Chemistry and Physics of Lipids 142 (2006) 103–110 Galactose oxidase action on galactose containing glycolipids—a fluorescence method Michaela Fortelius, Peter Mattjus ∗ Department of Biochemistry and Pharmacy, ˚ Abo Akademi University, Artillerigatan 6A, FI-20520 ˚ Abo/Turku, Finland Received 6 February 2006; received in revised form 15 March 2006; accepted 15 March 2006 Available online 30 March 2006 Abstract Features that alter the glycolipid sugar headgroup accessibility at the membrane interface have been studied in bilayer lipid model vesicles using a fluorescence technique with the enzyme galactose oxidase. The effects on oxidation caused by variation in the hydrophobic moiety of galactosylceramide or the membrane environment for galactosylceramide, monogalactosyldiacylglycerol and digalactosyldiacylglycerol were studied. For this study we combined the galactose oxidase method for determining the oxidizability of galactose containing glycolipids, and the fluorescence method for determining enzymatic hydrogen peroxide production. Exposed galactose residues with a free hydroxymethyl group at position 6 in the headgroup of glycolipids were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 10-acetyl-3,7- dihydroxyphenoxazine (Amplex Red). Amplex Red reacts with hydrogen peroxide in the presence of horseradish peroxidase with a 1:1 stoichiometry to form resorufin. With this coupled enzyme approach it is also possible to determine the galactolipid transbilayer membrane distribution (inside–outside) in bilayer vesicles. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: MGDG; DGDG; Vesicle; Membrane; Galactosylceramide; Sphingomyelin; Phosphatidylcholine; Enzyme; Amplex Red 1. Introduction Galactose oxidase (d-galactose: oxygen 6-oxidore- ductase, E.C. 1.1.3.9) specifically oxidizes the C-6 hydroxymethyl group of free galactose as well as galactosyl derivatives such as N-acetylgalactosamine and glycolipids carrying a galactose residue in the terminal position (Avigad et al., 1961; Masserini et al., 1982; Suzuki and Suzuki, 1972). The enzymatic Abbreviations: DPPC, dipalmitoyl-phosphatidylcholine; HRP, horseradish peroxidase; PGalCer, palmitoyl-galactosylceramide; POPC, palmitoyl-oleoyl-phosphatidylcholine; PSM, palmitoyl-sphingomyelin; SGalCer, stearoyl-galactosylceramide ∗ Corresponding author. Tel.: +358 2 2154745; fax: +358 2 2154271. E-mail address: Peter.Mattjus@abo.fi (P. Mattjus). reaction has been employed previously successfully for the preparation of radioactive tracers of glycopro- teins or glycolipids by labeling of galactosyl and/or N-acetylgalactosaminyl residues (Agranoff et al., 1962; Blumenfeld et al., 1963; Lampio et al., 1988; Lingwood, 1979; Rosen et al., 1964; Suzuki and Suzuki, 1972). The interaction of galactose oxidase with glycol- ipid substrates has been shown to be dependent on the intrinsic characteristics of the membrane matrix embed- ding the glycolipid (Masserini et al., 1982). Stewart and Boggs (1993) found that there is no difference in the ability of galactose oxidase to oxidize either PGalCer or SGalCer as pure entities in a tetrahydrofuran/water mixture. Detergents (Triton X-100 and bile acids) and phospholipid vesicles do not alter the kinetics of galac- tose oxidase action on pure galactose, shown previously 0009-3084/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2006.03.007