Initial Rate Determination at Low Substrate Concentrations

As in the case of any assay procedure, determination of the rate of product formation becomes difficult at the lower limits of substrate concentration. However, changes that can be made both in the assay system and in the chromatographic equipment can alleviate this problem.

The first change is, of course, to increase the sensitivity of the detector. Most HPLC detectors contain range switches that make this a simple matter. When range switching is carried out, it is useful to determine whether calibration curves constructed at one range setting are still valid at another.

Next, the amount of product being detected may be increased by increasing the volume of the reaction mixture that is injected for analysis. With fixed-loop injectors this requires changing the loop, a rather simple procedure. If the loop is changed, the additional volume must be entered when the total volume of the incubation mixture is calculated.

There is an upper limit to the volume of an injection, usually around 200 fiL. The injection of larger volumes results in spreading of the peak, a phenomenon that decreases resolution. However, it is possible to make loops of any volume and thus control this upper volume limit. If separation is adequate and resolution is not a problem, the loop volume may be increased.

At times, obtaining enough reaction product requires the concentration of an entire reaction mixture. Following concentration, the residue is resuspended in a small volume of buffer and analyzed.

Finally, it is always possible to increase sensitivity by using as substrates analogs, such as radiochemicals, whereupon the amount of radioactive product that has formed is determined. For experiments of this type, the eluent may be carried through a radiochemical detector; in the absence of such an instrument, fractions may be collected and their radioactive content determined.

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