Armstrong Shayn E, Bell Donald R
Center for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208-3479, USA.
Anal Biochem. 2002 Sep 15;308(2):255-64. doi: 10.1016/s0003-2697(02)00239-7.
The size of hyaluronan in solid tissue was measured using a combination of agarose gel electrophoresis and a radiometric assay. Radiolabeled hyaluronan binding proteins, used in the radiometric assay, were also used to detect hyaluronan after transfer to a nylon membrane following gel electrophoresis. Lane intensity on the autoradiograph was linearly related to the amount applied to the gel between 10 and 100ng. The intensity was independent of the hyaluronan molecular weight for standards with molecular weights equal to or greater than 790,000. The radiometric assay was used to measure hyaluronan irrespective of size, while gel electrophoresis was used to measure hyaluronan with molecular weights greater than 0.79x10(6) or 4x10(6). Deferoxamine was used to inhibit depolymerization during the digestion of tissue samples with protease. The molecular weight pattern was similar for skin, skeletal muscle, heart, lung, small intestine, and large intestine despite large differences in hyaluronan content. For all tissues, 58% of the hyaluronan had a molecular weight greater than 4million. All tissues showed an absence of hyaluronan with a molecular weight below 790,000. The procedure can be used to study changes in hyaluronan size in tissue during inflammation and other pathological states.
采用琼脂糖凝胶电泳和放射测定法相结合的方法测量实体组织中透明质酸的大小。放射测定法中使用的放射性标记透明质酸结合蛋白，在凝胶电泳后转移至尼龙膜上后，也用于检测透明质酸。放射自显影片上的条带强度与加样至凝胶上的量在10至100纳克之间呈线性相关。对于分子量等于或大于790,000的标准品，强度与透明质酸分子量无关。放射测定法用于测量各种大小的透明质酸，而凝胶电泳用于测量分子量大于0.79×10⁶或4×10⁶的透明质酸。在使用蛋白酶消化组织样品期间，使用去铁胺抑制解聚。尽管透明质酸含量存在很大差异，但皮肤、骨骼肌、心脏、肺、小肠和大肠的分子量模式相似。对于所有组织，58%的透明质酸分子量大于400万。所有组织均未显示分子量低于790,000的透明质酸。该方法可用于研究炎症和其他病理状态下组织中透明质酸大小的变化。
