Sulfuric acid magnesium salt (1:1) → N-Acetyl-D-Mannosamine

Participants

Reaction Steps

To 1 ml of 0.1M tris-hydrochloric acid buffer solution of pH 7.5 were added 1 μmol of uridine diphosphate-N-acetylglucosamine and 40 μmol of magnesium sulfate. Incubation was conducted at 37° C. Thereto was added 0.2 ml (protein amount: 2 mg) of a crude enzyme liquid obtained from extracts of a living liver (Biochem. Biophys. Acta Vol. 252, 1971, p. 357). Reaction was conducted for 30 min to form N-acetylmannosamine. The reaction mixture was heated for 2 minutes at 100° C. Thereafter, the mixture was freed from the precipitate, then passed through a column packed with 2 ml of an ion exchange resin of Dowex-1 acetic acid type, and washed with water. The liquid obtained was adjusted to 5 ml in total. Of this volume, 2.5 ml was placed in an oxygen monitor which is an oxygen determination vessel. Thereto was added 0.5 ml of 0.2M tris-hydrochloric acid buffer solution containing 0.2 mole of glycine. The mixture was kept at 37° C. for 10 min. Then, an oxygen electrode was inserted into the vessel and the vessel was sealed. With stirring the contents at 37° C., 9 unit (50 μl) of N-acetylhexosamine oxidase was added. The amount of oxygen consumed was determined in the same manner as in Examples 1 and 4. As a result, an oxygen absorption of 8.0 (reading on recording sheet) was detected. By using a calibration line obtained by subjecting standard N-acetylmannosamine solutions to the same reaction procedure as above, the amount of N-acetylmannosamine present in the sample and formed by uridine diphosphate-N-acetylglucosamine-2'-epimerase was found to be 0.16 μmol. ##STR5##

Provenance & Evidence