DISULFIDE BOND MAPPING OF PROTEINS BY CYANYLATION

通过氰基化对蛋白质进行二硫键图谱

• 批准号: 6258782
• 负责人: JACK T WATSON
• 金额: $ 0.24万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6258782
• 关键词: bacteria; biological products; biomedical equipment development; biomedical resource; drug screening /evaluation; technology /technique development

A knowledge of protein structure requires a determination of which cysteine is connected to which cysteine. We cyanylate and cleave the peptide bond at the N-terminus of free cysteine residues. From the known sequence, we can anticipate the mass of degraded peptides for a given disulfide bond connectivity. We presently use cyanodiaminopyridium (CDAP) tetrafluoroborate, which cyanylates the protein at pH 3 to avoid disulfide bond exchange. We have established conditions for partial reduction at low pH which also avoids disulfide bond exchange. We have used this approach with ribonuclease A, which gives four singly reduced isomers, each having a different disulfide bond reduced. These isoforms were cyanylated and cleaved for analysis by MALDI-MS allowing us to verify the pairing of the disulfide bonds. We are also exploring the applicability of alternate cyanylating reagents to provide us with some flexibility in the chemical approach to solving a structural problem. We have synthesized a new reagent, 2-thiocyanopyridine, which will be characterized for its potency in cyanylating model compounds such as ?-lactoglobulin and others.

蛋白质结构的知识需要确定 半胱氨酸与哪个半胱氨酸连接。 我们氰化并切割 游离半胱氨酸残基N末端的肽键。 从 已知序列，我们可以预测降解肽的质量， 考虑到二硫键连接性。 我们目前使用 氰基二氨基吡啶（CDAP）四氟硼酸盐，其使 蛋白质在pH 3以避免二硫键交换。 我们建立 在低pH下部分还原的条件，这也避免了二硫化物 债券交易所 我们已经用这种方法与核糖核酸酶A， 得到四种单一的还原异构体，每种都有不同的二硫化物 债券减少。 将这些同种型氰基化并切割用于分析 通过MALDI-MS使我们能够验证二硫键的配对。 我们还在探索交替氰化的适用性 为我们提供了一些灵活的化学方法 解决一个结构性问题。 我们合成了一种新试剂， 2-硫氰基吡啶，其将表征其在以下方面的效力： 氰化模型化合物，如？-乳球蛋白等。