Chaiken I M, Miller T H, Sequeira R P, Kanmera T
Anal Biochem. 1984 Dec;143(2):215-25. doi: 10.1016/0003-2697(84)90656-0.
Both ion-exchange and reverse-phase HPLC protocols for micromapping of neurophysins have been examined and the structural relationships among the major isoforms identified in the maps have been characterized. Reverse-phase HPLC was found to be especially useful for obtaining fingerprints of the isoforms within each of the two major families of neurophysins, I (oxytocin-related) and II (vasopressin-related), for both bovine and human neurophysins from posterior pituitary sources. From fractionation of the bovine proteins on octylsilyl columns, at least four neurophysins I were identified, one of which corresponds to the intact sequence of 93 residues and three of which vary from the parent by various degrees of carboxyl-terminal truncation. For bovine neurophysin II, two isoforms were identified in the reverse-phase HPLC maps, both of 95 residues, which vary from one another by the residue, either Ile or Val, at position 89. Isoforms were also detected for human neurophysins, including a carboxyl-terminal truncated form of human neurophysin II. All of the major neurophysin isoforms and several of the minor forms were shown to be functionally active as expressed by their binding to peptide ligand affinity matrices. Reverse-phase HPLC mapping on the octylsilyl matrix allowed neurophysin fingerprinting of crude tissue extracts by providing a narrow "window" within which the neurophysins elute but many other polypeptides expected to be present are excluded. The reverse phase HPLC method provides a useful way to obtain isolated neurophysin isoforms for physicochemical characterizations now usually carried out with mixtures of isoforms obtained by ion-exchange chromatography. The method also has characteristics amenable both for high-sensitivity fingerprinting of neurophysin isoforms, from different species and anatomical sources, and as a prelude to microstructural and -functional characterization of the isoforms so isolated.
对用于神经垂体激素运载蛋白微测绘的离子交换和反相高效液相色谱法（HPLC）方案均进行了研究，并对图谱中鉴定出的主要亚型之间的结构关系进行了表征。发现反相HPLC对于从垂体后叶来源获取牛和人神经垂体激素运载蛋白的I（与催产素相关）和II（与加压素相关）这两个主要家族中每个家族内亚型的指纹图谱特别有用。通过在辛基甲硅烷基柱上对牛蛋白进行分级分离，鉴定出至少四种神经垂体激素运载蛋白I，其中一种对应于93个残基的完整序列，另外三种与亲本相比羧基末端有不同程度的截短。对于牛神经垂体激素运载蛋白II，在反相HPLC图谱中鉴定出两种亚型，均为95个残基，它们在第89位残基处分别为异亮氨酸或缬氨酸，彼此不同。在人神经垂体激素运载蛋白中也检测到了亚型，包括人神经垂体激素运载蛋白II的羧基末端截短形式。所有主要的神经垂体激素运载蛋白亚型和几种次要形式都表现出功能活性，这通过它们与肽配体亲和基质的结合得以体现。在辛基甲硅烷基基质上进行反相HPLC测绘，通过提供一个狭窄的“窗口”，使神经垂体激素运载蛋白在其中洗脱，但排除了许多预期存在的其他多肽，从而实现了对粗组织提取物的神经垂体激素运载蛋白指纹图谱分析。反相HPLC方法为获得分离的神经垂体激素运载蛋白亚型提供了一种有用的途径，以便进行现在通常用离子交换色谱法获得的亚型混合物进行的物理化学表征。该方法还具有适用于对来自不同物种和解剖来源的神经垂体激素运载蛋白亚型进行高灵敏度指纹图谱分析的特性，并且可作为对如此分离的亚型进行微观结构和功能表征的前奏。
