Fortelius Michaela, Mattjus Peter
Department of Biochemistry and Pharmacy, Abo Akademi University, Artillerigatan 6A, FI-20520 Abo/Turku, Finland.
Chem Phys Lipids. 2006 Jul;142(1-2):103-10. doi: 10.1016/j.chemphyslip.2006.03.007. Epub 2006 Mar 30.
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.
利用半乳糖氧化酶荧光技术，在双层脂质模型囊泡中研究了改变膜界面糖脂糖头基团可及性的特征。研究了半乳糖神经酰胺疏水部分的变化或半乳糖神经酰胺、单半乳糖二酰基甘油和二半乳糖二酰基甘油的膜环境对氧化的影响。在本研究中，我们结合了用于测定含半乳糖糖脂氧化能力的半乳糖氧化酶方法和用于测定酶促过氧化氢产生的荧光方法。糖脂头基团中6位带有游离羟甲基的暴露半乳糖残基用半乳糖氧化酶氧化，随后通过辣根过氧化物酶和10-乙酰基-3,7-二羟基吩恶嗪（Amplex Red）的组合测定产生的过氧化氢。Amplex Red在辣根过氧化物酶存在下与过氧化氢以1:1的化学计量比反应形成试卤灵。通过这种偶联酶方法，还可以确定双层囊泡中半乳糖脂的跨膜分布（内外）。
