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。     

糖化血红蛋白在糖尿病中的应用价值分析

目的:研究糖化血红蛋白的检测在糖尿病患者中的诊断价值及对代谢指标的影响。方法:对126例临床确诊为糖尿痛患者进行糖化血红蛋白及空腹血糖检测,并且将糖化血红蛋白分为A组(HbA1c≤10%)和B组(HbA1c>10%),比较两组TG,TC,LDL.HD等指标。结果:FPG在B组患者的水平明显高于A组患者的水平,差异有显著的统计学意义(P<0.01),而2hPG,TC,LDL B组患者的水平明显高于A组患者的水平,差异有明显的统计学意义(P<0.05)。结论:检测糖化血红蛋白在糖尿病患者具有重要的临床价值。     

连续自由流电泳样机的应用

用连续自由流电泳(continuous flow electrophoresis,CFE)成功地将细胞色素c(Cyt c)和牛血红蛋白(Hb)两种模式蛋白分开,电泳分离后的样品经过紫外分光光度计和聚丙烯酰胺凝胶电泳检测.样品的分离结果与众多性能参数有关,如:分离室间隙,样品流速,缓冲液流量、pH、电导率及分离功耗等.     

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。     

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。     

南昌地区汉族新生儿胎儿血红蛋白中γ与γ比值的测定

用改良的含Triton X-100和尿素的酸性聚丙烯酸胺凝胶电泳法测定了503例南昌地区汉族新生儿胎儿血红蛋白中^Gγ和^Aγ二比值。结果低GY组（34-46%）占1%,高^Gγ组（83-91%）占1.2%,其余为正常组（59-79%)。此结果提示,在南昌地区汉族新生儿中,异常γ基因的频率可能低于其它东亚黄种人     

活体红细胞内血红蛋白的电泳释放

当今的蛋白质组学研究,都是先裂解细胞放出蛋白质,然后对蛋白质溶液进行各种分析.对于红细胞来说,它的裂解产物也称＂溶血液＂,其中主要成分有血红蛋白A1,A2,A3和碳酸酐酶（CA）等.本实验室用未裂解的完整的活体红细胞直接进行电泳,观察其释放出来的血红蛋白（hemoglobin,Hb）,建立了淀粉-琼脂糖混合凝胶中红细胞的电泳释放实验.电泳释放可分为＂初释放＂（一次通电完成电泳,此时有Hb释放出来）和＂再释放＂（电泳过程中断电-再通电,又有Hb释放出来）.本实验室在＂初释放＂实验中发现了＂HbA2现象＂,并通过Hb交叉电泳发现了HbA2与HbA1的相互作用;利用初释放型双向对角线电泳发现红细胞内HbA2与HbA1结合存在;对电泳释放出来的＂HbA2现象＂成分做SDS-PAGE及质谱分析,发现Prx-2（Peroxiredoxin-2）可能参与＂HbA2现象＂的形成;在研究＂再释放＂实验中发现了＂Hb多带再释放现象＂,在此基础上创建等渗再释放、低渗再释放、等低渗全程再释放及再释放型双向对角线电泳;两种红细胞（全血中的红细胞和由它分离出来的游离红细胞）再释放的比较研究;血浆成分对红细胞再释放的影响等.以上研究方法的建立为活体细胞内蛋白质存在状态的研究提供了基础,并开辟了新的研究途径和领域.     

血红蛋白200g/L以上紫绀型先天性心脏病患者手术效果分析

目的:明确血红蛋白200 g/L以上紫绀型先天性心脏病患者的手术效果。方法:选取2009年3月至2012年3月于我中心就诊手术治疗的紫绀型先天性心脏病患者,按血红蛋白计数≥200 g/L和200 g/L分为A组和B组,完善术前检查后进行手术治疗。记录患者手术效果和随访情况;观察比较两组患者手术中情况包括:手术方式、手术时间、体外循环时间、心脏停搏时间、心脏自动复跳情况;记录并比较两组患者手术后恢复情况,包括机械通气时间、监护室滞留时间、手术后24小时内出血量、二次开胸止血例数和血管活性药物评分,以及监护室内肝肾功能异常和肺部并发症发生例数。结果:A组死亡3例(5.2%),23例术后3个月随访效果良好;B组死亡2例(5.8%),12例术后3个月随访效果良好。两组患者手术方式、手术时间、体外循环时间和心脏停搏时间、自动复跳例数均无明显差异(P0.05)。与B组比较,A组患者术后机械通气和监护室滞留时间长,术后24小时出血量多,血管活性药物使用评分高,肝肾功能异常例数和肺部并发症发生例数较多有统计学意义(P0.05),两组间二次开胸止血例数无统计学差异(P0.05)。结论:血红蛋白200 g/L以上紫绀型先天性心脏病患者与其他紫绀型先天性心脏病患者手术效果相似,但手术后恢复慢,并发症较多。     

血红蛋白种间分子杂交以及树鼩进化地位的探讨

为了探讨树鼩(Tupaia belangeri chinensis)的分类地位及其与灵长类动物的亲缘关系,本文利用血红蛋白种间分子杂交技术,以人类血红蛋白HbA为标准,分析比较了树鼩血红蛋白与原始灵长类动物懒猴(Nycticebus coucang)血红蛋白的结构异同。在碱性条件下,三种血红蛋白的电泳迁移率大小为:人>懒猴>树鼩。树鼩Hb(α_2~(Tup)和β_2~(Tup))和HbA(α_2~Aβ_2~A)杂交可以产生两种杂种分子 (α_2~(Tup)β_2~A和α_2~Aβ_2~(Tup))。HbA和懒猴Hb (α_2~(Nyc)β_2~(Nyc)) 杂交也可产生两种杂种分子(α_2~(Nyc)β_2~A和α_2~Aβ_2~(Nyc))。结果表明,树鼩Hb与懒猴Hb的α链净电荷相似。认为树鼩与懒猴的分类地位相近。     

Bioelectrocatalytic detection of glycated hemoglobin (HbA1c) based on the competitive binding of target and signaling glycoproteins to a boronate-modified surface

We developed an electrochemical glycated hemoglobin (HbA(1c)) biosensor for diagnosing diabetes in whole human blood based on the competitive binding reaction of glycated proteins. Until now, no studies have reported a simple and accurate electrochemical biosensor for the quantification of HbA(1c) in whole blood. This is because it is very difficult to correctly distinguish HbA(1c) from large amounts of hemoglobin and other components in whole blood. To detect glycated hemoglobin, we used electrodes modified with boronic acid, which forms a covalent bond between its diol group and the cis-diol group of the carbohydrate moiety of glycated proteins. For accurate HbA(1c) biosensing, we first removed blood components (except for hemoglobin) such as glycated proteins and blood glucose as they interfere with the boronate-based HbA(1c) competition analysis by reacting with the boronate-modified surface via a cis-diol interaction. After hemoglobin separation, target HbA(1c) and GOx at a predetermined concentration were reacted through a competition onto the boronate-modified electrode, allowing HbA(1c) to be detected linearly within a range of 4.5-15% of the separated hemoglobin sample (HbA(1c)/total hemoglobin). This range covers the required clinical reference range of diabetes mellitus. Hence, the proposed method can be used for measuring %HbA(1c) in whole human blood, and can also be applied to measuring the concentration of various glycated proteins that contain peripheral sugar groups.     

Identification of inherited protein variants in individual erythrocytes

Inherited electrophoretic variants of hemoglobin, carbonic anhydrase, and glucose-6-phosphate dehydrogenase in individual erythrocytes were separated by electrophoresis in ultrathin agar gels. By staining the electropherograms with specific fluorescein-conjugated antibodies against hemoglobins, relative proportions of two hemoglobins within individual erythrocytes can be estimated. The findings suggested that the intracellular proportions of HbA and HbS in heterozygotes are heterogeneous within a given population of cells. By this method cells containing hemoglobin F (F cells) as well as a minor variant of hemoglobin F were identified. This tool potentially offers an approach to monitoring distribution of inherited variants in individual erythrocytes for a large number of proteins.This work was supported by a grant from the John A. Hartford Foundation and from the National Institutes of Health, Grant No. HL 15160.     

Structural basis of peroxide-mediated changes in human hemoglobin: a novel oxidative pathway

Hydrogen peroxide (H(2)O(2)) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H(2)O(2) to highly purified human hemoglobin (HbA(0)) induced structural changes that primarily resided within beta subunits followed by the internalization of the heme moiety within alpha subunits. These modifications were observed when an equal molar concentration of H(2)O(2) was added to HbA(0) yet became more abundant with greater concentrations of H(2)O(2). Mass spectrometric and amino acid analysis revealed for the first time that betaCys-93 and betaCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA(0) was treated with H(2)O(2). Oxidation of further amino acids in HbA(0) exclusive to the beta-globin chain included modification of betaTrp-15 to oxyindolyl and kynureninyl products as well as betaMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the beta subunits as a result of the H(2)O(2) attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two alpha-peptide fragments (alpha128-alpha139) and a heme moiety with the loss of iron, cross-linked between alphaSer-138 and the porphyrin ring. The novel oxidative pathway of HbA(0) modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.     

A new method for the determination of alpha1-protease inhibitor (alpha1-antitrypsin) phenotypes based on the formation of alpha1-protease inhibitor allele product-elastase complexes.

Up until now it has been assumed that the protease-binding property of alpha1-protease inhibitor (alpha1PI) was destroyed by acid starch gel electrophoresis (pH 4.9). Analyses on acid starch gel blocks for pH and conductivity changes during and following a typical electrophoretic run showed that it was unlikely that the separating alpha1PI would be exposed to pH values lower than 6.2, and that the allele products, following the passage of the buffer front, were in an environment of constant pH(6.3), extremely low conductivity and high field strength. These results strongly suggested the likelihood that alpha1-PI would be chemically and physically unchanged as a result of exposure to acid starch gel electrophoresis. In order to test this likelihood, human serum was electrophoretically separated in acid starch gel and following electrophoresis, was immersed in 0.1 M diethylbarbiturate buffer, pH 8.6, containing 20 mug/ml of pancreatic elastase. The pH-adjusted (8.15) and elastase-impregnated starch gel layer was superimposed on hemoglobin-agar for 2.5 h at 37 degrees C followed by immersion of the hemoglobin-agar layer in 1% NaCl overnight, distilled water for 2 h, drying under filter paper and staining. The results showed zones of undigested hemoglobin indicating, unequivocally, that the separated alpha1PI allele products are capable of forming complexes with proteases and that alpha1PI is not inactivated following exposure to acid starch gel electrophoresis. Densitometric analysis of the transparent stained zones on a clear agar gel background offers an alternative to analysis of the acid starch gel-separated zones by antigen-antibody crossed electrophoresis and as such is suitable for identification of alpha1-protease inhibitor phenotypes. Further, the method is specific for alpha1PI and a densitometric scan provides direct information relative to the protease-binding capacity of the sample as well as the contribution of each alpha1PI allele product to that capacity.     

基于气相色谱法分析T2DM患者粪便中SCFAs水平与HbA1c的相关性

目的 研究2型糖尿病患者粪便中6种短链脂肪酸水平与糖化血红蛋白的相关性。 方法 采用气相色谱法检测粪便中短链脂肪酸的含量,并对方法学进行考察。选取2018年在本院查体的2型糖尿病患者57例,根据糖化血红蛋白水平将其分为高糖化组(糖化血红蛋白>7.0,28例)与低糖化组(糖化血红蛋白≤7.0,29例)。应用气相色谱法检测两组患者粪便中6种短链脂肪酸水平,并分析6种短链脂肪酸水平与糖化血红蛋白的相关性。 结果 低糖化组患者粪便中乙酸、丙酸、丁酸、戊酸水平显著高于高糖化组(均P0.05)。Pearson相关性分析结果表明:在2型糖尿病患者中,糖化血红蛋白与粪便中乙酸呈负相关(r=-0.540 1,P结论 2型糖尿病患者粪便中短链脂肪酸水平与糖化血红蛋白存在一定的相关性,短链脂肪酸水平的下降可能是影响血糖控制不佳的主要原因。     

Fructose-induced structural and functional modifications of hemoglobin: implication for oxidative stress in diabetes mellitus

Increased fructose concentration in diabetes mellitus causes fructation of several proteins. Here we have studied fructose-induced modifications of hemoglobin. We have demonstrated structural changes in fructose-modified hemoglobin (Fr-Hb) by enhanced fluorescence emission with excitation at 285 nm, more surface accessible tryptophan residues by using acrylamide, changes in secondary and tertiary structures by CD spectroscopy, and increased thermolability by using differential scanning calorimetry in comparison with those of normal hemoglobin, HbA(0). Release of iron from hemoglobin is directly related with the extent of fructation. H2O2-induced iron release from Fr-Hb is significantly higher than that from HbA(0). In the presence of H2O2, Fr-Hb degrades arachidonic acid, deoxyribose and plasmid DNA more efficiently than HbA(0), and these processes are significantly inhibited by desferrioxamine or mannitol. Thus increased iron release from Fr-Hb may cause enhanced formation of free radicals and oxidative stress in diabetes. Compared to HbA(0), Fr-Hb exhibits increased carbonyl formation, an index of oxidative modification. Functional modification in Fr-Hb has also been demonstrated by its decreased peroxidase activity and increased esterase activity in comparison with respective HbA(0) activities. Molecular modeling study reveals Lys 7alpha, Lys 127alpha and Lys 66beta to be the probable potential targets for fructation in HbA(0).     

Preparation and chemical characterization of the three chains of the major hemoglobin of the sea snake, Pelamis platurus.

One of the 2 main hemoglobins of the sea snake Pelamis platurus, (the Yellow-bellied sea snake) comprising about 70% of the total hemoglobin, was separated by DEAE-Sephadex column chromatography. From results of gel filtration and iron content determination, both intact sea snake hemoglobin, and the isolated major hemoglobin, were concluded to be composed of 4 subunits with a molecular weight of 66,000-67,000 daltons. Separation of the chains of globin of the major hemoglobin by CM 52 column chromatography gave 3 peaks, named, chains a, b, and c. The approximate molecular weights of chains a, b, and c were deduced by SDS gel electrophoresis to be 14,000, 16,000, and 20,000 daltons, respectively. The peptide maps and amino acid compositions of the three chains were distinctly different. N-Terminal and C-terminal amino acid sequence studies reveal that chains a, b, and c represented the alpha-chain, beta-chain, and beta'-chain differing from the normal beta-chain in having a C-terminal sequence of -Arg-Leu-His-Tyr. From the peak areas of the 3 chains obtained by CM 52 column chromatography, and the peak sizes of the 3 bands separated by SDS gel electrophoresis, it was concluded that the sea snake hemoglobin is composed of a hybrid tetramer, alpha2betabeta'.     

Agar Gel Electrophoresis: an Advantageous Technique for the Investigation of Certain Biological Stains

The agar gel method can be used to study direct dyes, for which paper can not be used because such dyes have a high affinity for the paper. A 1% gel made up with a buffer in the range of pH 9-4, of ionic strength 0.05, and spread on 8 × 10 cm lantern slides provides suitable conditions. Dyes to be tested are placed in 1.5 mm wells made in the agar and subjected to a current of 2.5 ma/cm width, at a potential of about 115 v. Separations, if any, occur in about 20 min. Mobility is affected by ionic strength; values above 0.05 may be less satisfactory by reducing mobility and allowing excessive diffusion of the dye. The method allows resolution of dyes whose molecular charges differ by only one unit. Photographic recording is sharp, since the gel is transparent. The method can be recommended as generally useful for studying both acid and direct dyes.     

Novel approach for the analysis of glycated hemoglobin using capillary isoelectric focusing with chemical mobilization

In this work, a novel CIEF methodology for the analysis of the glycated hemoglobin, HbA(1c), in dimethylpolysiloxane coated fused-silica capillaries (DB-1, 50 microm I.D., 27 cm, 0.20 microm coating thickness), using a narrow pH ampholyte mixture (4% pH 6-8:pH 3-10, 10:1, v/v) in 0.30% methylcellulose, was developed. In the focusing procedure, a 0.100-mol l(-1) phosphoric acid solution was used as anolyte and a 0.040-mol l(-1) NaOH solution was used as catholyte. During method development, two types of mobilization of the focused hemoglobins were tested: pressure and chemical mobilization. Chemical mobilization performed better, allowing the complete baseline resolution of the hemoglobin of interest, HbA(1c), from its adjacent peak, HbA, in less than 8 min. In the chemical mobilization procedure, the catholyte was replaced by a 0.040-mol l(-1) NaOH solution containing 0.080 mol l(-1) NaCl. The proposed methodology was applied to the analysis of 31 hemolysate samples and validated with respect to the selectivity, inter-assay and intra-assay precision (both migration time and hemoglobin percentage concentration). In addition, HbA(1c) determinations were compared for the CIEF method and a chromatographic standardized procedure using cation-exchanger columns (Variant, Bio-Rad), adopted in a local clinical laboratory, showing excellent correlation (r(2)=0.872, n=31). The slope was found to be statistically equal to one but the intercept differed from zero. Also the Bland-Altman plot indicates bias, implying that the CIEF method yields HbA(1c) concentration higher than the reference method. The separation of the hemoglobins HbA, HbA(2), HbF and HbA(1c) and the variants HbS and HbC was also demonstrated (8 min run). The resolving power of the proposed CIEF method allowed baseline resolution of hemoglobins with a pI difference as small as ca. 0.03, as it is the case for the pairs HbC/HbA(2) and HbA/HbA(1c).     

Studies on subfractions of hemoglobin A1b and hemoglobin A1c in diabetic subjects

Hemoglobin (Hb) obtained from the hemolysate of normal subjects and diabetic patients was separated into HbA1a1, HbA1a2, HbA1b, HbA1c and HbA0 (major Hb) by Bio-Rex 70 cation exchange column chromatography. The glycosylated Hbs were further separated reproductively by cation exchange high performance liquid chromatography (HPLC), using 50 mM sodium phosphate buffer pH 5.80 with 0-0.2 M NaCl linear gradient system. HbA1b and HbA1c were separated into two subfractions (HbA1b1 and HbA1b2) and three subfractions (HbA1c1, HbA1c2, HbA1c3), respectively. The percentages of each subfraction except HbA1c1 in diabetic patients were significantly higher than those in normal subjects. Furthermore, HbA1c1, HbA1c2 and HbA1c3 correlated well with fasting blood glucose levels in the prior 5 month period, while subfractions in HbA1b revealed no significant correlation with blood glucose levels. The percentages of each subfraction of HbA1c in patients either with diabetic cataracts or with diabetic neuropathy were almost the same as those in the patients without complications. However, the percentages of each of the three groups were markedly higher than those of the normal subjects. These results suggest that glycosylation of hemoglobin in diabetic patients may be increased in various sites of the molecule in parallel with the blood glucose levels during the preceding 4-5 months.