Enzyme assay of CMP-Neu5Ac hydroxylase

Authors：

Category

Glycosyltransferases & related proteins

Protocol Name

Authors

Takematsu, Hiromu
Laboratory of Biochemistry, Human Health Sciences, Graduate School of Medicine, Kyoto University

KeyWords

Reagents

●	CMP-Neu5Ac

Instruments

●	HPLC system

Methods

Reaction

1)	40 μL of Sample or the sample buffer (10 mM Tris-HC1 buffer, pH 7.5 containing 0.1 mM DTT).	Comment 1

2)	Start the reaction by adding 10 μL of the reaction mixture (10 mM Tris-HC1 buffer, pH 7.5 containing 34 nmol of NADH (or NADPH), 2 nmol of CMP-Neu5Ac and 5 mM DTT) and incubating at 37°C.	Comment 0

3)	Stop the reaction by mixing with 0.3 mL of ice-cold ethanol.	Comment 0

4)	Incubate the reaction tubes on ice for 15 min.	Comment 0

Centrifuge the reaction tubes at 12,000 × g for 5 min in a refrigerated centrifuge.

Comment 0

Analysis

1)	Analyze aliquots of the supernatant directly by HPLC on a reversed-phase column (4.6 mm i.d. × 250 mm, TSK-gel ODS-80TM; Tosoh Corp., Tokyo, Japan) using the elution buffer (50 mM NH₄H₂PO₄) at the flow rate of 0.5 mL/min.	Comment 0

2)	Monitor CMP-Neu5Ac and CMP-Neu5Gc at 271 nm (Fig. 1)	Comment 0

Calculate the enzyme activity.

Comment 1

Figure & Legends

Fig. 1. A typical example of the elution profile of the reaction product.

Copyrights

Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses

Date of registration：2014-11-26 15:52:02