Spectrophotometric assay for B-lactamases

benedik at uh.edu benedik at uh.edu
Wed Aug 16 14:24:39 EST 1995

In article <40pugt$pro at lantana.singnet.com.sg>
anomie at singnet.com.sg (Poon Wai Mun) writes:

> Hi ! I'm a student of Ngee Ann Polytechnic. My classmates and I are doing a project on
> characterisation of Beta- lactamases.
> We need a standard protocol for the spectrometric assay of b-lactamases
> DESPERATELY!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
> Any kind person who have the protocol, PLEASE reply this note.
> Please include the substrate to use, buffer,pH of buffer, amount of substrate and enzyme to add,
> the wavelength to scan (lamda maximun) etc.
> We shall be eternally grateful to you.
Here is the section from a thesis of a recent student of mine. You can
also get a colorimetric compound, nitrocefin, but it is pretty

 b-Lactamase activity was measured by adding a 10 µl sample (culture
supernatant or sonic extract) to 90 µl 50 mM potassium phosphate pH 6.6
with 0.75 mg/ml ampicillin. The decrease of absorbance at 240 nm was
monitored using the kinetics program on a Milton Roy Spectronic 3000

Michael Benedik               benedik at uh.edu
Biochemical Sciences
University of Houston
Houston, TX 77204-5934

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