Sources of interference in the use of 2,3-diaminonaphthalene for the fluorimetric determination of nitric oxide synthase activity in biological samples

被引:24
作者
Fernández-Cancio, M [1 ]
Fernández-Vitos, EM [1 ]
Centelles, JJ [1 ]
Imperial, S [1 ]
机构
[1] Univ Barcelona, Fac Quim, Dept Bioquim & Biol Mol, E-08028 Barcelona, Spain
关键词
nitric oxide synthase; 2,3-diaminonaphthalene; interference; fluorimetric assay;
D O I
10.1016/S0009-8981(01)00625-8
中图分类号
R446 [实验室诊断]; R-33 [实验医学、医学实验];
学科分类号
1001 ;
摘要
The use of 2,3-diaminonalihthalene (DAN) for the fluorimetric determination of nitric oxide synthase (NOS) activity in rat brain extracts has been re-examined. Two types of interference were observed, due either to components of the reaction mixture or to the enzymatic sample itself, One of (he substrates (NADPH) and some cofactors (FADH(2), FMNH2) required for the enzyme activity interfere in the assay by quenching the fluorescence produced. Interference was minimized by using lower FADH(2), FMNH2 and NADPH concentrations (1 mu mol/l) and a NADPH recycling system in the reaction mixture. The addition of bovine serum albumin or hemoglobin to the sample quenched fluorescence intensity, but these protein interferences could be reduced by filtering the samples after reaction. We conclude that the DAN fluorimetric assay as originally described is not suitable for the determination of NOS activity in crude extracts such as rat brain cytosolic fraction, due to the presence of interfering substances. Nevertheless, DAN could be used for the determination of enzyme activity after reducing protein interference by filtering, or in less complex samples such as cell cultures (e.g. activated macrophages), or in chromatographic fractions obtained during the purification of the enzyme. A careful use of the commercial kits based on the use of DAN for the determination of NOS activity is recommended. (C) 2001 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:205 / 212
页数:8
相关论文
共 24 条
[1]   Elimination of matrix-based interferences to a fluorescent nitrite/nitrate assay by a simple filtration procedure [J].
Bedwell, DW ;
Rivera, VR ;
Merrill, GA ;
Pusateri, AE .
ANALYTICAL BIOCHEMISTRY, 2000, 284 (01) :1-5
[2]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[3]   NITRIC-OXIDE - A PHYSIOLOGICAL MESSENGER MOLECULE [J].
BREDT, DS ;
SNYDER, SH .
ANNUAL REVIEW OF BIOCHEMISTRY, 1994, 63 :175-195
[4]  
GREEN LC, ANAL BIOCH, V26, P131
[5]  
GRIESS JP, 1879, VER DTSCH CHEM GES, V12, P426
[6]  
Griess P., 1864, Philosophical Transactions of the Royal Society of London, V154, P667
[7]  
Heiduschka P, 1998, NEUROREPORT, V9, P4051
[8]   Intense exercise causes decrease in expression of both endothelial NO synthase and tissue NOx level in hearts [J].
Iemitsu, M ;
Miyauchi, T ;
Maeda, S ;
Yuki, K ;
Kobayashi, T ;
Kumagai, Y ;
Shimojo, N ;
Yamaguchi, I ;
Matsuda, M .
AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY, 2000, 279 (03) :R951-R959
[9]  
Ignarro L. J., 1996, Nitric Oxide, P111
[10]   Up-regulation of endothelial nitric oxide synthase through β2-adrenergic receptor -: The role of a β-blocker with NO-releasing action [J].
Jayachandran, M ;
Hayashi, T ;
Sumi, D ;
Thakur, NK ;
Kano, H ;
Ignarro, LJ ;
Iguchi, A .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2001, 280 (03) :589-594