Enzyme Reaction
Mannose-1-phopsphate + GTP (substrates)
Mannose-1-phosphate Guanylyltransferase (enzyme)
GDP-mannose + PPi (Products)
To prove this enzyme assay by using HPLC, I used GTP as standard. The conditions at which i got GTP peak was,
Mobile Phase----------------> Kh2PO4
Stationary Phase ----------> C18 column
UV detector wavelength------> 259nm.
But problem is that i could not detect GDP-mannose (product) peak by using above mentioned condition. To publish enzyme activity results, there should be two peaks. One is the decrease peak of substrate (GTP) and second is appearance of product (GDP-mannose) peak.
So can someone could suggest me such conditions by which i would be able to detect both GTP (substrate) and GDP-mannose (Product) at same time??
Thank you
Hi there,
Obviously Reverse Phase -HPLC is not the most described approach for measuring this activity. It is rather anion exchange HPLC or Ion pair RP-HPLC which are used. Article One pot synthesis of GDP-mannose by a multi-enzyme cascade f...
https://academic.oup.com/glycob/article/20/7/865/1987880
Hi, the first answer was right. You can use all the material you have, making an ion-pair chromatography. Eluent: methanol–water (20:80, v/v) containing 75 mM KH2PO4, 100 mM NH4Cl and 1 mM 1-octylamine (pH 4.55) at a flow rate of 1.0 ml/min on C18 column (4.6 × 250 mm). I did this myself for the analysis of nucleotides and it worked well, however, the half-life of the column was somewhat shortened.
Paper: "An improved HPLC method for the quantitation of 3-phosphoadenosine 5-phosphate (PAP) to assay sulfotransferase enzyme activity in HepG2 cells"
Good luck, Bernhard
A long time ago, I used an ion-pairing RP-HPLC method to separate nucleotides. I don't know whether it will separate the ones you are interested in, but it probably will. Here is the reference:
http://www.jbc.org/content/265/8/4340.full.pdf
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes