Purification, Crystallization, and Preliminary X-Ray Diffraction Studies on Hemoglobin from the Angora Goat (Capra Aegagrus Hircus).

IF 1.1 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

Protein and Peptide Letters Pub Date : 2025-07-14 DOI:10.2174/0109298665370730250708065019

Farheen Pervez, Shobana Nagaraj, Mpho Setshedi, Arshad Mather, Ponnuswamy Mondikalipudur Nanjappa Gounder, Yasien Sayed, Ramesh Pandian

{"title":"Purification, Crystallization, and Preliminary X-Ray Diffraction Studies on Hemoglobin from the Angora Goat (Capra Aegagrus Hircus).","authors":"Farheen Pervez, Shobana Nagaraj, Mpho Setshedi, Arshad Mather, Ponnuswamy Mondikalipudur Nanjappa Gounder, Yasien Sayed, Ramesh Pandian","doi":"10.2174/0109298665370730250708065019","DOIUrl":null,"url":null,"abstract":"Introduction: Angora goats are a distinct breed that differs significantly from common goats and shares a similar appearance to sheep. In Angora goats, only the level of glutathione (GSH) is elevated during under-stimulated conditions, as well as after the period of hypoxic stress; however, no changes are found in 2,3-diphosphoglycerate (2,3-DPG) levels, which are commonly present in the red blood cells (RBCs) of most mammals. We chose the Angora goat for our investigation because no previous studies have been conducted on the structural and functional aspects of hemoglobin (Hb). In addition, no sequence or structural information is currently available in any database.Methods: Angora goat Hb was isolated and purified by anion-exchange chromatography, followed by crystallization using various methods. X-ray data collection for Angora goat Hb was performed under a liquid nitrogen cryo-stream using a Bruker D8 Venture Bio Photon III 28-pixel array area detector system.Results: Good diffracting crystals were obtained using the hanging-drop vapor-diffusion method with polyethylene glycol (PEG) 3350 as the precipitant in water, without the addition of any salt or buffer. The Angora goat Hb diffracted to a resolution of 1.85 Å, and the structure solution was obtained by the molecular replacement method, using the structure of domestic goat Hb as the starting model.Discussion: The solved structure of Angora goat crystallized in the monoclinic space group P21, consisting of one whole biological molecule in the asymmetric unit, with unit cell dimensions of a = 52.08 Å, b = 76.70 Å, c = 74.08 Å, and β = 91.77 °. The solvent content and Matthews coefficient (Vm) for the Angora goat Hb are 49.05% and 2.41 Å3/Da, respectively, and are within the normal range for protein crystals.Conclusion: Purification, crystallization, and preliminary X-ray diffraction studies of Angora goat Hb were performed successfully. Structural refinement and biophysical characterization of Angora goat Hb are in progress in the absence and presence of GSH and 2,3-DPG.","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":""},"PeriodicalIF":1.1000,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein and Peptide Letters","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.2174/0109298665370730250708065019","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

Introduction: Angora goats are a distinct breed that differs significantly from common goats and shares a similar appearance to sheep. In Angora goats, only the level of glutathione (GSH) is elevated during under-stimulated conditions, as well as after the period of hypoxic stress; however, no changes are found in 2,3-diphosphoglycerate (2,3-DPG) levels, which are commonly present in the red blood cells (RBCs) of most mammals. We chose the Angora goat for our investigation because no previous studies have been conducted on the structural and functional aspects of hemoglobin (Hb). In addition, no sequence or structural information is currently available in any database.

Methods: Angora goat Hb was isolated and purified by anion-exchange chromatography, followed by crystallization using various methods. X-ray data collection for Angora goat Hb was performed under a liquid nitrogen cryo-stream using a Bruker D8 Venture Bio Photon III 28-pixel array area detector system.

Results: Good diffracting crystals were obtained using the hanging-drop vapor-diffusion method with polyethylene glycol (PEG) 3350 as the precipitant in water, without the addition of any salt or buffer. The Angora goat Hb diffracted to a resolution of 1.85 Å, and the structure solution was obtained by the molecular replacement method, using the structure of domestic goat Hb as the starting model.

Discussion: The solved structure of Angora goat crystallized in the monoclinic space group P21, consisting of one whole biological molecule in the asymmetric unit, with unit cell dimensions of a = 52.08 Å, b = 76.70 Å, c = 74.08 Å, and β = 91.77 °. The solvent content and Matthews coefficient (Vm) for the Angora goat Hb are 49.05% and 2.41 Å3/Da, respectively, and are within the normal range for protein crystals.

Conclusion: Purification, crystallization, and preliminary X-ray diffraction studies of Angora goat Hb were performed successfully. Structural refinement and biophysical characterization of Angora goat Hb are in progress in the absence and presence of GSH and 2,3-DPG.

查看原文本刊更多论文

安哥拉山羊血红蛋白的纯化、结晶及x射线衍射初步研究。

安哥拉山羊是一个独特的品种，与普通山羊有很大不同，与绵羊有相似的外观。在安哥拉山羊中，只有谷胱甘肽（GSH）水平在低刺激条件下以及缺氧应激期后升高；然而，在大多数哺乳动物的红细胞（红细胞）中普遍存在的2,3-二磷酸甘油酸（2,3- dpg）水平没有发现变化。我们之所以选择安哥拉山羊作为我们的研究对象，是因为之前没有对血红蛋白（Hb）的结构和功能方面进行过研究。此外，目前在任何数据库中都没有序列或结构信息。方法：采用阴离子交换色谱法分离纯化安哥拉山羊血红蛋白，采用多种方法进行结晶。利用Bruker D8 Venture Bio Photon III 28像素阵列区域探测器系统，在液氮低温流下采集安哥拉山羊Hb的x射线数据。结果：以聚乙二醇（PEG） 3350为沉淀剂，在不添加任何盐或缓冲液的情况下，采用悬滴气相扩散法获得了良好的衍射晶体。安哥拉山羊Hb衍射分辨率为1.85 Å，以家山羊Hb结构为起始模型，采用分子置换法获得结构溶液。讨论：求解的安哥拉山羊结构结晶于单斜空间群P21中，由一个完整的生物分子在不对称单元中组成，单位细胞尺寸为a = 52.08 Å， b = 76.70 Å， c = 74.08 Å， β = 91.77°。安哥拉山羊Hb的溶剂含量和马修斯系数（Vm）分别为49.05%和2.41 Å3/Da，在蛋白质晶体的正常范围内。结论：成功地完成了安哥拉山羊血红蛋白的纯化、结晶和初步的x射线衍射研究。在GSH和2,3- dpg不存在和不存在的情况下，安哥拉山羊Hb的结构改进和生物物理特性正在进行中。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Protein and Peptide Letters 生物-生化与分子生物学

CiteScore

2.90

自引率

0.00%

发文量

审稿时长

2 months

期刊介绍： Protein & Peptide Letters publishes letters, original research papers, mini-reviews and guest edited issues in all important aspects of protein and peptide research, including structural studies, advances in recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, and drug design. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallization and preliminary structure determination of biologically important proteins are considered only if they include significant new approaches or deal with proteins of immediate importance, and preliminary structure determinations of biologically important proteins. Purely theoretical/review papers should provide new insight into the principles of protein/peptide structure and function. Manuscripts describing computational work should include some experimental data to provide confirmation of the results of calculations. Protein & Peptide Letters focuses on: Structure Studies Advances in Recombinant Expression Drug Design Chemical Synthesis Function Pharmacology Enzymology Conformational Analysis Immunology Biotechnology Protein Engineering Protein Folding Sequencing Molecular Recognition Purification and Analysis