Hello
I am working with recombinant β-lactamase enzymes (350-400 aa) with C-terminal his tag and the tag is not cleaved. I know nitrocefin is a chromogenic cephalosporin for β-lactamase activity assay. However, wondering if I could do kinetic analysis with nitrocefin. I performed a couple of test-run with 50 mM phosphate buffer and took absorbance at A490. Every run given me data which was abnormal (very high or very low). Anyone can help me, please?
Thanks in advance.
You should be able to do kinetic analysis if you have the instrumentation to do a time course. Just be sure you are taking time points early on so that substrate (Nitrocefin) does not become limiting or so that your absorbance reading is within the linear range of your spectrophotometer. I don't think the buffer should matter much so if you prefer Tris or something else it should be fine.
You don't have to use phosphate buffer, of course, but the buffer is probably not the reason you are having trouble, unless your enzyme is a metallo-beta-lactamase (MBL). Because MBLs require zinc for activity and zinc phosphate has low solubility, it would be better to use a different buffer, such as HEPES. Also, include zinc sulfate in the buffer, and experiment to find the optimal concentration. It may be only a few micromolar.
If your enzyme is a serine beta-lactamase, phosphate buffer is fine.
Make sure your enzyme concentration is consistent. If you are freezing and thawing the solution, make sure you mix it gently but thoroughly after thawing. When refreezing the solution, do it rapidly using dry ice-ethanol, dry ice-acetone, liquid nitrogen, or my favorite, powdered dry ice. Rapid freezing helps reduce separation of solutes as the water freezes. Small volume aliquots (100 µl or less) are preferred because they freeze and thaw quickly.
Titrate the enzyme concentration to get a suitable signal. Monitor the absorbance change continuously, as suggested by Michael J. Benedik
Nitrocefin has a fairly limited solubility in buffer. Try using 100 µM in your reaction. That generally gives a useful absorbance change before the reaction starts to slow down. Later you can titrate the nitrocefin concentration to get the Km.
Not all beta-lactamase enzymes use nitrocefin as a substrate, by the way. Hopefully yours does.
Hi,
We are usually using HEPES buffer and it works well for metallo and non-metallo beta lactamase kinetic studies.
Good luck,
P.
- Badges
- Science method