Myelin with galactose oxidase, oxidation

Treatment with galactose oxidase. Oxidation of myelin with galactose oxidase was performed as described previously for similar oxidation of rat spinal cord preparations (4). Typically, myelin containing 0.2-1.1 mg protein is incubated with 100-500 units of galactose oxidase in 1-3 ml of phosphate buffer (10-100 mM, pH 7.2-7.4) with or without catalase. After the incubation at room temperature to 30°C for the duration of 30 min to overnight, myelin is recovered by centrifugation, washed, and lyophilized. Total lipids were extracted from the dried residue and the oxidized cerebroside as well as unaltered cerebrosides were analyzed as described above. Alternatively, the incubation was stopped by the addition of 5 volumes of chloroform/methanol (2/1, v/v) and mixed. The lower layer after centrifugation of the mixture is washed and then evaporated to dryness, and the total lipids obtained were analyzed as described above. 

We obtained unexpected findings using this method to study myelin the oxidation by galactose oxidase of myelin-bound cerebrosides could not be detected. The oxidation did not occur either with the intact spinal cord preparation, with isolated myelin, or even with lyophilized myelin. In one experiment, lyophilized myelin containing 5 mg each of dry weight was incubated with 100, 200, and 500 units of galactose oxidase for 60 min at room temperature, and no cerebroside oxidation occurred. 

One possible explanation for the lack of oxidation by galactose oxidase was thought to be steric hindrance. To investigate this possibility, lyophilized myelin containing 5.45 mg protein in 5 ml 0.1 M phosphate buffer, pH 7.4, was mixed with 0.5 ml of the same buffer solution containing 1400 units of trypsin and incubated for 1 h at 37°C. 5 mg Of trypsin inhibitor in 1 ml of... 

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