Kinetics and apparent activation energy of the reaction of the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde with ovalbumin

被引:23
作者
Pinto, D
Arriaga, EA
Schoenherr, RM
Chou, SSH
Dovichi, NJ [1 ]
机构
[1] Univ Washington, Dept Chem, Seattle, WA 98195 USA
[2] Natl Res Council Canada, Inst Marine Biosci, Halifax, NS B3H 3Z1, Canada
[3] Univ Minnesota, Dept Chem, Minneapolis, MN 55455 USA
来源
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES | 2003年 / 793卷 / 01期
基金
美国国家卫生研究院;
关键词
kinetics; apparent activation energy; 5-furoylquinoline-3-carboxaldehyde; ovalbumin;
D O I
10.1016/S1570-0232(03)00368-4
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 degreesC, 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. (C) 2003 Elsevier B.V. All rights reserved.
引用
收藏
页码:107 / 114
页数:8
相关论文
共 21 条
[1]   FLUORESCENCE REAGENTS FOR HIGH-SENSITIVITY CHROMATOGRAPHIC MEASUREMENTS OF PRIMARY AMINES [J].
BEALE, SC ;
SAVAGE, JC ;
WIESLER, D ;
WIETSTOCK, SM ;
NOVOTNY, M .
ANALYTICAL CHEMISTRY, 1988, 60 (17) :1765-1769
[2]   Single-molecule detection in capillary electrophoresis: Molecular shot noise as a fundamental limit to chemical analysis [J].
Chen, DY ;
Dovichi, NJ .
ANALYTICAL CHEMISTRY, 1996, 68 (04) :690-696
[3]   SUBATTOMOLE AMINO-ACID ANALYSIS BY CAPILLARY ZONE ELECTROPHORESIS AND LASER-INDUCED FLUORESCENCE [J].
CHENG, YF ;
DOVICHI, NJ .
SCIENCE, 1988, 242 (4878) :562-564
[4]   Multiple labeling of proteins [J].
Craig, DB ;
Dovichi, NJ .
ANALYTICAL CHEMISTRY, 1998, 70 (13) :2493-2494
[5]  
Herbert B, 1999, ELECTROPHORESIS, V20, P660, DOI 10.1002/(SICI)1522-2683(19990101)20:4/5<660::AID-ELPS660>3.0.CO
[6]  
2-Q
[7]   QUANTUM EFFICIENCY INDEPENDENCE OF TIME INTEGRATED EMISSION FROM A FLUORESCENT MOLECULE [J].
HIRSCHFELD, T .
APPLIED OPTICS, 1976, 15 (12) :3135-3139
[8]   DENATURATION CAPACITY - A NEW QUANTITATIVE CRITERION FOR SELECTION OF ORGANIC-SOLVENTS AS REACTION MEDIA IN BIOCATALYSIS [J].
KHMELNITSKY, YL ;
MOZHAEV, VV ;
BELOVA, AB ;
SERGEEVA, MV ;
MARTINEK, K .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1991, 198 (01) :31-41
[9]   Picomolar analysis of proteins using electrophoretically mediated microanalysis and capillary electrophoresis with laser-induced fluorescence detection [J].
Lee, IH ;
Pinto, D ;
Arriaga, EA ;
Zhang, ZR ;
Dovichi, NJ .
ANALYTICAL CHEMISTRY, 1998, 70 (21) :4546-4548
[10]   Homogeneous fluorescent derivatization of large proteins [J].
Liu, HJ ;
Cho, BY ;
Krull, IS ;
Cohen, SA .
JOURNAL OF CHROMATOGRAPHY A, 2001, 927 (1-2) :77-89