Ž . Clinica Chimica Acta 312 2001 205–212 www.elsevier.comrlocaterclinchim Sources of interference in the use of 2,3-diaminonaphthalene for the fluorimetric determination of nitric oxide synthase activity in biological samples Monica Fernandez-Cancio, Eva Marıa Fernandez-Vitos, Josep Joan Centelles, ´ ´ ´ ´ Santiago Imperial ) Departament de Bioquımica i Biologia Molecular, Facultat de Quımica, UniÕersitat de Barcelona, Martı i Franques 1, ´ ´ ´ ` 08028 Barcelona, Spain Received 13 March 2001; received in revised form 2 July 2001; accepted 14 July 2001 Abstract Ž . Ž . The use of 2,3-diaminonaphthalene 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 Ž . Ž . mixture or to the enzymatic sample itself. One of the substrates NADPH and some cofactors FADH , FMNH required 2 2 for the enzyme activity interfere in the assay by quenching the fluorescence produced. Interference was minimized by using Ž . lower FADH , FMNH and NADPH concentrations 1 mmolrl and a NADPH recycling system in the reaction mixture. 2 2 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 Ž 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. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Nitric oxide synthase; 2,3-Diaminonaphthalene; Interference; Fluorimetric assay AbbreÕiations: NO, nitric oxide; NOS, nitric oxide synthase; DAN, 2,3-diaminonaphthalene; NADPH, nicotine adenine dinu- cleotide phosphate; FADH , flavine adenine dinucleotide; 2 FMNH , flavine mononucleotide. 2 ) Corresponding author. Tel.: q 34-93-402-1215; fax: q 34-93- 402-1219. Ž . E-mail address: imperial@sun.bq.ub.es S. Imperial . 1. Introduction Ž . Nitric oxide NO has for some years been con- sidered a toxic pollutant. However, in the last decade many studies have shown that mammalian cells syn- thesize NO and that this molecule acts as a modu- lator in different physiological processes in the car- w x diovascular, immune and nervous systems 1–3 . In 0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. Ž . PII: S0009-8981 01 00625-8