牛胰臟去氧核糖核酸水解酶中雙硫鍵之生物功能及其N與C端片段參與活性蛋白摺疊之重要性

Abstract

牛胰臟去氧核糖核酸水解酶 (bpDNase) 是研究得最為透徹的一種DNase，它會水解雙股DNA，但不具序列專一性。它也是第一個被發現且以傳統蛋白質定序技術決定出胺基酸序列之DNase，目前已由前人得到其解析度達2.0 Å之X-光繞射三維晶體結構。在本論文中，我們針對bpDNase中雙硫鍵之生物功能及其N-與C-端片段參與活性蛋白摺疊之重要性進行深入的探討。 我們以定位突變法分別製備了bpDNase中非必需 (C101-C104) 及必需雙硫鍵(C173-C209) 之突變株酵素 [brDNase(C101A)、brDNase(C173A) 與brDNase(C209A)]，以研究其生化功能；另外也分析了一株具有新增第三對雙硫鍵之突變株酵素[brDNase(F192C/A217C)] 的特性。在缺乏鈣離子保護下，bpDNase與brDNase(C101A)會被胰蛋白

Bovine pancreatic deoxyribonuclease (bpDNase) is the best-characterized DNase which cleaves double stranded DNA with no sequence specificity. It was the first DNase discovered and sequenced with the conventional protein sequencing technique and the X-ray structure was resolved at 2.0 Å with refinement. In this dissertation, the biological functions of the disulfides in bpDNase and the involvement of its N- and C-terminal fragments in active protein folding were demonstrated. The biochemical functions of the small non-essential (C101-C104) and the large essential (C173-C209) disulfides in bpDNase have been characterized using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. In addition, we have characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca2+ protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin while brDNase(F192C/A217C) remained active. With Ca2+, all forms of DNase, except for brDNase(C101A), were protected against trypsin. All forms of DNase, after being dissolved in 6 M guanidine-HCl, were fully reactivated by diluting into a Ca2+-containing buffer. However, when diluted into a Ca2+-free buffer, bpDNase and brDNase(C101A) remained inactive, but 60% of the bpDNase activity was restored with brDNase(F192C/A217C). When heated, bpDNase was inactivated at a transition temperature of 65