Lee Whitaik David, Gawri Rahul, Shiba Toshikazu, Ji Ae-Ri, Stanford William L, Kandel Rita A
1 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
2 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Cartilage. 2018 Oct;9(4):417-427. doi: 10.1177/1947603517690856. Epub 2017 Mar 16.
Inorganic polyphosphates (polyP) play a multitude of roles in mammalian biology. PolyP research is hindered by the lack of a simple and sensitive quantification method. The aim of this study was to develop a robust method for quantifying the low levels of polyP in mammalian tissue such as cartilage, which is rich in macromolecules that interfere with its determination.
Native and in vitro formed tissues were digested with proteinase K to release sequestrated polyP. The tissue digest was loaded on to silica spin columns, followed by elution of bound polyP and various treatments were assessed to minimize non-polyP fluorescence. The eluent was then quantified for polyP content using fluorometry based on DAPI (4',6-diamidino-2-phenylindole) fluorescence shift occurring with polyP.
Proteinase K pretreatment reduced the inhibitory effect of proteins on polyP recovery. The eluent was contaminated with nucleic acids and glycosaminoglycans, which cause extraneous fluorescence signals. These were then effectively eliminated by nucleases treatment and addition of concentrated Tris buffer. PolyP levels were quantified and recovery ratio determined using samples spiked with a known amount of polyP. This silica spin column method was able to recover at least 80% of initially loaded polyP, and detect as little as 10 mol.
This sensitive, reproducible, easy to do method of quantifying polyP will be a useful tool for investigation of polyP biology in mammalian cells and tissues. Although the protocol was developed for mammalian tissues, this method should be able to quantify polyP in most biological sources, including fluid samples such as blood and serum.
无机多聚磷酸盐（多聚P）在哺乳动物生物学中发挥着多种作用。多聚P的研究因缺乏简单灵敏的定量方法而受到阻碍。本研究的目的是开发一种可靠的方法，用于定量哺乳动物组织（如富含干扰其测定的大分子的软骨）中低水平的多聚P。
将天然组织和体外形成的组织用蛋白酶K消化，以释放被隔离的多聚P。将组织消化物加载到硅胶旋转柱上，然后洗脱结合的多聚P，并评估各种处理以尽量减少非多聚P荧光。然后使用基于多聚P时发生的4',6-二脒基-2-苯基吲哚（DAPI）荧光位移的荧光法对洗脱液中的多聚P含量进行定量。
蛋白酶K预处理降低了蛋白质对多聚P回收的抑制作用。洗脱液被核酸和糖胺聚糖污染，这会导致额外的荧光信号。然后通过核酸酶处理和添加浓Tris缓冲液有效地消除了这些信号。使用添加了已知量多聚P的样品对多聚P水平进行定量并确定回收率。这种硅胶旋转柱方法能够回收至少80%最初加载的多聚P，并能检测低至10摩尔的量。
这种灵敏、可重复、易于操作的多聚P定量方法将成为研究哺乳动物细胞和组织中多聚P生物学的有用工具。尽管该方案是针对哺乳动物组织开发的，但这种方法应该能够定量大多数生物来源中的多聚P，包括血液和血清等液体样品。
