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

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 subs trates (NADPH) and some cofactors (FADH(2), FMNH2) required for the enzyme activity interfere in the assay by quenching the fluorescence produced. Int erference was minimized by using lower FADH(2), FMNH2 and NADPH concentrati ons (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 fluor escence intensity, but these protein interferences could be reduced by filt ering the samples after reaction. We conclude that the DAN fluorimetric ass ay as originally described is not suitable for the determination of NOS act ivity in crude extracts such as rat brain cytosolic fraction, due to the pr esence of interfering substances. Nevertheless, DAN could be used for the d etermination of enzyme activity after reducing protein interference by filt ering, or in less complex samples such as cell cultures (e.g. activated mac rophages), or in chromatographic fractions obtained during the purification of the enzyme. A careful use of the commercial kits based on the use of DA N for the determination of NOS activity is recommended. (C) 2001 Elsevier S cience B.V. All rights reserved.