Mathematical models of the oxygen-binding function of intact human hemoglobin and hemoglobin modified by UV-radiation in the presence of carbon oxide

The influence of carbon oxide and UV-radiation in doses of 151-453 J/m2 on the physiological properties of human oxyhemoglobin has been studied. Mathematical models of the oxygen-binding function of intact and modified hemoprotein have been developed. It has been found that saturation of human hemoglobin with oxygen obeys the logistic dependence. In the presence of carboxyhemoglobin, the oxygenation parameters change and saturation curves are described by the equations of degree dependence. It has been shown that UV light had the stimulating influence on the functional properties of human hemoglobin modified by carbon oxide if the concentration of carboxyform of the hemoprotein in solution was no higher than 10 per cent. The disturbance of the oxygen-binding ability of hemoglobin by the action of higher concentrations of carboxyhemoglobin was irreversible and was not corrected by UV-radiation.     

Correlation of the internal microviscosity of human erythrocytes to the cell volume and the viscosity of hemoglobin solutions

The microviscosity of the cytoplasm of human erythrocytes as well as of membrane-free hemoglobin solutions was investigated measuring the rotation of the small spin-label molecule, Tempone. The dependence of the intracellular microviscosity on the extracellular pH and osmotic pressure which was varied by NaCl or sucrose was sufficiently explained on the basis of alterations of the red blood cell volume. The intracellular microviscosity depended exclusively on the hemoglobin concentration. It did not differ from that of comparable membrane-free hemoglobin solutions. It was not necessary to take into account long-range interactions between hemoglobin molecules. The conclusion therefore was that the intracellular viscosity is not modified by cytoplasmic structures or the cell membrane. Above a hemoglobin concentration of 6 mM the viscosity of hemoglobin solutions increased much faster than the microviscosity. From measurements obtained with different spin-labels it followed that also the charge of these molecules is of importance.     

The effect of organic compounds on the crystal habit and crystallization of normal and sickle cell hemoglobin in phosphate buffer

Comparative studies were made on the effect of numerous organic compounds in promoting the crystallization of human hemoglobin in 1.9 m phosphate, pH 7.0. It was found that alicyclic or benzenoid structures are essential for promoting crystallization of hemoglobin under these conditions. Hemoglobin crystals prepared in the presence of toluene differed in habit from crystals prepared in its absence. It is suggested that steric factors determine the effectiveness of organic substances in promoting the crystallization of hemoglobin and that the heme group is the binding site involved in the complex formation.The solubility of homozygous sickle cell hemoglobin HbS was found to be less than the heterozygous hemoglobins AS and AC or normal hemoglobin HbA in the presence of organic substances promoting the crystallization of hemoglobin.     

Binding of acellular, native and cross-linked human hemoglobins to haptoglobin: enhanced distribution and clearance in the rat

It is well established that hemoglobin resulting from red cell lysis binds to haptoglobin in plasma to form a complex. The increased molecular size precludes its filtration by the kidneys, redirecting it toward hepatocellular entry. Chemically cross-linked hemoglobins are designed to be resistant to renal excretion, even in the absence of haptoglobin. The manner in which binding to haptoglobin influences the pharmacokinetics of acellular cross-linked and native hemoglobins was investigated after intravenous injection of radiolabeled native human hemoglobin and trimesyl-(Lys82)beta-(Lys82)beta cross-linked human hemoglobin, at trace doses, into rats. Under these conditions, there is sufficient plasma haptoglobin for binding with hemoglobin. In vitro binding assayed by size-exclusion chromatography for bound and free hemoglobin revealed that, at <8 muM hemoglobin, native human hemoglobin was completely bound to rat haptoglobin, whereas only approximately 30% of trimesyl-(Lys82)beta-(Lys82)beta cross-linked hemoglobin was bound. Plasma disappearance of low doses (0.31 mumol/kg) of native and cross-linked hemoglobins was monoexponential (half-life = 23 and 33 min, respectively). The volume of distribution (40 vs. 19 ml/kg) and plasma clearance (1.22 vs. 0.4 ml.min(-1).kg(-1)) were higher for native than for cross-linked hemoglobin. Native and cross-linked human hemoglobins were found primarily in the liver, and not in the kidney, heart, lung, or spleen, mostly as degradation products. These pharmacokinetic findings suggest that the binding of hemoglobin to haptoglobin enhances its hepatocellular entry, clearance, and distribution.     

Studies of rhesus monkey (Macaca mulatta) hemoglobin.

One hemaglobin was found in the adult rhesus monkey (Macaca mulatta) by the technique of isoelectric focusing. In addition to the adult hemoglobin, one fetal hemaglobin was observed in neonates of the same species. The alpha and non-alpha globins of rhesus monkey hemoglobins showed similar electrophoretic mobilities as those of human hemoglobin A by cellulose acetate membrane electrophoresis. It appeared that the rhesus monkey hemoglobin system is a good model system for the study of human hemoglobin development.     

Affinity chromatography: specific binding of hemoglobin on agarose linked haptoglobin

Human haptoglobin was coupled to agarose and used as affinity adsorbant to bind human hemoglobin. The optimal conditions of hemoglobin binding and dissociation were defined. It was found that the haptoglobin adsorbant removed effectively and specifically free hemoglobin from hemolysed sera.     

Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane

The hemoglobin binding sites on the inner surface of the erythrocyte membrane were identified by measuring the fraction of hemoglobin released following selective proteolytic or lipolytic enzyme digestion. In addition, binding stoichiometry to and fractional hemoglobin release from inside-out vesicle preparations of human and rabbit membranes were compared since rabbit membranes differ significantly from human membranes only in that they lack glycophorin. Our results show that rabbit inside-out vesicles bind about 65% less human or rabbit hemoglobin under conditions of optimal and stoichiometric binding, despite being otherwise similar in composition. We suggest that this difference is either directly or indirectly due to the absence of glycophorin in rabbit membranes. Further supportive evidence includes demonstrating (a) that neuraminidase treatment of human membranes did not affect hemoglobin binding and (b) that reconstitution of isolated glycophorin into phospholipid vesicles increased the hemoglobin binding capacity in a manner proportional to the fraction of glycophorin molecules oriented with their cytoplasmic sides exposed to the exterior of the vesicle. Proteolysis of human inside-out vesicles either before or after addition of hemoglobin reduced the binding capacity by about 25%. This is consistent with the known proportion of total hemoglobin binding sites involving band 3 protein and the selective lability of the cytoplasmic aspect of band 3 protein to proteolysis. Phospholipid involvement in hemoglobin binding was determined using various phospholipase C preparations which differ in their reactivity profiles. Approximately 38% of the bound hemoglobin was released upon cleavage of phospholipid headgroups. These results suggest that the predominant sites of binding for hemoglobin on the inner surface of the red cell membrane are the two major integral membrane glycoproteins.     

The interaction of organic phosphates with human and chicken hemoglobin.

In this study of the binding properties of inositol hexaphosphate and 2,3-bisphosphoglycerate to chicken and human deoxyhemoglobin and carboxyhemoglobin were compared. It appeared that in all cases the binding to chicken hemoglobin is much stronger than to human hemoglobin. This is very probably due to the fact that 4 out of the 12 residues, responsible for the binding of phosphates in chicken hemoglobin, are arginines. These are absent in human hemoglobin, where the binding site is made up to only 8 residues. For chicken hemoglobin one strong binding site could be observed in both unliganded and liganded hemoglobin. From these observations we conclude that the same binding site is involved in both the oxy- and deoxy structure showing different affinity to phosphates in the two conformational states. For human hemoglobin we reached the same conclusion.     

Functional consequences of ligand-linked dissociation in hemoglobin from the sea cucumber Molpadia arenicola.

It has been established that Molpadia hemoglobin tends to dissociate into subunits as oxygen is bound. The kinetics and equilibria of carbon monoxide and ethylisocyanide binding reported here show a dependence on protein concentration that supports the conclusion that the aggregated hemoglobin has a lower ligand affinity than the dissociated subunits. This is true for the isolated D-chain as well as for the unfractionated hemolysate that contains four distinct polypeptide chains (A-D). This indicates that even homopolymers of Molpadia hemoglobin have lower ligand affinity than the dissociated subunits. At high protein concentration hemolysates of Molpadia hemoglobin show slight cooperativity. The time course of ligand binding to the deoxy D-chain also suggests cooperative interactions. The low affinity of the aggregated state may have a different molecular explanation than in human hemoglobin where tetramers of identical subunits (as in Hb H) show high oxygen affinity. The absence of tyrosine and histidine at the C-terminal of the Molpadia D-chains also suggests a different stabilization of the low affinity deoxy state. An additional functional difference between Molpadia hemoglobin and human hemoglobin is that organic phosphates do not alter the ligand affinity of the sea cucumber hemoglobin.     

Aging of the erythrocyte. XI. Membrane glycosylation

It has been demonstrated that glucose reacts nonezymatically with blood proteins, especially albumin (1) and hemoglobin (2, 3). The level of albumin-bound and hemoglobin-bound glucose can be a long-term measure of blood-glucose concentration (1–3). The content of glycosylated hemoglobin increases with age of human erythrocytes (4). The aim of this study was to determine whether a cell-age-dependent glycosylation of membrane constituents can be found in the erythrocytes.     

Hemoglobin types of gibbons and leaf-monkeys

Electrophoretic patterns of hemoglobin were observed in 61 samples from gibbons and in 29 samples from leaf-monkeys. In gibbons, three hemoglobin types, involving two main molecules with a minor component, were found. The two gibbon hemoglobins were presumed to differ in their corresponding beta chain. It is characteristic that each of the three hemoglobin types was present in three different species of gibbons. In three species of leaf-monkey, only one hemoglobin was observed, showing electrophoretic mobility very similar to human Hb-A.     

共振瑞利散射测试血清蛋白酸碱度影响的探讨

提出了一种基于共振瑞利散射（RRS）原理测量人体血清蛋白的新方法。在缓冲溶液的作用下,把配制好的人体血清蛋白稀释液按比例与四羧基酞菁锌混合,经过化学作用后在波长为400 nm左右蓝色波段强光照射下,散射出480 nm左右的共振瑞利散射光强信号。考察在不同pH对共振瑞利散射光强信号与混合物中的血清蛋白反应线性关系的影响。结果表明,pH在6.0～8.0范围内混合溶液共振瑞利散射光强信号与血清蛋白的线性关系良好。     

A simulation study of the anomalous osmotic behavior of red cells

The factors responsible for movements of water across cell membranes were described mathematically and incorporated into a model which simulates water balance in the cell. Included in the model are a variable charge and osmotic coefficient of hemoglobin, a Na/K pump whose rate varies with ionic concentrations, and the standard electroneutrality and osmotic equilibrium assumptions. The model was used to investigate the phenomena whereby human red cells placed in media of varying tonicities exhibit steady state volume changes less than those predicted by van't Hoff's Law. The model results showed that this anomalous osmotic behavior was primarily due to changes in the osmotic coefficient of hemoglobin as its concentration in the cell varied. A second factor accounting for a part of this behavior was the alteration in the rate of the Na/K pump due to intracellular ionic concentration changes as cell volume varied. The effect of variable electrical charge on the hemoglobin molecule was found to be in the wrong direction to account for the observed osmotic behavior. Also, this effect was seen to produce relatively large changes in cell membrane potential, a result inconsistent with experimental data. It was concluded from the model results that the anomalous osmotic behavior of human red cells is primarily due to the variation in the osmotic coefficient of hemoglobin as the cell volume changes, and that the variable charge effect on the hemoglobin molecule, if it exists, does not play a role in this response.     

Cellular Responses of Congenital Immunity in the Starfish <Emphasis Type="Italic">Asterias rubens</Emphasis>

The work deals with analysis of changes of cellular defense factors in the starfish Asterias rubens in response to injection of human erythrocytes (HE). The number of circulating coelomocytes, dynamics of their production of active oxygen forms, activity of peroxidase, and dynamics of elimination of human hemoglobin from coelomic fluid were estimated before immunization with HE as well as at 6–144 h. The number of coelomocytes was counted in Goryaev chamber, production of active oxygen forms was determined in the test of spontaneous and zymosan-induced reduction of Tetrazolium Nitro Blue, peroxidase activity—in a color enzymatic reaction. Time of human hemoglobin elimination from the coelomic fluid was determined by spectrophotometric method by hemoglobin binding with acetone cyanohydrin with formation of a colored product. It is revealed that injection of human erythrocytes into the starfish Asterias rubens leads to a decrease of the number of coelomocytes in 24–96 h and to an increase of their specific production of active oxygen forms in 96–120 h after the HE injection. In coelomic fluid of Asterias rubens the presence of peroxidase activity is established. The circulation time of human hemoglobin released from erythrocytes in coelomic fluid of these animals does not exceed 24 h. It is suggested that the cellular defense reactions are the major factor of the starfish congenital immunity.__________Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 2, 2005, pp. 107–113.Original Russian Text Copyright © 2005 by Kudryavtsev, D’yachkov, Kazakov, Kanaikin, Kharazova, Polevshchikov.     

Functional consequences of ligand-linked dissociation in hemoglobin from the sea cucumber molpadia arenicola

It has been established that Molpadia hemoglobin tends to dissociate into subunits as oxygen is bound. The kinetics and equilibria of carbon monoxide and ehtylisocyanide binding reported here show a dependence on protein concentration that supports the conclusions that the aggregated hemoglobin has a lower ligand affinity than the dissociated subunits. This is true for the isolated D-chain as well as for the unfractionated hemolysate that contains four distinct polypeptide chains (A-D). This indicates that even homopolymers of Molpadia hemoglobin have lower ligand affinity than the dissociated subunits. At high protein concentration hemolysates of Molpadia hemoglobin show slight cooperativity. The time course of ligand binding to the deoxy D-chain also suggests cooperative interactions, The low affinity of the aggregated state may have a different molecular explanation than in human hemoglobin were tetramers of identical subunits (as in Hb H) show high oxygen affinity. The absence of tyrosine and histidine at the C-tremini of the Molpadia D-chains also suggests a different stabilization of the low affinity deoxy state. An additional functional difference between Molpadia hemoglobin and human hemoglobin is that organic phosphate do not alter the ligand affinity of the sea cucumber hemoglobin.     

Broad-spectrum antimicrobial activity of hemoglobin

While hemoglobin is one of the most well characterized proteins due to its function in oxygen transport, few additional properties of hemoglobin have been described. While screening serum samples for novel antimicrobial factors, it was found that intact hemoglobin tetramers, including that from human, exhibited considerable activity against gram-positive and gram-negative bacteria, and fungi. To further characterize this surprising activity, the antimicrobial potency of sections of human hemoglobin was tested against a panel of microorganisms. In all cases separate testing of the alpha and beta subunits provided activity at least as potent as the intact tetramer. This activity is derived from the protein portion of hemoglobin since removal of the heme prosthetic group did not lead to decreases in potency. In addition, cyanogen bromide cleavage of both subunits provided fragments that still contained substantial antimicrobial activity. It has been possible to map specific regions of the human hemoglobin molecule that are responsible for significant antimicrobial activity. The carboxyl terminal thirty amino acids of the beta subunit, which form a cationic alpha-helix based on the crystal structure of the intact tetramer, were active against Escherichia coli, Staphylococcus aureus and Candida albicans. In view of the fact that different hemoglobin-derived peptide fragments exhibit diverse antibiotic activities, it is conceivable that, in addition to its role in oxygen transport. hemoglobin functions as an important multi-defense agent against a wide range of microorganisms.     

Amino acid sequences of the alpha and beta chains of adult hemoglobin of the brown lemur, Lemur fulvus fulvus.

Globin prepared from hemoglobin of the brown lemur (Lemur fulvus fulvus) was separated into alpha and beta chains by chromatography on a CM 52 column. The S-aminoethylated alpha and beta chains were each digested with trypsin and resulting peptides were isolated. The amino acid sequences of the tryptic peptides were established. The ordering of these peptides in the alpha and beta chains was deduced from the homology of their amino acid sequences with that of human adult hemoglobin. The primary structure of brown lemur hemoglobin thus obtained differs from that of human hemoglobin in 15 amino acids in the alpha chain and 26 in the beta chain.     

Kinetics of the Bohr effect of menhaden hemoglobin, Brevoortia tyrannus.

The kinetics of proton release on ligation of menhaden hemoglobin was studied by flash photolysis over a range of pH. In contrast to all previous kinetic work with human hemoglobin, a nonlinear relationship between proton release and CO binding was found. Proton uptake was also observed in the course of O₂ replacement by CO at low pH. It follows that at least part of the proton release is associated with quaternary rather than tertiary conformational changes i.e. this result is consistent with a two-state model in which L is a function of pH.     

The interaction between phosphate and protein, and the respiration of the llama, the human fetus and the horse (author's transl)

The sequence analysis of llama (Lama glama, Camelidae) hemoglobin is described. The chains were separated, cleaved by trypsin as previously described, quantitatively characterized and sequenced in the sequenator. The llama hemoglobin differs from the human hemoglobin in that it has 25 different amino acids in the alpha chain and 24 different amino acids in the beta chain. The interaction between protein and phosphate is discussed. The earlier finding that the O2 affinity of the llama hemoglobin is dependent on its content of 2, 3-bisphosphoglycerate is interpreted here as a mutation of the 2, 3-bisphosphoglycerate contact position beta2 His in human hemoglobin to beta2 Asn in llama hemoglobin, whereby one of the four 2, 3-bisphosphoglycerate contact points is interrupted. This interruption gives rise to a diminished reduction of intrinsic oxygen affinity in the hemoglobin molecule and explains, on a molecular basis, the increased oxygen affinity of the llama hemoglobin, and consequently, the high-altitude respiration of the llama. By analogy, the increased O2 affinity of human fetal hemoglobin is interpreted according to previous physiological investigations on blood and fetal hemoglobin by the inactivation of the phosphoglycerate contact point beta143 His in the adult hemoglobin by mutation to gamma 143 Ser in the fetal hemoglobin. With respect to respiration in horses (2, 3-bisphosphoglycerate contact beta2 Gln), measurement of atomic parameters show that the amido group of the glutamine is situated close enough to the 2, 3-bisphosphoglycerate oxygen to build a hydrogen bond with the phosphate. Consequently, the explanation of the low-altitude respiration of the horse lies in the fact that glutamine and histidine fulfill sterochemically an identical function.