反応 #1178398

反応方程式

NaCl

UDP-GalNAc

→

GalNAc

溶媒

water

反応条件

後処理

1. 1
   その他obtained by chemical desulfation of CS
2. 2
   その他a K4CP enzyme solution (recombinant enzyme obtained
3. 3
   workup.ADDITIONcorresponding to 37.5 μg protein) was added
4. 4
   workup.WAITthermal inactivation was performed for 1 minute
5. 5
   その他the reaction mixture was subjected to ethanol precipitation
6. 6
   workup.DISSOLUTIONthe residue was dissolved again in 200 μl of distilled water
7. 7
   ろ過The solution was subjected to gel filtration chromatography in a Superdex 75 HR10/30 column
8. 8
   その他were collected every 1 minute (1 ml)
9. 9
   workup.DISSOLUTIONeach of the lyophilized fractions was dissolved again in 10 μl of distilled water
10. 10
    その他From a part of the solution, 10 μl of a 1 nmole/μl solution was prepared
11. 11
    その他to prepare a sample for MALDI-TOF-MS

実験手順

To an enzymatic reaction mixture (500 μl) containing CH obtained by chemical desulfation of CS and having different non-reducing ends (1 mg, average molecular weight: 10,000, Seikagaku Corporation), UDP-GalNAc (3 μmol) 50 mM Tris-HCl buffer, pH 7.2, 20 mM MnCl2 and 0.15 M NaCl, a K4CP enzyme solution (recombinant enzyme obtained according to the procedure of the example of Japanese Patent Laid-open Publication (KOKAI) No. 2003-199583; corresponding to 37.5 μg protein) was added. A reaction was performed at 30° C. for 18 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 75 HR10/30 column using 0.2 M ammonium acetate as a developing buffer. The solution 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. From a part of the solution, 10 μl of a 1 nmole/μl solution was prepared, and the solution was loaded on a small amount of Dowex 50 XW8 (H+ form) gel to prepare a sample for MALDI-TOF-MS.

出典