Pinto Devanand, Arriaga Edgar A, Schoenherr Regine M, Chou Shirley Shinn-Huey, Dovichi Norman J
National Research Council of Canada, Institute for Marine Biosciences, 1411 Oxford St, Nova Scotia B3H 3Z1, Halifax, Canada.
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Aug 5;793(1):107-14. doi: 10.1016/s1570-0232(03)00368-4.
Incomplete labeling of proteins by a derivatizing reagent usually results in the formation of a large number of products, which can produce unacceptable band broadening during electrophoretic analysis. In this paper, we report on the reaction of the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ) with the lysine residues of ovalbumin. Mass spectrometry was first used to determine the distribution in the number of labels attached to the protein. At room temperature, 3.6+/-1.9 labels were attached after 30 min. The reaction rate and number of labels increased at elevated temperatures. At 65 degrees C, 6+/-2.5 labels were attached after 5 min. The apparent activation energy for this reaction is estimated as 48+/-17 kJ/mol. Based on the mass spectrometry study, the labeling reaction was assumed to consist of two steps. In the first, the protein unfolds to make lysine residues accessible. In the second, the reagents react with the epsilon -amine of the lysine residues. To test this hypothesis, submicellar capillary electrophoresis and laser-induced fluorescence were used to characterize the reaction mixture. The apparent activation energy was measured for the labeling reaction; the apparent activation energy was 57+/-12 kJ/mol for reaction performed in the separation buffer. Denaturing agents were added to the reaction mixture. The addition of 2 M thiourea with 6 M urea to the reaction resulted in a modest decrease in the apparent activation energy to 42+/-2 kJ/mol. The addition of 2.5 M or higher concentration of ethanol decreased the apparent activation energy to 32+/-2 kJ/mol. We conclude that the apparent activation energy for protein labeling is dominated by denaturation of the protein, and that the addition of suitable denaturing reagents can eliminate this contribution to the reaction chemistry.
衍生试剂对蛋白质的标记不完全通常会导致形成大量产物，这些产物在电泳分析过程中会产生不可接受的谱带展宽。在本文中，我们报道了荧光试剂5-呋喃甲酰喹啉-3-甲醛（FQ）与卵清蛋白赖氨酸残基的反应。首先使用质谱法确定附着在蛋白质上的标记数量分布。在室温下，30分钟后附着了3.6±1.9个标记。在升高的温度下，反应速率和标记数量增加。在65℃时，5分钟后附着了6±2.5个标记。该反应的表观活化能估计为48±17 kJ/mol。基于质谱研究，标记反应被认为由两个步骤组成。第一步，蛋白质展开使赖氨酸残基可及。第二步，试剂与赖氨酸残基的ε-胺反应。为了验证这一假设，使用亚胶束毛细管电泳和激光诱导荧光对反应混合物进行表征。测量了标记反应的表观活化能；在分离缓冲液中进行的反应的表观活化能为57±12 kJ/mol。向反应混合物中加入变性剂。向反应中加入2 M硫脲和6 M尿素会使表观活化能适度降低至42±2 kJ/mol。加入2.5 M或更高浓度的乙醇会使表观活化能降低至32±2 kJ/mol。我们得出结论，蛋白质标记的表观活化能主要由蛋白质的变性决定，并且加入合适的变性试剂可以消除这种对反应化学的影响。
