Fluorescent assay for studying the substrate specificity of neuraminidase

Toyopearl 650 M was purchased from Tosoh (Japan). The 10-~1 pipette tips with filter (cat. no. FI08-96R-I0) and thin-well 0.2-ml PCR microtubes (cat. no. 430-01XNBZ-Q) with caps were obtained from Porex. Black polystyrene 96-well microtiter plates were purchased from Nunc (Denmark, cat. no. 437111). Vibrio cholerae neuraminidase (3.2.1.18) was purchased from Sigma (USA). 3'SiaLac and 6'SiaLac were purchased from Lectinity (Russia). ro-Aminospacered 3'SiaLac, 3'SiaLacNAc (Neu5Ac Cl2-3GaI131-4GlcNAc), and 6'SiaLacNAc (Neu5Ac Cl2-6GaI131-4GlcNAc) were synthesized previously [5-7]. ro-Aminospacered trisaccharide (5 ~ol), followed by triethylamine (5 ~l), was added to BODIPY FLSE (5.5 ~ol) in dimethylformamide (0.5ml). The reaction mixture was stirred at room temperature for 2-3 h and was subjected to gel filtration on a Sephadex LH-20 column equilibrated with acetonitrile/water (1:1, v/v). The yield of BODIPYlabeled trisaccharides was 80-90%. (Complete lH nuclear magnetic resonance data (500 MHz, DzO) are available from www.carbohydrate.ru/docs/nmrdata_OOl.rtf.) Virus NBulgaria/120/82 (HINl) and virus A!FPV/ Rostock/34 (H7Nl) were obtained from the virus repository of the Ivanovsky Institute of Virology in Moscow, Russia, and were propagated in 10-day-old embryonated chick~n eggs. A Madin-Darby canine kidney (MDCK)derived variant of human influenza virus A/Hongkong/ 1143/99 (H3N2) was obtained from the Institute of Applied Microbiology in Vienna, Austria. Primary isolation, propagation of human influenza virus, and purification of all viruses were done as described previously [8]. A titer of viruses was determined in a hemagglutination reaction with human erythrocytes Neuraminidase, or sialidase, is an exoglycosidase that hydrolyzes tX-ketosidic linkage between sialic acid and adjacent sugar residue. There are several methods of neuraminidase activity determination, usually based on fluorescent or chemiluminescent detection of an aglycon formed due to neuraminic acid cleavage [1-3]. Substrate specificity of neuraminidase is usually defined as the enzyme ability to distinguish the type of bond between neuraminic acid and galactose residues. As a rule, 3'SiaLac (Neu5ActX2-3Galf31-4Glc)! and 6'SiaLac (Neu5Ac tX2-6Galf31-4Glc) are used as substrates, and the amount of released neuraminic acid is measured [4]. In this article, we propose a simple and sensitive fluorescent method for determining the neuraminidase specificity using BODIPY -labeled trisaccharides as substrates. The method is based on the quantitative separation of neutral disaccharide-BODIPY from negatively charged uncleaved substrate using an anion exchange microcartridge, with the reaction product being registered by its fluorescence (Scheme 1). BODIPY FLSE (4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-propionic acid) was purchased from Molecular Probes (The Netherlands). All other chemicals used were obtained from Fluka (Switzerland). All chemicals used were of analytical grade. DEAE-