以毛細管區帶電泳結合等速電泳線上濃縮法監測核糖核酸還原酵素之活性

Online preconcentration by transient isotachophoresis with capillary zone electrophoresis for monitoring the activity of ribonucleotide reductase

黃世緯(Shi-Wei Huang)

指導教授 ： 曾惠芬

國立暨南國際大學/科技學院/應用化學系/碩士(2008年)

https://doi.org/10.6837/NCNU.2008.00087

摘要

DNA合成的第一步驟是將核糖核苷二磷酸(NDP)還原成去氧核糖核苷二磷酸(dNDP)，此步驟是由核糖核苷酸還原酶所催化。因此，核糖核苷酸還原酶之活性會關係到細胞週期之進行以及DNA之穩定度。在本研究中，將以毛細管區帶電泳結合等速電泳線上濃縮法，分析核糖核苷酸還原酶之酵素活性溶液，以提升偵測靈敏度。等速電泳線上濃縮法並不限於低導電度樣品溶液的使用，因此很適合用於酵素分析溶液等高鹽溶液。在實驗中分別對於前導電解質濃度、樣品緩衝溶液pH值和濃度、電滲流抑制劑濃度等參數進行探討及最佳化。前導電解質濃度的改變對於樣品堆積效率以及分離效果皆有影響。電泳緩衝溶液pH值會影響樣品間解析狀況，而電泳緩衝溶液濃度則會關係到樣品滯留於等速電泳模式中的時間，進而改變分離效果。電滲流抑制劑的使用可減少樣品區帶的擴散現象。所有參數以得到最佳堆積效果及分離效率為方向進行最佳化。以最佳化條件進行實驗，在6分鐘之內即可完成分析，進樣體積可比一般毛細管區帶電泳方式增加150倍，且不損失解析度。dCDP的線性範圍在2∼40 µM之間，相關係數為0.9999，偵測極限可達0.44 µM。此方法將可應用於核糖核苷酸還原酶之活性測定以增加偵測靈敏度。

關鍵字

核糖核苷酸還原酶等速電泳 ； 毛細管區帶電泳

並列摘要

The first step of DNA synthesis is the reduction of ribonucleoside 5’-diphosphates to form 2’-deoxyribonucleoside 5’-diphosphates, which is catalyzed by ribonucleotide reductase. Therefore the activity of ribonucleotide reductase is associated with not only cell cycle but also the stability of DNA synthesis. In this study, the enzyme assay mixture was analyzed by capillary zone electrophoresis coupled with isotachophoresis as online preconcentration technique to improve the sensitivity. This preconcentration method can be applied to the sample buffer even with high conductivity, so it is suitable for the direct injection of the enzyme assay mixtures. Several parameters including the concentration of leading electrolyte, the pH and the concentration of running buffer, and the concentration of EOF suppresser were optimized. All these parameters were adjusted to get the optimal stacking and separation results. By using the optimal condition, the good separation of dCDP and dADP were achieved in 6 minutes, and the injection volume could be increased to 150-fold of that used in the normal CZE mode without losing the resolution. The linear range of this method was 2-40 µM for dCDP, and the correlation coefficient was 0.9999. The detection limit of dCDP was 0.44 µM. This method could be applied to increase the sensitivity of ribonucleotide reductase assays.

並列關鍵字

ribonucleotide reductase ； isotachophoresis ； capillary zone electrophoresis

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