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Spectrophotometric methods

Spectrophotometric methods, and particularly enzyme-coupled assays, are the classic way to measure intermediates of metabolism. They are reliable methods. Usually they are as specific as the coupling enzyme - often very specific. In contrast to HPLC-based methods, chiral specificity usually comes as standard, rather than as an extra achieved at great expense (special column materials). The down side is that each assay will usually measure at most two or three metabolites; this is hardly a method suitable for broad-range profiling or metabolomics.

Nevertheless, recently the concept of the cycling assay has been resurrected. These assays use the metabolite of interest as a catalyst rather than something to be consumed. As a result they are phenomenally sensitive - unbelievably so. They are technically challenging to set up, and the controls must be planned carefully. For further information consult these references:

Gibon Y, Vigeolas H, Tiessen A, Geigenberger P, Stitt M (2002) Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system. PLANT JOURNAL 30: 221-235
Lowry OH, Passonneau JV (1972) A flexible system of enzymatic analysis. Academic Press, New York

Assay methods

Although as a metabolite service we don't intend to make much use of spectrophotometric methods, we have some expertise and are in contact with people with much more expertise. If you are a JIC group with no experience in coupled enzyme assays, but a need to use them, we will be happy to help.

If you are disappointed with this page and would like a full explanation of how these work, or if it would help you to have more examples of real assays here, I may be interested in expanding this page - but only if there is sufficient demand (within JIC or from outside). Please contact us.

Freeby software for spectrophotometry

I have software for Shimadzu spectrophotometers (specifically the UV2401PC, but the file formats apply to other models too) to streamline metabolite and enzyme assays. Most modern specs. now come with good software, but few manufacturers understand the need to measure baselines before adding the substrate in an enzyme assay, or the occasional need to allow for a sloping baseline in a metabolite assay. If you need an automated version of the old-fashioned ruler and chart-recorder paper, this is it.

Download "lkn3x6" now

The programme is written for DOS, but don't worry, it uses a mouse and has nice graphics. You should unzip the downloaded file to any convenient directory, and make yourself an icon for the lkn3x6.exe file. If you have any problems or comments, please contact us.