Wisniewski Hans-Georg, Sweet Moshe H, Stern Robert
Department of Microbiology, School of Medicine, New York University, NY 10016, USA.
Anal Biochem. 2005 Dec 1;347(1):42-8. doi: 10.1016/j.ab.2005.09.038. Epub 2005 Oct 21.
A simple, rapid, and reproducible microtiter-based chondroitinase (CSase) assay is reported here, based on the competition of chondroitin sulfate (CS) with immobilized hyaluronan (HA) for the binding of TSG-6 protein, the product of TNF-inducible gene 6. Although the catabolic reaction of bacterial and other prokaryotic CSase enzymes, often referred to as the chondroitin lyases, can be followed by tracking the generation of unsaturated bonds by the spectrophotometrical determination of the absorbance at 232 nm, no rapid, sensitive, and simple assay has been devised to date for measuring the activity of the vertebrate enzymes that cleave their substrate exclusively by hydrolysis. We provide data demonstrating that the CSase assay described here is suitable for the determination of the activities of both classes of enzymes. For the bacterial enzyme CSase ABC, both the determination of the absorbance at 232 nm and the assay based on TSG-6 binding are suitable using the same range of enzyme activities. However, for testicular hyaluronidase, considerably higher enzyme activities were needed to cleave CS than to cleave HA. Using the HA-binding domain of aggrecan for a comparison, we determined that the interaction between TSG-6 and chondroitin sulfate is uniquely suited for this CSase assay.
本文报道了一种基于微量滴定板的简单、快速且可重复的软骨素酶（CSase）检测方法，该方法基于硫酸软骨素（CS）与固定化透明质酸（HA）竞争结合肿瘤坏死因子诱导基因6的产物TSG-6蛋白。尽管细菌和其他原核CSase酶（通常称为软骨素裂解酶）的分解代谢反应可通过分光光度法测定232nm处的吸光度来追踪不饱和键的生成，但迄今为止，尚未设计出快速、灵敏且简单的方法来测量仅通过水解作用切割底物的脊椎动物酶的活性。我们提供的数据表明，本文所述的CSase检测方法适用于测定这两类酶的活性。对于细菌酶CSase ABC，在相同的酶活性范围内，232nm处吸光度的测定和基于TSG-6结合的检测方法均适用。然而，对于睾丸透明质酸酶，切割CS所需的酶活性远高于切割HA。通过使用聚集蛋白聚糖的HA结合结构域进行比较，我们确定TSG-6与硫酸软骨素之间的相互作用特别适用于这种CSase检测方法。
