanoscale hydrogel particles swelled as the temperature decreased from 40 to 29 degrees C, which suggests expansion of the hydrogel matrix and reduced resistance to oxygen transport.     

Quaternary structure has little influence on spin states in mixed-spin human methemoglobins

A key feature of the Perutz stereochemical model for cooperativity in hemoglobin is a strong coupling between quaternary structure and the spin state of the heme iron [Perutz, M. F. (1979) Annu. Rev. Biochem. 48, 327-386]. While this coupling appears to be present for carp azide methemoglobin, it should also be present for all liganded forms of human methemoglobin that exhibit a thermal high-spin in equilibrium low-spin equilibrium. To test this hypothesis, we have measured the changes in spin equilibria upon conversion of six mixed-spin forms of human methemoglobin from the R (high-affinity) to the T (low-affinity) quaternary structure by addition of inositol hexaphosphate. These experiments were done with a sensitive superconducting magnetic susceptibility instrument on solutions at 20 degrees C in 20 mM maleate buffer, pH 6. The data show zero or small increases in high-spin content upon switching from R to T, changes that are equivalent to a relative stabilization of the high-spin form by only 0-300 cal mol-1 heme-1. These changes in energy are far less than the 1200 cal mol-1 heme-1 predicted from the Perutz stereochemical model [Cho, K. C., & Hopfield, J. J. (1979) Biochemistry 18, 5826-5833]. That is, these data do not support a view that the low affinity of the T state is due to restraints acting through the iron-proximal histidine linkage. The mechanistic implications of these results and the differences between species and ferric ligands are discussed.     

The freeze-dried preservation of liposome encapsulated hemoglobin: a potential blood substitute

In this report, the ability of carbohydrates (trehalose, sucrose, and glucose) to preserve the blood substitute liposome-encapsulated hemoglobin (LEH) in the freeze-dried state is examined. The water-free stabilization of individual components of this blood substitute and LEH is reported. Lyophilization of hemoglobin solutions in the absence of carbohydrates results in significant oxidative degradation of Hb as measured by a large increase (approximately 60%) in methemoglobin. Hb samples lyophilized in increasing carbohydrate concentrations show reduced levels of methemoglobin, and at 0.5 M trehalose, sucrose, or glucose, these levels are reduced to nearly the same levels as unlyophilized controls. Storage of lyophilized Hb samples following rehydration at 4 degrees C shows the same rate of methemoglobin formation regardless of whether carbohydrates are present. This suggests that carbohydrates prevent Hb oxidation in the dry state but are less effective at retarding oxidative damage to Hb in solution. The addition of 0.25 M trehalose or sucrose to LEH results in the maintenance of liposomal size following lyophilization. In these experiments, glucose was least effective at inhibiting dehydration-induced LEH fusion. Lyophilization of LEH in 0.25 M trehalose or sucrose also results in significantly greater retention of the encapsulated hemoglobin following lyophilization and rehydration. These results suggest that the long-term stabilization of LEH in the dry state is a realizable goal.     

Reduction of methemoglobin by cobaltocytochrome c catalyzed by mediators.

The reduction of methemoglobin by cobaltocytochrome c (Cocyt c) has been measured using nine mediators of different half-reduction potentials, Em, 7. The rate increases with the increase of Em, 7 for the mediator but dropped precipitously when it becomes more positive than the Em, 7 for the methemoglobin/hemoglobin couple. The reaction is most efficient with phenzaine methosulfate, therefore it was studied in detail. The reaction is first order in the concentrations of Cocyt c and phenazine methosulfate. The average second-order rate constant for Cocyt c + phenazine methosulfate (M) k1 leads to Cocyt c+ M-. is 2.9 x 10(4) M-1 s-1 at 25 degrees C, 0.1 M phosphate pH 7.0. There is a slight negative temperature dependence of k1 at low temperature; at higher temperatures the process has deltaH not equal to approximately 27 kJ mol-1 and deltaS not equal to approxmately - 75 J mol-1 K-1. The effect of anions reflects the dependence of Em, 7 for the methemoglobin/hemoglobin couple with various anions. There is no significant effect on k1 by the addition of inositol hexakisphosphate. The variation of k1 with pH is complicated. The experimental rate constants are compared with values calculated with the theory of nonadiabatic multiphonon process of electron tunneling.     

The nicotinamide adenine dinucleotides as allosteric effectors of human hemoglobin

The oxygen binding properties of human hemoglobin are appreciably altered by the nicotinamide dinucleotides NADH, NADP+, and NADPH. These cofactors are important in the control of many metabolic pathways and in providing reductive potential for a number of enzymatic reactions, including in vivo reduction of methemoglobin. Specific binding of these cofactors to hemoglobin and their potential for acting as allosteric modifiers of hemoglobin function have not been previously recognized. Detailed oxygen binding studies utilizing a thin-layer method suggest that the nicotinamide dinucleotides bind with high affinity to the deoxyhemoglobin tetramer at the beta chain anion-binding site and stabilize the low affinity "T-state" conformation. Stripped Hb A in 0.05 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 6.5, at 20 degrees C is half-saturated at a pO2 of 1.6 mm Hg. In the presence of 0.5 mM NADH, NADP+, or NADPH, the P50 is raised to 3.8, 7.1, and 12.5 mm Hg, respectively. The Bohr factor for stripped Hb A in 0.05 M HEPES buffer is sensitive to these effectors and is raised from 0.25 to about 0.65 by the addition of NADPH. The data suggest that routine use of these effectors in studies of human hemoglobin variants or the allosteric mechanism of Hb A be considered carefully. The relatively low intraerythrocytic levels of the nicotinamide dinucleotides in relation to hemoglobin dictate that these cofactors cannot significantly affect in vivo oxygen delivery. However, the converse is theoretically possible. The binding of the cofactors to hemoglobin and the preferential binding of their reduced forms may affect cofactor-dependent metabolic processes in red blood cells.     

Phospholipid vesicles increase the survival of freeze-dried human red blood cells

In a previous report [Z. T?r?k, G. Satpathy, M. Banerjee, R. Bali, E. Little, R. Novaes, H. Van Ly, D. Dwyre, A. Kheirolomoom, F. Tablin, J.H. Crowe, N.M. Tsvetkova, Preservation of trehalose loaded red blood cells by lyophilization, Cell Preservation Technol. 3 (2005) 96-111.], we presented a method for preserving human red blood cells (RBCs) by loading them with trehalose and then freeze-drying. We have now improved that method, based on the discovery that addition of phospholipid vesicles to the lyophilization buffer substantially reduces hemolysis of freeze-dried RBCs after rehydration. The surviving cells synthesize 2,3-DPG, have low levels of methemoglobin, and have preserved morphology. Among the lipid species we studied, unsaturated PCs were found to be most effective in suppressing hemoglobin leakage. RBC-vesicle interactions depend on vesicle size and structure; unilamellar liposomes with average diameter of less than 300 nm were more effective in reducing the hemolysis than multilamellar vesicles. Trehalose loaded RBCs demonstrated high survival and low levels of methemoglobin during 10 weeks of storage at 4 degrees C in the dry state when lyophilized in the presence of liposomes.     

Dependence of human erythrocyte methemoglobin reductase on temperature

The effect of temperature on the activities of cytoplasmic and membrane-bound fractions of NADH-cytochrome beta 5 reductase on the total activity of methemoglobin reductase in intact human erythrocytes was studied within the temperature range of 20-50 degrees C. The above three activities showed a break in the Arrhenius plots at 42 degrees C which was attributed to irreversible inactivation of the enzymes. Thermal inactivation of methemoglobin reductase in erythrocytes was found to increase the methemoglobin content concomitantly with a decrease in the osmotic stability and activation of spontaneous cell hemolysis.     

Methemoglobin reduction under near physiological conditions

Pure methemoglobin was prepared from fresh red cells and was used as substrate for methemoglobin reduction reaction. Two sources of methemoglobin reductase were used: (a) red cell hemolysate which was prepared by freezing and thawing of unwashed red cells; (b) purified methemoglobin reductase from bank blood. Methemoglobin reduction rate was measured in a mixture of pure methemoglobin (substrate) and hemolysate (enzyme). In other experiments the rate of methemoglobin reduction was measured in the above mixture with the addition of various other compounds such as NADH, cytochrome b5, and pure methemoglobin reductase. Only the addition of pure enzyme accelerated the rate of methemoglobin reduction. In other experiments, the rate of methemoglobin reduction was measured when the reduction reaction was carried out in the presence of various amounts of deoxyhemoglobin, globin, or albumin. It was shown that all proteins tested here decreased the reduction rate. It is concluded that (a) in the red cell, under normal conditions, only the activity of the methemoglobin reductase controls the speed of methemoglobin reduction, and (b) the inhibition of methemoglobin reduction by reduced hemoglobin is mostly nonspecific suggesting a noncompetitive reaction.     

Cyanate binding to the ferric heme octapeptide from cytochrome c. A model for anion binding to high spin ferric hemoproteins

Equilibrium constants for the binding of cyanate to the ferric heme c octapeptide in 50% ethylene glycol, 50% aqueous buffer were measured spectrophotometrically. Equilibrium constants measured at several temperatures from -20 degrees C to 0 degrees C exhibited an apparent van't Hoff relationship yielding thermodynamic values of delta Ho = -1.3 X 10(3) +/- 0.9 X 10(3) J/mol (-3.1 X 10(2) +/- 2 X 10(2) cal/mol), delta So = -3 +/- 3 J/K X mol (-0.6 +/- 0.8 cal/K X mol). The equilibrium constant for cyanate binding at 25 degrees C and pH 7.4 is 1.21 which is approximately 2 to 3 orders of magnitude lower than that observed for cyanate binding to methemoglobin and metmyoglobin. Krel, the ratio of the hemoprotein to model heme octapeptide binding constants, for NCO- is smaller than Krel for N3- suggesting that hydrogen bonding between the terminal ligand atoms and the distal histidine in hemoglobin and myoglobin does not contribute to the increased protein ligand stabilization observed for these anions relative to the model. A donor-acceptor interaction between the distal histidine and the electrophilic middle atoms of these bound ligands is proposed.     

The mechanism of superoxide anion generation by the interaction of phenylhydrazine with hemoglobin.

The mechanism by which superoxide anion is generated by the interaction of phenylhydrazine with either oxy- or methemoglobin was investigated. Rather than superoxide anion generation resulting from an accelerated autooxidation of oxyhemoglobin, it was found that both oxy- and methemoglobin function as peroxidases toward phenylhydrazine with the resultant oxidation of this compound to phenyldiazine. Generation of phenyldiazine from the oxidation of phenylhydrazine by hemoglobin or by the hydrolysis and subsequent decarboxylation of methyl phenylazoformate (C6H5N=NCOOCH3) resulted in the production of superoxide anion. It is suggested that under certain conditions hemoglobin may function as a drug-metabolizing peroxidase.     

The special behavior of equine erythrocytes connected with the methemoglobin regulation

Erythrocytes from thoroughbred horses were submitted to total (80-90%) and partial (25-40%) oxidation of hemoglobin by sodium nitrite. The ability of these cells to reduce methemoglobin to hemoglobin in the presence of either glucose, glucose plus methylene blue or lactate was investigated. The results were compared with those ones obtained for human erythrocytes. Under total oxidation: the horse erythrocytes need longer incubation time with glucose or glucose plus methylene blue than human erythrocytes for reducing the methemoglobin; methylene blue did not enhance methemoglobin reduction in the equine erythrocytes, as occurred in human erythrocytes; for horses, lactate was a more efficient substrate in promoting methemoglobin reduction. The reduction of methemoglobin by equine erythrocytes under partial oxidation was very quick in any of the incubation media. The results can explain the incongruity between the previously reported inability of equine erythrocytes to reduce methemoglobin and the lack of methemoglobinemias in equine veterinary practice.     

The effect of crosslinking by bis(3,5-dibromosalicyl) fumarate on the autoxidation of hemoglobin

Bis(3,5-dibromosalicyl) fumarate was used to crosslink hemoglobin both in the oxy and deoxy states. This double headed diaspirin was known to crosslink oxy Hb A selectively between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxy Hb A between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R. Y., et al. (1986) J. Biol. Chem. 261, 9929). The autoxidation at 37 degrees C of oxy alpha 99 crosslinked hemoglobin was found to be 1.8 times as fast as that of Hb A while that of the oxy beta 82 crosslinked hemoglobin was only 1.2 times as fast. After 5 hours the formation of methemoglobin in the alpha crosslinked Hb A is 21.3% compared to 10.8% in beta crosslinked Hb A and 6.4% in Hb A. These results may effect the proposed use of alpha 99 crosslinked hemoglobin as a blood substitute by demonstrating the need for protection from autoxidation during storage.