PURIFICATION AND CHARACTERIZATION OF HYALURONAN FROM SYNOVIAL-FLUID
PURIFICATION AND CHARACTERIZATION OF HYALURONAN FROM SYNOVIAL-FLUID
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DOI：
10.1006/abio.1993.1230
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发表时间：
1993-05-15
影响因子：
2.9
通讯作者：
PAOLETTI, S
中科院分区：
文献类型：
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作者：
KVAM, C;GRANESE, D;PAOLETTI, S
研究了一种简便、快速的从滑液中分离透明质酸的方法。在苯酚-氯仿萃取去除蛋白质之前，通过疏水过滤器过滤去除脂质。通过三种不同的方法测量，透明质酸回收率为95%。透明质酸的平均分子量在纯化过程中没有变化。此外，研究发现，在过滤之前，对病理人体滑液进行优化的酶蛋白消化产生的透明质酸回收率高达五倍。此外，由于聚集物的存在，未被蛋白酶E消化的滑液中透明质酸的分子量明显较高。透明质酸纯化后（约15分钟），一个粒径排除层析步骤可以同时测定其浓度，合理估计其平均分子量和分子量分布曲线。在分子量为2.0 × 106 ~ 1.0 × 104的范围内，透明质酸的分子量对数值与排粒径层析洗脱量呈线性关系。与未经处理的透明质酸和蛋白质峰部分重叠的样品相比，去除蛋白质可以测定相当低分子量的透明质酸组分。
An easy and rapid method for the purification and characterization of hyaluronan from synovial fluid has been developed. Lipids were removed by filtration through a hydrophobic filter prior to the removal of proteins by phenol-chloroform extraction. The hyaluronan recovery was 95%, as measured by three different methods. The average molecular weight of hyaluronan did not change during the purification. Furthermore, it was found that an optimized enzymatic protein digestion of pathological human synovial fluid prior to filtration yielded up to five times more hyaluronan recovered. In addition, the molecular weight of hyaluronan from synovial fluid not digested with pronase E was apparently higher because of the presence of aggregates. After the purification of hyaluronan (ca. 15 min), a single size-exclusion chromatography step allowed the simultaneous determination of its concentration and the reasonable estimate of its average molecular weight and molecular weight distribution curve. The logarithm of the molecular weight showed a linear dependence on the size-exclusion chromatography elution volume for hyaluronan in the molecular weight range 2.0 × 106- 1.0 × 104. The removal of proteins allowed the determination of fairly low-molecular-weight fractions of hyaluronan, compared to untreated samples for which hyaluronan and protein peaks partially overlap.