载氧血红蛋白纯化工艺研究

目的改善心脑血管缺氧载氧药物是一种创新药物。由于它半径比红细胞小400~1 000倍,易于通过毛细血管,给缺血组织及时供氧,迅速缓解或纠正缺氧状态,达到治疗抢救目的。血红蛋白的纯化工艺是载氧药物研制的重要工艺步骤。方法本研究建立了一套通过热敏法分离纯化人脐带血血红蛋白的工艺以及较为完善的纯化血红蛋白质量检测指标。结果与现有的纯化方式相比,热敏法操作简便,仪器设备造价低廉,纯化与病毒灭活同时进行,得到的纯化产品损失少,纯度高,各项理化指标达到国际水平。结论本工艺适用于规模制备纯化血红蛋白,为进一步研制治疗心脑血管缺氧载氧药物创造了有利条件。     

Kinetics of the alkaline tetramer leads to dimer dissociation in liganded human hemoglobin: a laser light-scattering stopped-flow study.

The first-order dissociation of tetrameric HbCO to the dimer has been studied overthe pH range 10.30-11.57 in a light-scattering stopped-flow apparatus using argon-ion laser excitation. The first-order dissociation rate constant varies from 0.25 sec-1 to 24.0 sec-1over this pH interval. A semilogarithmic plot of k versus pH has a slope of 2.56 at pH 11.07, the midpoint. The pH dependence of the dissociation of the tetramer is consistent with progressive titration of alpha1-alpha2 and beta1-beta2 salt bridges. At pH 10.66, the dissociation rates of HbO2, HbCO, methemoglobin, and HbCN vary less than 20% from their mean value. A study of the dissociation kinetics as a function of protein concentration allows one to obtain both association and dissociation rate constants, and hence equilibrium constants, for the tetramer in equilibrium dimer reaction. In this manner, equilibrium constants were obtained on protein solutions with less than 15 sec of exposure to dissociating conditions.     

The oxygen affinity of concentrated human hemoglobin solutions and human blood.

The log P50 of normal human blood at 37 degrees C, PCO2 = 0, 21, 42, AND 57 MM Hg in the absence and in the presence of 2,3-DPG and ATP, has been determined in the pH range 7.0 to 7.6. Similar data have been obtained for human hemoglobin isotonic solutions at different protein concentrations in the presence of various amounts of each of the cofactors which are known to affect hemoglobin oxygen affinity in blood. It has been found that the addition of KC1, organic phosphates, magnesium ions, and CO2 confers to a 32% human hemoglobin solution the same oxygen affinity (over the entire physiological pH range) of whole blood. Thus there is no room for significant effects caused by some other unidentified molecules or ions.     

Multi-effective properties of homogentisic acid revealed in reactions with human hemoglobin and human erythrocytic hemoglobin

Homogentisic acid (HGA) causes oxidation of human oxyhemoglobin and reduction of methemoglobin. The rate of oxidation of oxyhemoglobin by HGA is greatly accelerated in the presence of myo-inositol hexakis-phosphate (P6-inositol) or superoxide dismutase (SOD), but is inhibited in the presence of catalase. The reduction rate of methemoglobin by HGA is accelerated in the presence of P6-inositol but is greatly inhibited in the presence of SOD. It is suggested that the semiquinone and quinone form of HGA and oxygen radicals may be involved in the mechanism of oxido-reductive reactions of human hemoglobin with HGA. In addition, a new anodic hemoglobin found by isoelectric focusing electrophoresis was produced during the reaction of oxyhemoglobin with HGA. When human erythrocytes were exposed to HGA for several hours at 37 degrees C (pH 7.4), the anodic oxyhemoglobin (HGA-modified hemoglobin) and its half met-form hemoglobin [(alpha3+beta2+)2 of HGA-modified hemoglobin] were produced in significant amounts. HGA-modified hemoglobin was stably purified and showed increased oxygen affinity, absence of titratable sulfhydryl groups, and the absorption spectrum of normal oxyhemoglobin. Our results demonstrate that HGA shows multiple effects on human hemoglobin and erythrocytic hemoglobin, which is consistent with the evidence that HGA is involved in various pathological conditions such as arthritis and carcinogenesis in humans.     

Acylation of hemoglobin by glutarylsalicylamide and its effect on oxygen transport properties

Hemoglobin A was modified in vitro with 0.02-0.03 M glutarylsalicylamide for two hours at pH 7.2 and 37 degrees C. The extent of modification was about 30-50%, as estimated by visual comparison after electrophoretic separation. A substantial decrease in oxygen affinity of modified hemoglobin solutions was observed. Similar results were also obtained for dilute cell suspensions of washed red blood cells and whole blood after GSM modification. Other properties such as cooperativity, Bohr effect and 2,3-DPG dependence remained essentially unchanged. Athough the site(s) of modification have not been determined, it is unlikely that they would involve any amino acid residue contributing to the above allosteric properties.     

Preparation, properties, and plasma retention of human hemoglobin derivatives: comparison of uncrosslinked carboxymethylated hemoglobin with crosslinked tetrameric hemoglobin.

Human hemoglobin A has been crosslinked by diisothiocyanatobenzenesulfonate to give a limited number of products in a yield of approximately 70%. The predominant product was crosslinked between subunits within a tetramer and had a Mr of 64,000; no higher Mr species were formed. This product had one crosslink per tetramer located between the NH2 termini of its alpha chains, as established by HPLC analysis, amino acid analysis, Edman degradation, and mass spectrometry. This crosslinked derivative had a slightly increased oxygen affinity [P50 = 9 mmHg (1 mmHg = 133 Pa); P50 for unmodified hemoglobin = 11 mmHg], and the retention time of this derivative in the circulation of rats was 2.9 and 3.3 hr at two hemoglobin concentrations (7 g/dl and 14 g/dl, respectively). The half-life of an uncrosslinked carboxymethylated derivative, which has a low oxygen affinity (P50 = 28 mmHg), was 0.6 and 0.7 hr under the same conditions. Therefore, prolongation of the plasma-retention time of infused hemoglobin is dependent on the crosslinking of the tetramer but independent of the oxygen affinity of the derivative.     

Thermodynamic aspects of the linkage between binding of chloride and oxygen to human hemoglobin

Oxygen isotherms of human hemoglobin measured in distilled water and in solutions of sodium chloride in the concentration range from 0.02 to 3.0 M indicate that the oxygen affinity decreases up to about 1 M salt and then begins to increase. The isotherms obtained in the range from 0.02 to 0.6 M sodium chloride, at 37 degrees and pH 7.4, have been analyzed in terms of changes in Gibbs free energy of heme ligation, resulting from the differential interaction between the chloride ion and the two forms of hemoglobin. The maximal theoretical change in Gibbs free energy that chloride ion can exert on the oxygen binding of hemoglobin amounts to 4.9 +/- 0.2 kcal/mol (21 +/- 0.8 kJ/mol) of hemoglobin tetramer. A plot of the logarithm of oxygen concentration at half saturation versus the logarithm of the chloride concentration has a slope of 0.40, suggesting 1.6 apparent chloride sites per hemoglobin tetramer. Because the interaction between chloride and hemoglobin is dependent on pH, the apparent thermodynamic linkage between chloride and oxygen binding will also include the salt dependence of the Bohr effect at pH 7.4. The fractional change in Gibbs free energy, measured as a function of the chloride concentration, can be approximated by the binding isotherm between a protein and a ligand, using an association constant of 11 M(-1). Thus, if the number of oxygen-linked chloride sites is more than one per hemoglobin tetramer, these sites must be considered independent.     

Reciprocal effects in human hemoglobin: direct measurement of the dimer-tetramer association constant at partial oxygen saturation.

An equilibrium gel permeation technique has been developed for determining as a function of oxygenation state the equilibrium constants for association of hemoglobin subunits. By using this method, the dimer-tetramer constant for human hemoglobin at a partial oxygenation state corresponding to 20% saturation for tetramers has been determined as 3.7 X 10(6) M-1 (dimers). Under the same conditions the corresponding constant for fully oxygenated hemoglobin is 4.1 X 10(5) M-1. These results are found to be in good agreement with the predicted behavior of the association reaction based upon oxygen binding curves measured as a function of protein concentration. Thus a high degree of consistency is found between the two independent experimental approaches to the reciprocal effects of this linkage system, lending support to the theory proposed earlier for these phenomena.     

Pressure applied during surgery alters the biomechanical properties of human saphenous vein graft

Pressure applied during harvesting of the saphenous vein (SV) graft in coronary artery bypass surgery might change its mechanical properties and thereby decrease the patency. This study was performed to assess the mechanical properties of the SV graft distended manually with different levels of pressure and to determine the pressure level that induces changes in its structure and mechanics. Saphenous vein graft segments, collected from 36 patients undergoing coronary artery bypass surgery, were distended with pressures of either 50–60, 75–100, or 130–150 mmHg. Grafts were tested for the stress–strain relationship; the Young’s moduli at the low- and high-strain regions were calculated, and their structures were examined by light and electron microscopy. Pressures of 50–60 mmHg did not influence the mechanics of the vein graft, whereas pressures of 75–100 mmHg elevated the elastic modulus of the vein at the low-strain region while pressures above 130 mmHg increased the elastic moduli at both low- and high-strain regions. There was a prominent loss of microfibrils at all distending pressure levels. The mechanical results suggest that distending pressures above 75 mmHg might play a role in graft failure. Furthermore, the absence of microfibrils surrounding elastin suggests that application of distending pressures, even as low as 50 mmHg, can cause degeneration of the elastic fibers following implantation, increasing the stiffness of the graft and thus impairing the graft’s function under its new hemodynamic conditions.     

Issues in the development of hemoglobin based oxygen carriers

Hemoglobin based oxygen carriers (HBOCs) have been developed as alternative oxygen transporting formulations for the acute treatment of anemia and ischemia. Efficacy has been demonstrated in a variety of preclinical models and selected human patients; however, a higher overall incidence of mortality and myocardial infarction in those dosed with HBOCs in later stage clinical trials has prevented widespread regulatory approval. Diagnosis of myocardial infarction is confounded by the fact that HBOCs interfere with troponin assays, as well as other clinical chemistry measurements. Analysis of data pertaining to potential toxicity mechanisms suggests that coronary vasoconstriction is an unlikely contributor, but promotion of intravascular thrombosis may occur by several mechanisms. In addition, fluid and anemia management in patients infused with HBOCs has been suboptimal. Elucidation of potential toxicity mechanisms, refinement of use protocols, and definition of improved patient inclusion/exclusion criteria remain active areas of inquiry in understanding the best manner in which to utilize HBOCs.     

Hematopoetic prostaglandin D synthase: an ESR1-dependent oviductal epithelial cell synthase

Oviductal disease is a primary cause of infertility, a problem that largely stems from excessive inflammation of this key reproductive organ. Our poor understanding of the mechanisms regulating oviductal inflammation restricts our ability to diagnose, treat, and/or prevent oviductal disease. Using mice, our objective was to determine the spatial localization, regulatory mechanism, and functional attributes of a hypothesized regulator of oviductal inflammation, the hematopoietic form of prostaglandin D synthase (HPGDS). Immunohistochemistry revealed specific localization of HPGDS to the oviduct's epithelium. In the isthmus, expression of HPGDS was consistent. In the ampulla, expression of HPGDS appeared dependent upon stage of the estrous cycle. HPGDS was expressed in the epithelium of immature and cycling mice but not in the oviducts of estrogen receptor α knockouts. Two receptor subtypes bind PGD?: PGD? receptor and G protein-coupled receptor 44. Expression of mRNA for Ptgdr was higher in the epithelial cells (EPI) than in the stroma (P < 0.05), whereas mRNA for Gpr44 was higher in the stroma than epithelium (P < 0.05). Treatment of human oviductal EPI with HQL-79, an inhibitor of HPGDS, decreased cell viability (P < 0.05). Treatment of mice with HQL-79 increased mRNA for chemokine (C-C motif) ligands 3, 4, and 19; chemokine (C-X-C motif) ligands 11 and 12; IL-13 and IL-17B; and TNF receptor superfamily, member 1b (P < 0.02 for each mRNA). Overall, these results suggest that HPGDS may play a role in the regulation of inflammation and EPI health within the oviduct.