Ikegami-Kawai Mayumi, Takahashi Tomoko
Institute of Medicinal Chemistry, Hoshi University, 2-4-41, Ebara, Shinagawaku, Tokyo 142-8501, Japan.
Anal Biochem. 2002 Dec 15;311(2):157-65. doi: 10.1016/s0003-2697(02)00425-6.
We established a rapid, sensitive polyacrylamide gel electrophoresis (PAGE) method for the analysis of hyaluronan (HA) oligosaccharides. Using mini-slab gels, but not large-slab gels so far reported, HA oligosaccharides of 5 to more than 50 repeating disaccharide units could be separated into discrete ladder-like bands in a short electrophoresis time of 45 min. Using a combined Alcian blue and silver staining protocol, the detection limit was less than 1 ng per band for 11 repeating disaccharide units, indicating 50 times higher sensitivity than that of an earlier-described sensitivity-enhanced PAGE method. Our PAGE method was applicable to the assay of hyaluronidase activity. When a total of multiple band intensities for 18-24 repeating disaccharide units was used as a measure of activity, as little as 3 x 10(-4) NFU of bovine testicular hyaluronidase was detectable on a 1-h incubation. This sensitivity permitted rapid measurements of human and rabbit serum hyaluronidases, the latter of which having never been detected even by a sensitive enzyme-linked immunosorbent assay (ELISA). Since this PAGE assay does not require specialized reagents and instruments and since it provides information on both the activity and the enzymatic HA degradation pattern, there may be many potential applications.
我们建立了一种快速、灵敏的聚丙烯酰胺凝胶电泳（PAGE）方法来分析透明质酸（HA）寡糖。使用迷你平板凝胶，而非迄今报道的大型平板凝胶，5至50多个重复二糖单元的HA寡糖能够在45分钟的短电泳时间内分离成离散的梯状条带。采用阿尔辛蓝和银染色相结合的方案，对于11个重复二糖单元，每条带的检测限低于1纳克，这表明灵敏度比先前描述的灵敏度增强PAGE方法高50倍。我们的PAGE方法适用于透明质酸酶活性的测定。当将18 - 24个重复二糖单元的多条带强度总和用作活性指标时，在1小时孵育后，低至3×10⁻⁴纳摩尔荧光单位（NFU）的牛睾丸透明质酸酶均可检测到。这种灵敏度使得能够快速测定人和兔血清中的透明质酸酶，其中兔血清透明质酸酶甚至通过灵敏的酶联免疫吸附测定（ELISA）都从未检测到过。由于这种PAGE测定不需要专门的试剂和仪器，并且它能提供关于活性和酶促HA降解模式的信息，因此可能有许多潜在应用。
