Galactose oxidase reduction method

Normal human fibroblasts grown in media with 12% fetal calf serum were treated with or without galactose oxidase and then subjected to reduction with potassium borotritiide. GSLg were isolated and subjected to acid-catalyzed methanolysis (62) (see Materials and Methods). Methyl glycosides, methyl sphingosine, and fatty acid methyl esters were isolated by extraction of the total hydrolysate with solvents as described previously (62). Radioactivity was measured in triplicate in aliquots of these extracts. 

Fibroblasts grown in media with 12% fetal calf serum were treated with galactose oxidase, followed by reduction with potassium borotritiide. The individual GSLs were isolated and the radioactivity was measured in triplicate (see Materials and Methods). 

Galactose is usually estimated quantitatively by the difference in the glucose oxidase determination and the total reducing substance as determined either by the copper reduction method (Nl) or the anthrone reaction (Al). 

After on-line dialysis of the milk sample, lactose is oxidized in the galactose oxidase reactor with release of hydrogen peroxide. The latter is detected by amperometric reduction of a mediator, oxidized by hydrogen peroxide in a peroxidase catalyzed reaction. The linear range of the method with 20 injection is in the range 0.05-300 mM. 

Albumins. — [ HjRaffinose (76) has been treated with D-galactose oxidase and then coupled to albumin by reductive amination (Scheme 15). The reaction provides a convenient method of labelling albumin for studies of the organ sites of catabolism of proteins in the circulation. 

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