Ehud Ilan

Oxygen-binding properties of extracellular hemoglobin from the leech, Hirudo medicinalis. Effects of pH, cations and temperature.

Electron microscopy of the extracellular hemoglobin from the medicinal leech, Hirudo medicinalis, revealed a typical annelid two-tiered hexagonal structure. The dimensions of the molecule were 30.2 +/- 1.6 nm corner to corner across the hexagon and 18.9 +/- 1.1 nm in height. The oxygenation properties of this high-molecular-weight hemoglobin have been thoroughly studied. The effects of hemoglobin concentration, pH, temperature and concentration of specific cations on the oxygen affinity and the degree of cooperativity have been investigated with special consideration given to physiological conditions. At near-physiological cation concentrations (6 mM Ca2+, 0.125 M Na+), the oxygen affinity and cooperativity tend to increase with increasing alkalinity. At physiological conditions ((pH 7.5) temperature 20 degrees C), they reach P50 values of 5.0 mmHg and n50 = 3.1. An alkaline Bohr effect with considerable magnitude (phi = -0.50) is observed at pH 7.0-8.0. The oxygen-binding properties are also affected by cations. Both oxygen affinity and cooperativity rise with increasing cation concentrations. Divalent cations, such as Ca2+ and Mg2+, bring about larger effects than monovalent ones. They begin to be effective at concentrations as low as 0.1 mM. The magnitude and position of the Bohr effect of H. medicinalis hemoglobin are controlled by cations to such an extent that this Bohr effect can be predominantly ascribable to the oxygen-linked binding of the physiologically available allosteric effectors Ca2+ and Na+. The results obtained indicate that protons and cations are effective allosteric regulators of achaete (leech) extracellular hemoglobins.

Bovine Hemoglobin Anaerobically Reacted with Divinyl Sulfone: a Potential Source for Hypothermic Oxygen Carriers

The bifunctional reagent divinyl sulfone was anaerobically reacted with bovine hemoglobin to give a noncrosslinked intramolecularly-modified new derivative (HbBv-DVS). By employing a high molar ratio of divinyl sulfone to HbBv-DVS, it was possible to effect anaerobic intermolecular crosslinkage. The polymerized material (Poly HbBv-DVS) was shown to consist of a mixture of modified intermolecularly-crosslinked hemoglobins characterized by molecular masses ranging from 130 to -500 kDa. Some functional properties of HbBv-DVS and Poly HbBv-DVS have been evaluated in vitro and in vivo. Viscosities of HbBv-DVS solutions at temperatures as low as 15 degrees C and concentrations up to 14.0 g/dl were proved to be much lower than that of normal human blood at 37 degrees C (-4 cp). Poly HbBv-DVS (14.0 g/dl) was iso-oncotic (COP = 23 mm Hg) with plasma, and had viscosities of 3.37 and 4.57 cp at 37 and 15 degrees C, respectively. The clearance of Poly HbBv-DVS from the circulation was significantly delayed (T1/2 = 270 min), compared with those of HbBv and HbBv-DVS (T1/2 = 80 and 100 min, respectively. The P50 values were substantially increased (P50 = 52 and 61 mm Hg at 37 degrees C, 0.15 M Cl- and pH 7.4 for HbBv-DVS and Poly HbBv-DVS, respectively). Due to their right-shifted oxygen equilibrium curves, these derivatives still yielded P50 values of about 20 mm Hg at the low temperature of 15 degrees C, as compared with only 7 mm Hg for native bovine hemoglobin. These properties make HbBv-DVS and Poly HbBv-DVS potential new candidates for low-temperature organ perfusion.

The anaerobic reaction of bovine hemoglobin with divinyl sulfone: structural changes and functional consequences

The anaerobic reaction of bovine hemoglobin with divinyl sulfone results in a non-cross-linked intramolecularly-modified new derivative. This chemically-modified hemoglobin is homogeneous with respect to its molecular mass (64 kDa) and electrophoretic properties. The absence of any 32 kDa band from its SDS-PAGE pattern proves the lack of intramolecular cross-linkage, while a single-peak high-performance gel-permeation chromatogram demonstrates the absence of intermolecular cross-linkage. The oxygen binding properties determined at 37 degrees C, 0.15 M Cl- and pH 7.4 display a P50 of 52 mmHg and a Hill coefficient n of 1.9. Under the same experimental conditions the oxygen affinity is not sensitive to chloride anions, suggesting that the covelant modification is in the beta-cleft. The maximum number of Bohr protons released is 0.8/tetramer, which is half that of normal bovine hemoglobin. The retention time in circulation, measured in rats, is similar to that of native bovine hemoglobin. Using a high molar ratio of divinyl sulfone to modified hemoglobin, it is feasible to effect anaerobic intermolecular cross-linkage. The polymerized material, isolated from a 24 h reaction, is a mixture of modified intermolecularly-crosslinked hemoglobins with a molecular mass range from 130 to approx. 500 kDa. The oxygen-transport characteristics of the polymerized material are similar to those of the modified non-cross-linked derivative, whereas its retention time in rats is increased three-fold with respect to native bovine hemoglobin.

Physicochemical features and oxygenation properties of extracellular hemoglobin from the leech, Macrobdella decora

Abstract 1. 1. Extracellular hemoglobin from the freshwater leech Macrobdella decora has a sedimentation coefficient of 57 S. Under the electron microscope, the molecule displays a two-layered hexagonal structure, characteristic of annelid extracellular hemoglobins, with dimensions 29.9 ± 1.8 nm (across the hexagon) and 19.2 ± 1.2 nm (height). M. decora hemoglobin contains one heme per 24,000 g of protein. 2. 2. The oxygen binding properties of this high molecular weight hemoglobin are characterized as a function of pH, cations and temperature. 3. 3. The oxygenation properties are affected by protons. At near-physiological cation concentrations, the oxygen affinity and cooperativity rise concomitantly with increasing alkalinity. At pH ∼ 8.0 and 20°C both these parameters attain maximal values of P 50 = 3.0 mm Hg and n 50 = 3.1. A normal alkaline Bohr effect ( φ = −0.33) at the pH range 6.5–7.5 is observed. 4. 4. The oxygen binding characteristics are also extremely sensitive to cations. At pH 7.75 and 20°C both oxygen affinity and cooperativity rise with increasing cation concentrations. Divalent cations such as Ca 2+ and Mg 2+ produce a larger effect than monovalent ones. They start to be effective at concentrations as low as 0.05 mM.

Structural characterization and oxygen binding properties of extracellular hemoglobin from the marine polychaete Eurythoe complanata

Abstract 1. 1. Extracellular hemoglobin from the polychaete Eurythoe complanata has a sedimentation coefficient of 54 S. In the electron microscope, the molecule exhibits a two-layered hexagonal appearance characteristic of annelid extracellular hemoglobins. 2. 2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol gave two main bands corresponding to mol. wts 21,000 and 34,000 and a faint band corresponding to mol. wt 25,000. 3. 3. In the pH range 4.6–10.5, oxygen binding showed little co-operativity (Hill coefficient ⩽1.7). Small alkaline ( φ = −0.09 ) and reverse acid ( φ = 0.04 ) Bohr effects were observed. 4. 4. The oxygen equilibrium properties of Eurythoe hemoglobin were compared with those of other polychaete hemoglobins.

Conversion of ammonia to glutamate by L-glutamic dehydrogenase, alcohol dehydrogenase and NAD+ immobilized within lipid-polyamide polyethyleneimine microcapsules.

The use of Albumin-Collodion coated Activated Charcoal (ACAC) hemoperfusion systems for treatment of end-stage renal failure has several advantages over the hemodialysis procedure. With coated charcoal ACAC hemoperfusion, the removal of uremic toxins, such as creatinine, uric acid, and middle molecules, is much more effective than with standard hemodialysis. However, the ACAC hemoperfusion system does not remove water, electrolytes or urea. As the removal of excess water can be achieved with the help of a small ultrafiltrator1,2, the remaining major problems are electrolytes and urea removal.

Structural and Oxygen-Binding Properties of Divinyl-Sulfone-Reacted Bovine Hemoglobin as a Function of Protein Ligation State and Reactant Concentration

Bovine hemoglobin (HbBv) was reacted with divinyl sulfone (DVS) at 2-4 degrees C and pH 7.4 for 24 h. Two procedures were employed: (1) low concentration (conc.) of oxygenated HbBv (ligand O2) and low molar ratio of DVS/oxyHbBv (25 ml of 8% oxyHbBv, 0.01 M DVS); (2) low conc. of deoxygenated HbBv (no ligand) and low DVS/deoxyHbBv molar ratio (25 ml of 8% deoxyHbBv, 0.01 M DVS). The nonpolymerized purified products of procedures 1 and 2 were designated oxyHbBv-DVS and HbBv-DVS. Utilizing a high conc. solution of oxyHbBv-DVS and a relatively high molar ratio of DVS/oxyHbBv-DVS (25 ml of 15% oxyHbBv-DVS, 0.03 M DVS) it was possible to aerobically polymerize the modified hemoglobin (procedure 3). Similarly it was possible to anaerobically polymerize HbBv-DVS (25 ml of 15% HbBv-DVS, 0.03 M DVS)(procedure 4). The polymerized products of procedures 3 and 4 were designated oxyPoly HbBv-DVS and Poly HbBv-DVS. The four isolated products were characterized structurally using SDS-PAGE and gel-permeation HPLC and functionally employing a Hemox-analyzer at 37 degrees C, pH 7.4 and 0.15 M Cl. Both oxyPoly HbBv-DVS (P50 = 13 mm Hg, n = 1.1) and Poly HbBv-DVS (P50 = 61 mm Hg, n = 1.6) were shown to be mixtures of intermolecularly-crosslinked hemoglobin. OxyHbBv-DVS (P50 = 13 mm Hg, n = 1.3) was shown to be an intramolecularly-crosslinked 64 kDa material, whereas HbBv-DVS (P50 = 52 mm Hg, n = 1.9) was found to be an intramolecularly-modified 64 kDa derivative, but not an intramolecularly-crosslinked one.(ABSTRACT TRUNCATED AT 250 WORDS)