反応 #1178400

反応方程式

UDP-GalNAc

UDP-GalNAc

UDP-GlcA

→

GalNAc

溶媒

water

反応条件

後処理

1. 1
   workup.ADDITIONadding the K4CP enzyme
2. 2
   workup.WAITthe reaction was performed again at 30° C. for 5 hours
3. 3
   workup.WAITThermal inactivation was performed for 1 minute
4. 4
   その他the reaction mixture was subjected to ethanol precipitation
5. 5
   workup.DISSOLUTIONthe residue was dissolved again in 200 μl of distilled water
6. 6
   ろ過The solution was subjected to gel filtration chromatography in a Superdex Peptide HR10/30 column
7. 7
   その他were collected every 1 minute (1 ml)
8. 8
   workup.DISSOLUTIONeach of the lyophilized fractions was dissolved again in 10 μl of distilled water
9. 9
   その他to obtain a sample for MALDI-TOF-MS

実験手順

A reaction was performed in the same manner as that of (1) mentioned above under conditions of 30° C. and pH 7.2 for 8 hours by using CH6, UDP-GalNAc and UDP-GlcA as substrates and adding the K4CP enzyme. Then, 300 nmole of UDP-GalNAc was added again, and the reaction was performed again at 30° C. for 5 hours. Thermal inactivation was performed for 1 minute in boiling water, the reaction mixture was subjected to ethanol precipitation, and the residue was dissolved again in 200 μl of distilled water. The solution was subjected to gel filtration chromatography in a Superdex Peptide HR10/30 column using 0.2 M ammonium acetate as a developing buffer. The mixture was loaded at a flow rate of 1 ml/minute, and fractions were collected every 1 minute (1 ml). The obtained fractions were lyophilized, and each of the lyophilized fractions was dissolved again in 10 μl of distilled water. The solution was loaded on a small amount of Dowex 50 XW8 (H+ form) gel to obtain a sample for MALDI-TOF-MS.

出典