STRUCTURE, PROPERTIES, AND FUNCTION OF UNSTABLE ANIMAL HEMOGLOBINS

不稳定动物血红蛋白的结构、性质和功能

• 批准号: 3937197
• 负责人: THOMAS A BORGESE
• 金额: --
• 依托单位: HERBERT H. LEHMAN COLLEGE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3937197
• 关键词: chemical aggregate; chemical association; chemical structure function; electron microscopy; electrophoresis; gel filtration chromatography; hemoglobin; ion exchange chromatography; oxidation reduction reaction

The purpose of this research is to investigate the mechanism(s) of hemoglobin association/dissociation. Its regulation in vivo and in vitro and its effect on hemoglobin function. The mollusc, Anadara ovalis, has erythrocytes which contain dimeric (HbMi) and tetrameric (HbMa) hemoglobins both of which dissociate to monomers upon oxidation with K3Fe(CN)6 (Borgese et al., 1987). In contrast, the marine teleost, Lophius amercanus (goosefish), has a single tetrameric hemoglobin which forms aggregates ranging from 160.000 to greater than 900,000 daltons when similarly oxidized (Borgese, 1986). While many species of elasmobranchs have multiple hemoglobins, some of which polymerize by disulfide bond formation, there have been no reports of hemoglobin aggregation among marine teleosts. Our specific aims are: 1) to determine the effects of disulfide reducing agents on the inhibition and reversal of the Hb aggregation (goosefish, ocean pout) and monomerization (Anadara ovalis). Hemoglobins will be isolated by DE-52 chromatography and molecular weights determined by gel filtration. 2) We will determine whether aggregation can occur in intacts cells by incubating cells with permeable oxidants such as NaN02 followed by electron microscopy of the packed cells. 3) The complete red cell organic phosphate profile will be determined by Ag 1x8 anion exchange chromatography; the effect of the major organic phosphates are hemoglobin function will be determined using the Hem-O-Scan oxygen dissociation analyzer. 4) We will determine whether cells with metabolically stimulated reductase activity have a greater capacity to inhibit aggregation. 5) We will determine the role of SH groups on thermal stability of goosefish and Anadara ovalis HbMa. 6) Urea gel electrophoresis and HPLC will be used to determine whether goosefish hemoglobin is a homo or heterotetramer. Amino acid composition of the isolated chains and the sequencing of these unusual hemoglobins (16 SH/tetramer-goosefish and monomer formation of Anadara HBs) are further steps in explaining struc- ture-function-property relationships. In addition to providing an explanation for apparent Hb polymorphism, alterations in the normal quaternary hemoglobin structure by polymerization or dissociation may have serious consequences if cell morphology and/or oxygen affinity are adversely affected.

本研究的目的是探讨 血红蛋白结合/解离。 其在体内的调节和 以及对血红蛋白功能影响。 软体动物， 卵圆阿纳达拉（Anadara ovalis），具有含有二聚体（HbMi）的红细胞， 四聚体（HbMa）血红蛋白，两者均解离成单体 在用K3 Fe（CN）6氧化时（Borgese等人，1987年）。 与此相反， 海洋硬骨鱼，Lophius amercanus（鹅鱼），有一个单一的 四聚体血红蛋白，其形成范围为160.000 当类似地氧化时大于900，000道尔顿（Borgese， 1986年）。 虽然许多种类的板鳃类有多个 血红蛋白，其中一些通过二硫键的形成而结合， 目前还没有关于海洋生物中血红蛋白聚集的报道。 硬骨鱼 我们的具体目标是：1）确定二硫化物的影响 还原剂对Hb的抑制和逆转 聚合（鹅，海洋pout）和单体化（Anadara 椭圆形）。 血红蛋白将通过DE-52色谱法分离， 通过凝胶过滤测定分子量。 2)我们将 通过以下方式确定聚集是否可以在intact细胞中发生： 用可渗透的氧化剂如NaNO 2孵育细胞， 电子显微镜下的压积细胞。 3)完整红细胞 将通过Ag 1x 8阴离子测定有机磷酸盐分布 交换层析;主要有机磷酸盐的影响 将使用Hem-O-Scan测定血红蛋白功能 氧离解分析仪 4)我们将确定细胞是否 具有代谢刺激的还原酶活性， 抑制聚集的能力。 5)我们将决定 SH基团对鹅和鹅肝鱼热稳定性的影响 HbMa。 6)尿素凝胶电泳和HPLC将用于 确定鹅血红蛋白是同源还是异源四聚体。 分离链的氨基酸组成和测序 这些不寻常的血红蛋白（16 SH/tetramer-goofish和单体 Anadara HB的形成）是解释结构的进一步步骤， ture-function-property关系。 除了提供对明显Hb的解释外， 多态性，正常四元血红蛋白的改变 通过聚合或解离形成结构可能具有严重的 如果细胞形态学和/或氧亲和力 受到不利影响。