红细胞外HbA_2与HbA间的相互作用

 1.为了阐明血红蛋白A_2现象的发生机制,研究了红细胞外Hb A_2与HbA有无相互作用。结果表明,这些血红蛋白在离开红细胞后仍能发生相互作用。 2.我们是用交叉电泳来验证这个问题,主要实验结果如下: (1)单向交叉电泳结果表明,溶血液HbA穿过另一溶血液的Hb A_2时,出现明显的交叉电泳图象。 (2)双向交叉电泳实验使上述结果更加明确。 (3)用提纯的Hb A做实验,证实了Hb A与Hb A_2间的相互作用。 (4)血浆白蛋白穿过Hb A_2、Hb A及CA时,没有看到交叉电泳图象。说明Hb A与Hb A_2之间的作用是特异的。 4.初步结论,不仅在缸细胞中Hb A_2是与Hb A结合存在,就是在离开红细胞的条件下,这两种血红蛋白之间仍能发生相互作用。我们认为,这就是血红蛋白A_2现象的发生机制,很可能如此。     

血红蛋白A_2现象发生机制的研究——“红细胞HbA_2”为HbA_2与HbA的结合产物

为了弄清血红蛋白A_2现象的发生机制,我们对“红细胞HbA_2”的化学组成进行了分析。“红细胞HbA_2”的双向电泳结果表明,它含有两种血红蛋白成分:一种相当于HbA,另一种很可能是溶血液HbA_2。其单向二次电泳结果也证明,它是由溶血液HbA_2和HbA所组成。结果初步说明,盘红细胞中HbA_2可能与HbA结合存在。两者可能有相互作用,也许这是产生血红蛋白A_2现象的原因。     

血红蛋白 A_2现象——Ⅰ.A_2现象的发现及其初步应用

1.本文报告作者实验室所发现的一种自然现象,并暂时命名为“血红蛋白A_2现象”。2.所谓“血红蛋白A_2现象”,就是溶血液中HbA_2与红细胞中“HbA_2”的差异。这里主要指的是电泳行为不同。3.将正常成人红细胞与其本身的溶血液并排电泳(薄层淀粉胶电泳)时,二者的HbA、碳酸酐酶电泳位置相同,只有HbA_2不一样。与溶血液中HbA_2相比,红细胞中的“HbA_2”电泳速度(向阳极)稍快。4.红细胞及溶血液中的HbF、HbN 包头、HbD 包头及HbE,都没有上述差异,而且它们对“HbA_2现象”没有影响。5.“HbA_2现象”的机制尚待阐明,但已经发现了它的应用。众所周知,HbE 与A_2的电泳行为及层析性质是相同或相近的,鉴别起来比较困难,现在我们可以利用“HbA_2现象”来区分这两种血红蛋白。如上所述,HbA_2有“HbA_2现象”,而HbE 本身则没有这类现象。     

血红蛋白A_2现象 Ⅰ.A_2现象的发现及其初步应用

1.本文报告作者实验室所发现的一种自然现象,并暂时命名为“血红蛋白A_2现象”。2.所谓“血红蛋白A_2现象”,就是溶血液中HbA_2与红细胞中“HbA_2”的差异。这里主要指的是电泳行为不同。3.将正常成人红细胞与其本身的溶血液并排电泳(薄层淀粉胶电泳)时,二者的HbA、碳酸酐酶电泳位置相同,只有HbA_2不一样。与溶血液中HbA_2相比,红细胞中的“HbA_2”电泳速度(向阳极)稍快。4.红细胞及溶血液中的HbF、HbN包头、HbD包头及HbE,都没有上述差异,而且它们对“HbA_2现象”没有影响。5.“HbA_2现象”的机制尚待阐明,但已经发现了它的应用。众所周知,HbE与A_2的电泳行为及层析性质是相同或相近的,鉴别起来比较困难,现在我们可以利用“HbA_2现象”来区分这两种血红蛋白。如上所述,HbA_2有“HbA_2现象”,而HbE本身则没有这类现象。     

6种丁香花挥发性成分的动态顶空吸附ATD-GC/MS分析

丁香是一种重要的芳香木本观赏植物,花开繁茂,芳香浓郁。该研究采用活体植物动态顶空套袋-吸附法收集了香雪丁香、花叶丁香、关东丁香、蓝丁香、小叶丁香、四季蓝丁香6种不同丁香花的挥发物,通过自动热脱附-气质联用分析技术分析丁香花的挥发物成分,为研究其花香释放机理、进一步开发利用丁香资源及新品种选育提供依据。结果表明：（1）在6种丁香花挥发物中共检测到80种成分,分别属于苯形烃、萜类、醇、醛、酮、脂肪烃、酯、酸及其它类。（2）花叶丁香花的挥发物释放量最高,且为释放量最低的四季蓝丁香的1.8倍。（3）除香雪丁香外其他5种丁香花的挥发物中苯形烃在9类挥发物成分中含量最多,而萜类是香雪丁香花挥发物的主要成分。（4）乙苯、间二甲苯、对二甲苯、二丁基羟基甲苯、2-乙基-1-己醇、5-乙基-2,2,3-三甲基庚烷这6种成分在不同丁香花挥发物成分中含量均较高,是6种丁香花挥发物的主要成分。     

血红蛋白A2现象发生机制的研究——“红细胞HbA2”为HbA2与HbA的结合产物

为了弄清血红蛋白A₂现象的发生机制,我们对“红细胞HbA₂”的化学组成进行了分析。“红细胞HbA₂”的双向电泳结果表明,它含有两种血红蛋白成分:一种相当于HbA,另一种很可能是溶血液HbA₂。其单向二次电泳结果也证明,它是由溶血液HbA₂和HbA所组成。结果初步说明,盘红细胞中HbA₂可能与HbA结合存在。两者可能有相互作用,也许这是产生血红蛋白A₂现象的原因。     

姜老师信箱

蛋白质研讨班学员问:姜老师,您好!非常感谢您上次的建议。我们用您说的方法重新尝试了2种不同的胞内表达蛋白,电泳后发现渗透冲击法能有效的把蛋白释放到上清中。虽然目前效率还没有达到100%(目测可能有50%-60%左右的目的蛋白释放到上清中),但相对之前始终没有蛋白的情况已经好了很多。现在我还有2个细节问题想请教一下:1.这种方法的效率能达到90%以上么?如果可以,我们实验的效率能否通过增加低渗液(水)的体积来进一步提高?     

完整人红细胞“发芽”释放无骨骼蛋白囊泡的观察

用Ca~2 -EDTA滴定法、加热法和室温静置法都能使人红细胞发芽并释放出囊泡,但发芽进程各不相同,囊泡大小也有差异。用微分干涉差显微镜和扫描电镜观察发现,发芽有时经芽胚红细胞这一中间阶段,但它决不是大多??数有关文献所提的棘细胞。又发现刚释放出来的囊泡具有“果蒂”样母体膜残留物。还发现发芽过程中常伴有细胞融合现象。据此认为芽胚红细胞极不稳定,它或者通过释放囊泡,或者通过细胞融合来达到稳定态。分析再次证明,发芽释放的囊泡缺失骨骼网络的主要成分,即收缩蛋白和肌动蛋白。     

在包头地区汉族中检出的一种快泳血红蛋白——血红蛋白包头(Hb Paotow)

1.在包头市约1,000名汉族中,发现一例快泳异常血红蛋白。2.家族调查结果表明,这种血红蛋白为遗传变异产物。3.分子杂交结果证明其为β链异常。4.这种血红蛋白在碱性pH下的电泳速度,是位于HbA与HbH之间。5.进行酸性琼脂电泳时,它泳向阴极,速度近似HbF。6.这种血红蛋白的紫外区吸收光谱,与HbA及HbF都不一样。7.鉴于它与已知血红蛋白都不相同,故命名为血红蛋白包头(Hb Paotow)以示区别,     

在包头地区汉族中检出的一种快泳血红蛋白——血红蛋白包头(Hb Paotow)

1.在包头市约1,000名汉族中,发现一例快泳异常血红蛋白。2.家族调查结果表明,这种血红蛋白为遗传变异产物。3.分子杂交结果证明其为p链异常。4.这种血红蛋白在碱性pH下的电泳速度,是位于HbA与HbH之间。5.进行酸性琼脂电泳时,它泳向阴极,速度近似HbF。6.这种血红蛋白的紫外区吸收光谱,与HbA及HbF都不一样。7.鉴于它与已知血红蛋白都不相同,故命名为血红蛋白包头(Hb Paotow)以示区别。     

Targeted O2 delivery by low-P50 hemoglobin: a new basis for O2 therapeutics

To assess O2 delivery to tissue by a new surface-modified, polyethylene glycol-conjugated human hemoglobin [MP4; Po2 at 50% saturation of hemoglobin (P50); 5.4 mmHg], we studied microcirculatory hemodynamics and O2 release in golden Syrian hamsters hemodiluted with MP4 or polymerized bovine hemoglobin (PolyBvHb; P50 54.2 mmHg). Comparisons were made with the animals' hemodiluted blood with a non-O2 carrying plasma expander with similar solution properties (Dextran-70). Systemic hemodynamics (arterial blood pressure and heart rate) and acid-base parameters were not correlated with microhemodynamics (arteriolar and venular diameter, red blood cell velocity, and flow). Microscopic measurements of Po2 and the O2 equilibrium curves permitted analysis of O2 release in precapillary and capillary vessels by red blood cells and plasma hemoglobin separately. No significant differences between the groups of animals with respect to arteriolar diameter, flow, or flow velocity were observed, but the functional capillary density was significantly higher in the MP4-treated animals (67%) compared with PolyBvHb-treated animals (37%; P < 0.05) or dextran-treated animals (53%). In the PolyBvHb-treated animals, predominant O2 release (both red blood cells and plasma hemoglobin) occurred in precapillary vessels, whereas in MP4 animals most of the O2 was released from both red blood cells and plasma hemoglobin in capillaries. Base excess correlated directly with capillary O2 release but not systemic O2 content or total O2 release. Higher O2 extraction of both red blood cell and plasma hemoglobin in capillaries represents a new mechanism of action of cell-free hemoglobin. High O2 affinity appears to be an important property for cell-free hemoglobin solutions.     

Hemolysis and ATP Release from Human and Rat Erythrocytes under Conditions of Hypoxia: A Comparative Study

Red blood cells are involved not only in transportation of oxygen and carbon dioxide but also in autoregulation of vascular tone by ATP release in hypoxic conditions. Molecular mechanisms of the ATP release from red blood cells in response to a decrease in partial oxygen pressure still remain to be elucidated. In this work we have studied effects of hypoxia on red blood cell hemolysis in humans and rats and compared the effects of inhibitors of ecto-ATPase and pannexin on the release of ATP and hemoglobin from rat erythrocytes. The 20-min hypoxia at 37°C increased hemolysis of red blood cells in humans and rats 1.5- and 2.5-fold, respectively. In rat erythrocytes a significant increase in hypoxia-induced extracellular ATP level was found only in the presence of ecto-ATPase inhibitor ARL 67156. In these conditions we observed a positive correlation (R² = 0.5003) between the increase in free hemoglobin concentration and the ATP release. Neither carbenoxolon nor probenecid, the inhibitors of low-selectivity pannexin channels, altered the hypoxia-induced ATP release from rat erythrocytes. The obtained results indicate a key role of hemolysis in the ATP release from red blood cells.     

A quantitative framework for the design of acellular hemoglobins as blood substitutes: implications of dynamic flow conditions

The delivery of oxygen to tissue by cell-free carriers eliminates intraluminal barriers associated with red blood cells. This is important in arterioles, since arteriolar tone controls capillary perfusion. We describe a mathematical model for O(2) transport by hemoglobin solutions and red blood cells flowing through arteriolar-sized tubes to optimize values of p50, Hill number, hemoglobin molecular diffusivity and concentration. Oxygen release is evaluated by including an extra-luminal resistance term to reflect tissue oxygen consumption. For low consumption (i.e., high resistance to O(2) release) a hemoglobin solution with p50=15 mmHg, n=1, D(HBO2)=3 x 10(-7) cm(2)/s delivers O(2) at a rate similar to that of red blood cells. For high consumption, the p50 must be decreased to 5 mmHg. The model predicts that regardless of size, hemoglobin solutions with higher p50 will present excess O(2) to arteriolar walls. Oversupply of O(2) to arteriolar walls may cause constriction and paradoxically reduced capillary perfusion.     

The induction of ferritin synthesis in circulating larval red blood cells.

The circulating red blood cells formed in bullfrog larvae, chicken embryos, and mouse embryos contain large amounts of ferritin and storage iron in excess of the need for hemoglobin. In contrast, the circulating red cells of adult animals contain little ferritin. Ferritin synthesis and iron storage are coordinated with differentiation and hemoglobin synthesis in the red cells of adults. In order to test the hypothesis that ferritin synthesis could be controlled independently of hemoglobin synthesis and differentiation in the red cells formed early in life, bullfrog larvae were injected with iron to determine if ferritin synthesis was increased in the circulating red cells. Within 17 h after the injection of iron, the synthesis of ferritin, assayed as the incorporation of [14C]leucine by cell suspensions prepared from circulating red cells, was increased from 2.9 to 10.2% of the total protein, and the specific activity of the ferritin synthesized increased from 1100 to 3000 cpm/A280. There was no change in the hematocrit of the animals nor in the specific activity of hemoglobin synthesized by suspensions of red cells (average, 720 cpm/A280). The results suggest that in mature, larval red cells, ferritin synthesis can be controlled by changes in the extracellular environment. The results also indicate that ferritin synthesis can be controlled independently of hemoglobin synthesis with which it is coordinated during erythroid differentiation in adult animals.     

Utilization of a differential hemoglobin elution procedure as a rapid assay for identification of embryonic and adult chicken red blood cells.

1. A hemoglobin elution-staining procedure has been developed for distinguishing embryonic chick red blood cells from adult chicken red blood cells. 2. Adult hemoglobin is eluted from red blood cells with 1.9 M potassium phosphate buffer, pH 7.2; whereas, embryonic hemoglobin is retained within the cells and gives positive staining with erythrosin B. 3. The hemoglobin elution-staining pattern during development can be correlated with two embryonic hemoglobins as detected by polyacrylamide gel electrophoresis. 4. The series of red blood cells staining with erythrosin B correspond to the primary erythrocyte series suggesting that hemoglobin expression during development is correlated with different cell populations.     

Heme redox properties of S-nitrosated hemoglobin A0 and hemoglobin S: implications for interactions of nitric oxide with normal and sickle red blood cells

S-Nitrosated hemoglobin is remarkably stable and can be cycled between deoxy, oxygenated, or oxidized forms without significant loss of NO. Here we show that S-nitrosation of adult human hemoglobin (Hb A(0)) or sickle cell Hb (Hb S) results in an increased ease of anaerobic heme oxidation, while anions cause redox shifts in the opposite direction. The negatively charged groups of the cytoplasmic domain of Band 3 protein also produce an allosteric effect on S-nitrosated Hb. Formation and deoxygenation of a SNO-Hb/Band 3 protein assembly does not in itself cause NO release, even in the presence of glutathione; however, this assembly may play a role in the migration of NO from the red blood cells to other targets and may be linked to Heinz body formation. Studies of the anaerobic oxidation of Hb S revealed an altered redox potential relative to Hb A(0) that favors met-Hb formation and may therefore underlie the increased rate of autoxidation of Hb S under aerobic conditions, the increased formation of Heinz bodies in sickle cells, and the decreased lifetime of red cells containing Hb S. A model for the interrelationships between the deoxy, oxy, and met forms of Hb A(0) and Hb S, and their S-nitrosated counterparts, is presented.     

Iodination-Coupled Tetrazonium and Ferric Ferricya-Nide Reduction Stains for Differentiating Red Blood Cells Containing Fetal or Adult Hemoglobin

Either the iodination-coupled tetrazonium reaction or the ferric ferricyanide reduction procedure can be used to differentiate red blood cells containing fetal hemoglobin (hemoglobin F) from those containing adult hemoglobin (hemoglobin A) in blood smears. Oxalated blood is diluted with 3 parts of physiological saline, and smears are made on slides. The air-dried slides are treated with absolute ethanol for 2 min, dried, and placed in phosphate-citrate buffer of pH 3.2-3.6 for 1 min at 37°C. They are then rinsed in distilled water, and dried for storage or stained at once by either the iodination-coupled tetrazonium or the ferric ferricyanide reduction procedure. Adult hemoglobin is extracted by the buffer, so that red blood cells containing fetal hemoglobin give a much darker stain than those containing adult hemoglobin. The hemoglobin S of patients with sickle-cell anemia behaves like adult hemoglobin.     

The accumulation of basic nonhemoglobin proteins during the differentiation of primitive (embryonic) red cells of amphibia

The preferential accumulation of hemoglobin is a characteristic of the differentiation of definitive (adult) red blood cells. Since primitive (embryonic or larval) red blood cells have many properties which contrast with definitive red cells, the accumulation of red cell proteins was analyzed during the differentiation of primtive red cells to determine whether or not hemoglobin was the only protein which showed a substantial increase in amount. Primitive red cells of amphibia were used because the mature circulating cell retains large amounts (13%) of specific, well characterized, nonhemoglobin proteins (CP). Preparations of primitive red cells enriched in immature cells were obtained from the circulation of bullfrog tadpoles recovering from phenylhydrazine-induced anemia. The amount of CP, determined by electrophoresis, imunodiffusion, and ion-exchange chromatography, was compared for red cells from normal animals and anemic animals. Mature cells contained three to six times the amount of CP and three times the amount of hemoglobin found in the population of red cells enriched in immature cells. The accumulation of CP during maturation of primitive red cells indicates that differentiation of primitive red cells is less restrictive than differentiation of definitive red cells. Since the primitive red cell is less specialized than the definitive red cell, it is possible that primitive red cells have several roles in the developing animal, in contrast to the single role of synthesizing and maintaining hemoglobin in the adult animal.     

Inhibition of the heme-induced hemolysis of red blood cells by the chlorite-based drug WF10

Excessive release of hemoglobin from red blood cells markedly disturbs the health status of patients due to cytotoxic effects of free hemoglobin and heme. The latter component is able to initiate novel hemolytic events in unperturbed red blood cells. We modeled this process by incubation of ferric protoporphyrin IX with freshly isolated red blood cells from healthy volunteers. The heme-induced hemolysis was inhibited in a concentration-dependent manner by the chlorite-based drug WF10, whereby the hemolysis degree was totally abolished at a molar ratio of 1:2 between chlorite and heme. Upon incubation of heme with WF10, the ultraviolet-visible spectrum changed, whereas the release of iron from heme and the appearance of fluorescent breakdown products of the porphyrin ring were negligible at this ratio, but increased with increasing excess of chlorite over heme. Thus, inhibition of hemolysis by WF10 takes already place at those chlorite concentrations, where no degradation of the porphyrin ring occurs. As WF10 is applied in form of an intravenous infusion to patients with severe inflammatory states, these data support the hypothesis that the beneficial WF10 effects are closely associated with inactivation of free heme.     

The binding of hemoglobin to membranes of normal and sickle erythrocytes.

The binding of hemoglobins A, S, and A2 to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with 14C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A2, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4-22 degrees C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied. An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites. These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.