Time control, enzyme analyzers

The simplest method of coupling enzymatic reactions to electrochemical detection is to monitor an off-line reaction using FIAEC or LCEC. The enzymatic reaction is carried out in a test tube under controlled conditions with aliquots being taken at timed intervals. These aliquots are then analyzed for the electroactive product and the enzyme activity in the sample calculated from the generated kinetic information. 

In order to use the stopped-flow technique, the reaction under study must have a convenient absorbance or fluorescence that can be measured spectrophotometri-cally. Another method, called rapid quench or quench-flow, operates for enzymatic systems having no component (reactant or product) that can be spectrally monitored in real time. The quench-flow is a very finely tuned, computer-controlled machine that is designed to mix enzyme and reactants very rapidly to start the enzymatic reaction, and then quench it after a defined time. The time course of the reaction can then be analyzed by electrophoretic methods. The reaction time currently ranges from about 5 ms to several seconds. 

Three time points were used to determine linear reaction velocities, and control reactions contained enzyme that had been boiled for 10 minutes. Five milliliters of y-Glu-Gly was added as internal standard. Next, 125 /xL of ice-cold 2.0 M KOH and 2.4 M KHCO3 was added. When gas evolution slowed, 200 /xL of freshly prepared 1.5% (v/v) solution of l-fluoro-2,4-dinitrobenzene in ethanol was added. The derivatization reaction was allowed to proceed in the dark for at least 8 hours at 22°C. The mixtures were filtered before 5 to 50 /xL was analyzed by HPLG... 

Fig. 1. Time dependency of ADP-ribosylation of actin by C2 toxin in rot basophilic leukemia (RBL) cells. Rbl cells were incubated with C2 holotoxin (lOOng/ml C2I + 200ng/ml C2II) for the times indicated. Controls were incubation with the enzyme component C2I (lOOng/ml) alone, or with the binding component C2II (200ng/ml) alone, for 120 min. Another control (-) was not treated with toxin. After incubation, the medium was removed and cells were scraped off in the presence of lysis buffer (2 mM MgCb, 0.1 mM phenylmethylsulfonyl fluoride, 10 ig/ml leupeptin, 25 mM triethanolamine, pH 7.5), sonicated five times for 5sec on ice and centrifuged for 10 min at 1000 g. The supernatants were incubated with 1 [ig/ml C2I and 5nM [ P]NAD for 15 min at 30°C. The proteins were separated by 12.5% SDS-polyacrylamide gel electrophoresis and analyzed by phosphorimaging...

The application of quality control procedures to ensure that satisfactory analytical performance of enzyme assays is maintained on a day-to-day basis is complicated by the tendency of enzyme preparations to undergo denaturation with loss of activity. This maltes it difficult to distinguish between poor analytical performance and denaturation as possible causes of a low result obtained for a control sample introduced into a batch of analyses. Assured stability within a defined usable time span is therefore the prime requirement for enzyme control materials, as it is for enzyme calibrators. However, specifications for the two types of materials can differ in other respects. Because the function of a calibrator is to provide a stated activity under defined assay conditions, it is not necessary for it to show sensitivity to changes in the assay system identical to those of the samples under test therefore within certain Umits, enzymes from various sources can be considered in the search for stability. However, it is the function of a control to reveal small variations in reaction conditions, so it must mimic the samples being analyzed. The preparation of enzymes from human sources is not by itself a guarantee of an effective control. For example, human placental ALP is very stable, but it differs significantly in kinetic properties from the liver and bone enzymes that contribute most of the ALP activity of human serum samples it is therefore not an ideal enzyme for use in control material for the determination of ALP. 

In most amperometric cytochrome b2 electrodes the reaction is followed by anodic oxidation of ferrocyanide at a potential of +0.25 V or above. The first of such sensors was assembled by Williams et al. (1970), who immobilized the enzyme (from baker s yeast) physically at the tip of a platinum electrode within a nylon net of 0.15 mm thickness. The large layer thickness resulted in a response time of 3-10 min. Owing to the low specific enzyme activity used, the sensor was kinetically controlled. Therefore the linear measuring range extended only up to 0.1 Km-A similar sensor has been applied by Durliat et al. (1979) to continuous lactate analysis. The enzyme was contained in a reaction chamber of 1 pi volume in front of the electrode. This principle has also been employed in the first commercial lactate analyzer using an enzyme electrode (Roche LA 640, see Section 5.2.3.3X With a sensor stability of 30 days and a C V below 5%, 20-30 samples/h can be processed with this device. 

The publications in this field indicate clearly improved performance of the physical side of the analysis, that is, shorter analysis times, better separation efficiencies and a dramatically reduced consumption of reagents. Furthermore, due to the minute volumes for internal connections, new types of combinations can be used, and small samples can be analyzed with success. TTie use of parallel [57, 70, 71] or multi-dimensional arrangements [72] would lead to even larger munbers of analyses per unit time, or to dramatically increased peak capacities (separation of > 1,000 components [73]). The trend to combine biological assays with separation methods, that is, protein protein interactions, enzymes or antibodies with CE [74, 75, 76], could lead to novel concepts for chemical sensing. Optically defined sample plugs allow for precise small volume injections for millisecond separations [55], which can be used for on-line or in-vivo monitoring experiments [77]. Novel approaches to control the flow, e.g., radial control of electroosmosis in capillaries [78] or inductive mechanical micro piunps [79, 80], will allow access to novel cyclic separation techniques. 

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