通过在硫酸皮肤素取代的1,6 - 二氨基己烷 - 琼脂糖4B上进行色谱法快速纯化牛睾丸透明质酸酶。
A rapid purification of bovine testicular hyaluronidase by chromatography on dermatan sulphate-substituted 1,6-diaminohexane--sepharose 4B.
作者信息
Lyon M, Phelps C F
出版信息
Biochem J. 1981 Nov 1;199(2):419-26. doi: 10.1042/bj1990419.
The binding of bovine testicular hyaluronidase to AH-Sepharose (1,6-diaminohexane--Sepharose) gels substituted with (1) dermatan sulphate, (2) desulphated dermatan sulphate, (3) heparin and (4) de-N/O-sulphated, re-N-acetylated heparin was investigated. Hyaluronidase was found to bind to (1) and (3), but not (2) and (4). On the basis of these observations a preparative scheme for the purification of testicular hyaluronidase was developed. This consisted of two steps: (i) chromatography on dermatan sulphate-substituted AH-Sepharose 4B; (ii) chromatography on acetylated AH-Sepharose 4B. This procedure gave hyaluronidase with a specific activity of 19.1 units (mumol/min)/mg in high yield. Polyacrylamide-gel electrophoresis at pH 4.3 revealed two components, both possessing hyaluronidase activity. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis likewise revealed two close bands with approximate molecular weights of 61000 and 67200.
研究了牛睾丸透明质酸酶与用以下物质取代的AH-琼脂糖凝胶（1,6-二氨基己烷-琼脂糖）的结合情况：（1）硫酸皮肤素；（2）脱硫酸皮肤素；（3）肝素；（4）去N/O-硫酸化、再N-乙酰化的肝素。发现透明质酸酶与（1）和（3）结合，但不与（2）和（4）结合。基于这些观察结果，制定了一种纯化睾丸透明质酸酶的制备方案。该方案包括两个步骤：（i）在硫酸皮肤素取代的AH-琼脂糖4B上进行色谱分离；（ii）在乙酰化的AH-琼脂糖4B上进行色谱分离。该方法以高产率得到了比活性为19.1单位（微摩尔/分钟）/毫克的透明质酸酶。在pH 4.3条件下的聚丙烯酰胺凝胶电泳显示有两个组分，两者均具有透明质酸酶活性。十二烷基硫酸钠聚丙烯酰胺凝胶电泳同样显示有两条紧密的条带，近似分子量分别为61000和67200。
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