Stepaniak Leszek
Department of Food Science, Agricultural University of Norway, As.
Prep Biochem Biotechnol. 2002 Feb;32(1):17-28. doi: 10.1081/PB-120013158.
Catalytic antibodies (abzymes) which hydrolyze RNA and DNA were isolated from bovine colostrum by sequential chromatography on Protein A Sepharose, denaturated DNA-cellulose, Mono Q, and gel permeation chromatography on Superose 12 at pH 2.3 after acidic shock. Metachromatic agar containing toluidine blue and yeast RNA was used to measure RNase activity. Electrophoresis in agarose showed DNase activity on plasmid DNA from Escherichia coli and DNA from calf thymus in fractions from all 4 purification steps. Gel permeation chromatography showed that the abzymes hydrolysed both a single-stranded polyadenylic acid (Poly A) and single-stranded polycitidylic acid (Poly C), while partially purified RNase from the colostrum hydrolysed Poly (C), but not Poly (A). Electrophoresis of purified abzymes under denaturing conditions showed protein bands of molecular mass corresponding to heavy and light chains of IgG. The abzymes immunoreacted with anti-bovine IgG. The RNase activity of the purified abzymes represented 0.022% of total RNase activity in the colostrum; acid shock and gel filtration at low pH reduced the specific RNase activity of abzymes 3.6-fold. The RNase activity of abzymes at pH 6.6 was reduced by 90% by heat treatment at 75 degrees C for 52 min.
通过在蛋白A琼脂糖凝胶、变性DNA纤维素、Mono Q上依次进行层析，并在酸性休克后于pH 2.3条件下在Superose 12上进行凝胶渗透层析，从牛初乳中分离出了能水解RNA和DNA的催化抗体（抗体酶）。使用含有甲苯胺蓝和酵母RNA的异染性琼脂来测定核糖核酸酶活性。琼脂糖凝胶电泳显示，在所有4个纯化步骤的组分中，来自大肠杆菌的质粒DNA和小牛胸腺DNA上均有脱氧核糖核酸酶活性。凝胶渗透层析表明，抗体酶能水解单链聚腺苷酸（Poly A）和单链聚胞苷酸（Poly C），而初乳中部分纯化的核糖核酸酶能水解Poly（C），但不能水解Poly（A）。在变性条件下对纯化的抗体酶进行电泳，显示出分子量与IgG重链和轻链相对应的蛋白条带。这些抗体酶与抗牛IgG发生免疫反应。纯化的抗体酶的核糖核酸酶活性占初乳中总核糖核酸酶活性的0.022%；酸性休克和低pH值下的凝胶过滤使抗体酶的比核糖核酸酶活性降低了3.6倍。在75℃下热处理52分钟后，抗体酶在pH 6.6时的核糖核酸酶活性降低了90%。
