Ein Verfahren zum spezifischen Nachweis kleinster Glykolylmengen in Glykoproteinen

Exploration of the Sialic Acid World

“…807 With respect to the suggestion of a direct oxidation of the N-acetyl group in the case of Neu5Gc, first experiments with homogenates of porcine submandibular glands did not show the formation of N-glycolylglucosamine (GlcNGc) from the sialic acid precursor GlcNAc. 270,802 This pointed to a sialic acid modification at a higher biosynthetic step. Incubation of surviving slices of porcine submandibular glands with N-[1-14 C]acetyl-glucosamine led to labeling of sialic acids, including Neu5Gc having a radioactive glycolyl group (for analysis of glycolyl groups, see Section 7.1).…”

“…In fact, a possible presence of Neu5,9Ac 2 was excluded, because the combination of "1 mol periodate consumption and a positive Aminoff test" was not found. The number of N-glycolyl groups was estimated using a microanalysis protocol for glycolic acid, 270 and the number of O-acetyl groups was determined with the colorimetric Hestrin assay (alkaline hydroxylamine/Fe(ClO 4 ) 3 ; see Section 7.1). 271 Note that all bovine sialic acids could also be released with the sialidases of V. cholerae and C. perfringens, although at different rates, but equine Neu4,5Ac 2 and di-O-acetylated Neu5Ac (presumably one of the O-acetyl groups attached at C4) were resistant to these enzymes (see Section 11.5.2).…”

“…271,333 Incubation of N,O-acylneuraminic acids with alkaline hydroxylamine affords acylhydroxamates that form red chromophores with Fe(ClO 4 ) 3 , the absorbance of which are measured at 520 nm. 334 O-Glycolyl (but also N-glycolyl) groups can be quantified using a protocol that includes 64,270,[334][335][336] The reddish chromophore that is formed (20 min, 100°C), represents the condensation product of 1 mol of formaldehyde (oxidative decarboxylation of glycolic acid) and 2 mol of 2,7-dihydroxynaphtalene, and yields an absorption at λ max 546 nm. Finally, the configuration and the amount of O-lactyl groups in N,O-acylneuraminic acids can be measured as lactic acid with L-lactate dehydrogenase and D-lactate dehydrogenase after release with NaOH (1 h, room temperature) and neutralization.…”

“…801 Elucidation of the biosynthesis of Neu5Gc was a tedious procedure; first attempts go back to 1967. 270,802 Theoretically, the origin of the N-glycolyl group is a glycolic acid residue, e.g., from coenzyme A, or it is generated by a direct oxidation of the N-acetyl group of the sialic acid precursors GlcNAc and ManNAc, or Neu5Ac itself. Evidence for the formation of glycolyl-CoA from hydroxypyruvate was demonstrated in porcine liver.…”

“…H 2 SO 4 . 64,270,[334][335][336] The reddish chromophore that is formed (20 min, 100°C), represents the condensation product of 1 mol of formaldehyde (oxidative decarboxylation of glycolic acid) and 2 mol of 2,7-dihydroxynaphtalene, and yields an absorption at λ max 546 nm. Finally, the configuration and the amount of O-lactyl groups in N,O-acylneuraminic acids can be measured as lactic acid with L-lactate dehydrogenase and D-lactate dehydrogenase after release with NaOH (1 h, room temperature) and neutralization.…”