I need a robust assay to determine enzymatic activity of my protein. The two small molecules are not UV-Vis unique. The two small chemical molecules are Naphthelene backbone and an isoprene tail. My enzyme puts the two together, but I can't find a robust assay for enzymatic analysis.
I'm sure those are not the substrates. What substrates (DMAPP?) or cofactors does your enzyme use? What are the products?
Naphtalene really doesn't absorb in UV? Or you mean the absorbance is not changed?
I faced similar problem when I was working with a cis-trans isomerase and the only option was HPLC. Luckily I could use runs short as 7 minutes in total.
HPLC is definitely a method you should use first. If you have clean conversion, you can always change to a phosphate detection assay (e.g. EnzChek as colorimetric, coupled assay) assuming that DMAPP is the co-substrate and pyrophosphate is released; PPi is easily converted to Pi using pyrophosphatase. The advantage of using this kind of assay is an easy and quick read-out. Disadvantages: any trace of phosphate in the buffer, from the enzyme preparation or arising from DMAPP hydrolysis will show up, so good controls, as always, are key. Also, if measuring kinetics: for any coupled assay you will need to be careful to ensure that you are measuring the rate of the reaction you're interested in rather than that of the coupled assay (depends on which reaction is rate-limiting); again, controls and method optimisation are required.
I would presume that reducing power is required as NADH or NADPH ? If so then you can use a spectrophotometric assay that looks at NADH/NADPH consumption as a function of one of the substrates. IF the enzyme uses ATP then you can use an ATP monitoring assay.
Alternatively, addition of the isoprene tail should alter the mobility of the fluorescent naphthalene in a simple TLC system - you can also use fluorescence to visualize the mobility shift.
HPLC and cofactor consumption sounds good, try that first!
If you don't have a HPLC available you can try one radio labeled substrate and resolve the reaction mix on a 60 cm high percentage polyacrylamide gel with 0.2mm thickness ( Everybody hates that, I know....)
This might be old-fashioned but did you consider TLC? Atleast as a first step. It is not as quantitative as HPLC (not easily anyway) but much cheaper if you don't have a lot of samples and you can process multiple samples on the same plate at one run.
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