Flora B, Gusman H, Helmerhorst E J, Troxler R F, Oppenheim F G
Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, MA 02118-2392, USA.
Protein Expr Purif. 2001 Oct;23(1):198-206. doi: 10.1006/prep.2001.1493.
Histatins, a family of small-molecular-weight, histidine-rich cationic salivary proteins, have been difficult to isolate in an efficient way by conventional procedures due to their anomalous interactions with chromatographic resins. In the present study we explored the possibility of developing a new isolation procedure based on recent observations that histatins associate with various metal ions, including zinc. Since solubility studies showed that histatin 5 forms precipitates with zinc under alkaline conditions, we investigated whether this characteristic could be exploited for the preparative isolation of histatins from salivary secretions. A fast and efficient two-step procedure was developed using zinc precipitation of histatins from human parotid secretion followed by final purification using reversed-phase high-performance liquid chromatography (HPLC). Analysis of zinc precipitates by Tricine-SDS-PAGE, cationic PAGE, HPLC, and mass spectrometry revealed the presence of the three major histatins, 1, 3, and 5, as well as statherin. The histatin yield obtained by the precipitation step was approximately 90%. Therefore, zinc precipitation of histatins from glandular salivary secretions is a novel, rapid, and effective means for the isolation of these proteins.
富组蛋白是一类小分子、富含组氨酸的阳离子唾液蛋白，由于它们与色谱树脂存在异常相互作用，采用传统方法难以高效分离。在本研究中，我们基于富组蛋白与包括锌在内的多种金属离子结合的最新观察结果，探索了开发一种新分离方法的可能性。由于溶解度研究表明，在碱性条件下，组蛋白5会与锌形成沉淀，因此我们研究了能否利用这一特性从唾液分泌物中制备分离富组蛋白。我们开发了一种快速有效的两步法，即先从人腮腺分泌物中通过锌沉淀法分离富组蛋白，然后使用反相高效液相色谱（HPLC）进行最终纯化。通过Tricine-SDS-PAGE、阳离子PAGE、HPLC和质谱对锌沉淀物进行分析，结果显示存在三种主要的富组蛋白，即富组蛋白1、3和5，以及富含脯氨酸的磷蛋白。沉淀步骤获得的富组蛋白产量约为90%。因此，从腺性唾液分泌物中通过锌沉淀法分离富组蛋白是一种分离这些蛋白质的新颖、快速且有效的方法。
