MONITORING BIOSYNTHETIC TRANSFORMATIONS OF N-ACETYLLACTOSAMINE USING FLUORESCENTLY LABELED OLIGOSACCHARIDES AND CAPILLARY ELECTROPHORETIC SEPARATION

Capillary electrophoresis with laser-induced fluorescence detection wa s used to monitor biosynthetic transformations of a labeled derivative of N-acetyllactosamine (beta Gal(1 --> 4)beta GlcNAc, LacNAc) in crud e microsomal extracts, For this purpose, six authentic standards inclu ding LacNAc-O-TMR itself (-TMR, a linker arm attached to tetramethylrh odamine), the tri-and tetrasaccharides that would form by either alpha (1 --> 2) or alpha(1 --> 3) fucosylation of LacNAc or both (i.e,, H-ty pe 2, LeX and LeY sequences) were chemically synthesized. The potentia l degradation products produced by galactosidase and hexosaminidase we re also prepared, Ah of the standards were kinetically competent subst rates for the enzymes present in HT-29 cells and could be baseline sep arated in a single run requiring 11 min, The action of competing enzym es acting on the common LacNAc sequence could thus be monitored in a s ingle run with sensitivity routinely as low as a few thousand molecule s. (C) 1995 Academic Press, Inc.