30 45 60
electrokinetically or hydrodynamically). When the potential is switched on, the components will mix and the reaction will be started. With few exceptions, the product and the substrate-enzyme complex will be transported at different velocities. Product formation will continue until the enzyme leaves the system. The theoretical elution profile will be as shown in Figure 8.9. Here it is assumed that the enzyme-substrate complex moves with a higher velocity than the product (Fig. 8.9.4). The first product detected at point A corresponds to the enzyme as it migrates past the detector. The spike at position B is that of the product formed during the short interval between introducing the enzyme into the system and switching on the potential. If the transport velocity of the product is higher than that of the enzyme-substrate complex, the electrophero-gram will be reversed, as shown in Figure 8.9B. At point A, we observe the artificial spike caused by the existence of the interval between introducing the enzyme and switching on the potential. From the position of the artificial peak on the electropherogram, it is possible to determine the relative migration velocities of the enzyme-substrate complex and the product. The level of the plateau indicated as C in Figure 8.9A,B is directly proportional to enzyme concentration at constant potential. Figure 8.9C depicts the situation that may occur with an isoenzyme mixture and a common product.
Switching the system to zero potential for a fixed period of time within the time window bracketing the passage of the enzyme by the detector would allow for product accumulation. When the power is switched on again, the enzyme will be separated from the product and, if the product has suitable detection properties, it will appear as a peak on the enzyme plateau. This approach is sometimes referred to as "parked reaction." A practical application featuring the activity assay of D-glucose-6-phosphate:NADPH oxidore-ductase is shown in Figure 8.10.
A less complex approach can be used for enzyme activity estimation; namely, fractions can be collected and enzymatic activity measured in these fractions. The practical realization involves a normal run before fraction collection to detect the migration time of the peak, which should be collected. The time Tc when the peak is going to leave the capillary can be estimated as retention time multiplied by the length of the capillary divided by the capillary length to the detector. When fractions are to be collected, the potential is switched off just before Tc. A new vial is placed at the end of the separation
Figure 8.7 Capillary gel electrophoresis profiles of collagen chains and chain polymers in 4% Polyacrylamide; 75 mm i.d. capillary, 45 cm long (35 cm to the detector). Tris-glycine buffer, pH 8.8, 10 kV. Detection by UV absorbance at 220 nm. (A) Complete sample. (B) a-Region proteins sampled from a preceding slab gel run. (C) As in (A), but 0-region excised from the slab gel. The appearance of the gel separation is shown at top (cathode on the right side of the gel). (From Deyl and MikSik, 1995, with permission.)
y- and higher polymers
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