So why bother using a separation method while assaying enzymes? Why not just stick the enzyme in with a fluorogenic substrate (= a "non-fluorescent" substrate that is converted into a highly fluorescent product) and measure the increase in signal with a fluorimeter?
2 reasons
1. Improved sensitivity
2. Improved selectivity
1. I have yet to see a "non-fluorescent" substrate that produces no fluorescent signal. If your detector is sensitive enough you will detect signal. When you place a cuvette filled with a suitable buffer into a fluorimeter you will get a background signal. If you include a substrate this background will be higher. Remember, backgound noise increases with the square root of the background signal. Since the presence of substrate increases the background signal it will also increase the noise (and no, zeroing the instrument will not fix this! When you zero an instrument you arbitrarily tell it what to call zero, you do not alter the noise). Limit of detection is proportional to noise. The higher the noise the higher the limit of detection (remember - you want a low limit of detection - ie be able to see a small amount). Therefore, as soon as you add the substrate your sensitivity will suffer. The trick is to detect the product in the absence of the substrate. If you use a separation method you separate the substrate and product prior to detecting them. This allows you to detect product in the absence of the substrate signal.
So what kind of detection limits can one pull off using capillary electrophoresis laser-induced fluorescence detection? We did an alkaline phosphatase assay. Mixed enzyme and substrate, let them incubate and analyzed the reaction mixture. We got a detection limit of 1.5x10-17 M. When one considers that our injection volume was 2.4x10-9 liters one realizes that this corresponds to a mass of 0.02 molecules of enzyme. Of course, nobody usually prepares nanoliter volume samples. In practical terms our mass detection limit is 9 molecules per microliter, with a microliter being the smallest sample size one would normally use. And yes, the instrument does work with 1 microliter volumes.
2. Suppose you have a substrate that can be acted upon by your enzyme to produce two different products, or there is another enzyme present as a contaminant that can act upon you substrate to form a second product. If you use a separation method you can separate out all the products and quantitate them individually.
What's the deal with single enzyme molecule assays?