Fig. 3.

Concentration of activity and ESI-MS analysis. a. Concentration of activity of RGXT1, RGXT2 & RGXT3 by applying i) simple dialysis of the media fractions or ii) liquid chromatography on a Phenyl Sepharose CL4 column (binding of enzyme in 50% saturated (NH₄)₂SO₄ to phenyl Sepharose columns followed by elution of bound protein and dialysis of eluate). Total activities are given relative to the total activity in one volume of crude media (100%), i.e. the phenyl Sepharose concentrated activities have to be multiplied with a factor of 4 for direct comparison on a μl to μl basis. Electrospray ionization mass spectrometry (ESI-MS) of xylosyltransferase assays containing dialysed F-sRGXT2 and 0.5 M L-fucose as acceptor b and dialysed sRGXT1-F and 0.005 M Me α-L-fucoside as acceptor c. The methyl α-D-xylosyl-α-L-fucoside disaccharide product of m/z 333 ([M+Na]⁺), the α-D-xylosyl-α-L-fucose disaccharide product of m/z 319 ([M+Na]⁺) and sodium acceptor adducts are indicated on the chromatograms