Galactose oxidase action on galactose containing glycolipids - a fluorescence method

A1 Journal article (refereed)


Internal Authors/Editors


Publication Details

List of Authors: Fortelius M, Mattjus P
Publication year: 2006
Journal: Chemistry and Physics of Lipids
Journal acronym: CHEM PHYS LIPIDS
Volume number: 142
Issue number: 1-2
Start page: 103
End page: 110
Number of pages: 8
ISSN: 0009-3084
eISSN: 1873-2941


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.


Keywords

Amplex Red, DGDG, enzyme, galactosylceramide, membrane, MGDG, phosphatidylcholine, sphingomyelin, vesicle

Last updated on 2019-19-10 at 03:17