LOCATION OF DISULFIDE BONDS IN PROTEINS BY FABMS

硫醇和二硫化物的相互转化对许多人来说很重要 生物功能，如蛋白质合成、神经递质 活性和酶结构。为了更好地理解这些 在分子水平上的功能，重要的是要知道 蛋白质中二硫键交联键的位置。所有的 目前用于定位二硫键的方法使用高度特异性 裂解反应产生二硫键结合的多肽。自.以来 这些切割反应通常是无效的，建议的 调查的重点将是开发定位方法 蛋白质中的二硫键，不能用 通常使用的高度特异的切割反应。 由模型的部分酸水解产生的多肽 蛋白质，蛋清溶菌酶，将被分馏 反相高效液相色谱分离及其分子鉴定 重量，这将由快速原子轰击确定 质谱仪(FABMS)。一个电化学探测器将是 用于揭示哪些组分含有二硫键多肽。 分子量信息的计算机辅助分析 将被用来将分子量与特定的片段联系起来 从而定位二硫键。 一种类似的方法是直接耦合微孔高效液相色谱 将开发FABMS系统来识别二硫键 水解物中的多肽。这种方法可以减少样本 一次高效液相色谱-火焰原子吸收光谱分析。 随着这些方法的发展，它们将被用于定位 生物重要蛋白质中的二硫键，如 黄体生成素和组织型纤溶酶原激活剂。 亮氨酸化激素，一种促性腺激素，直接参与 睾丸和卵巢的功能，这是因为 生产二硫键多肽的难度较大 对天然材料中的每个二硫键进行诊断。 组织型纤溶酶原激活剂是一种大型 目前通过重组DNA生产的蛋白质 技术

The interconversion of thiols and disulfides is important for many biological functions such as protein synthesis, neurotransmitter activity, and enzyme structure. To better understand these functions on the molecular level, it is important to know the locations of disulfide cross-linkages in proteins. All of the present methods for locating disulfide bonds use highly specific cleavage reactions to produce disulfide-bonded peptides. Since these cleavage reactions are often ineffective, the proposed investigation will focus on developing methods for locating disulfide bonds in proteins which cannot be cleaved with the commonly used highly specific cleavage reactions. Peptides produced from the partial acid hydrolysis of a model protein, hen egg-white lysozyme, will be fractionated by reversed-phase HPLC and identified from their molecular weights, which will be determined by fast atom bombardment mass spectrometry (FABMS). An electrochemical detector will be used to reveal which fractions contain disulfide-bonded peptides. Computer-assisted analysis of the molecular weight information will be used to relate the molecular weights to specific segments of the protein, and hence to locate the disulfide bonds. A similar approach is which a directly-coupled microbore HPLC FABMS system will be developed to identify disulfide-bonded peptides in the hydrolysate. This approach could reduce sample analysis to a single HPLC FABMS run. As these methods are developed, they will be used to locate disulfide linkages in biologically important proteins such as luteinizing hormone and tissue-type plasminogen activator. Leuteinizing hormone, a gonadotropin which is directly involved in testicular and ovarian functions, is of interest because of the difficulty in producing disulfide-bonded peptides which are diagnostic for each of the disulfide bonds in the native material. Tissue-type plasminogen activator is an example of a large protein which is presently produced via recombinant DNA technology.