Application of stopped-flow analysis with respect to dilution

The tubing configuration initially considered for the design of the stopped-flow analyser is shown in Figure 4.4 (p. 145). The objective is for the complete mixture to traverse the flow-through cuvette rather than further withdrawing a stream for analysis. The sample of the precipitated feed or supernatant is controlled by independent on-off valves. Diluent followed by assay mixture are pumped into the resultant stream, mixing coils positioned after the tees. An additional diluent pump is incorporated for runs of higher range activity. The diluent and clean fluid from the microcentrifiige are used in the wash cycle. A microcomputer associated with the unit will actuate the pumps and valves, and make readings of the optical density at 0.4 s intervals.

The peristaltic pumps and solenoid valves discussed in section 4.2 are selected because their application and installation have been proven for the microcentrifiige unit. By altering individual volumetric flow rates including that of the supernatant, the particular ratios are obtained. They must be repeatable but absolute accuracy is not as important if the proportions of sample and assay mixture to the total volume are the same for both precipitated feed suspension and supernatant. A maximum constraint of 50 ml/min is imposed for every pump to limit the overall pressure drop and conserve the materials consumption. For instance a combined flow rate of 150 ml/min should pass through a 1 mm i.d. 100 fil cuvette inlet at 3.2 m/s, equivalent to 25 volume changes /s. The minimum tube bore available and unlikely to block frequently is 0.5 mm with volumetric flow rates of 0.3, 1.5, 3.0 and 5.1 ml/min at 10, 50, 100 and 170 rpm respectively. A compromise is made between the largest possible variation of throughput ratios at low pump speed against the fastest flow rates, 50 rpm being chosen.

Dilution of the sample is vital to ensure the optical density is sufficiently in range for measurement. Phosphate buffer is added prior to the assay mixture and not with it as resolubilisation is desired to occur initially. The optical system deviation

from linearity may be assumed to become significant after an optical density of 1.0, where the dilution of clarified yeast homogenate in phosphate buffer was determined off-line to be 56.7 times. Hence the sample dilution must be greater unless there is calibration as the first reading would be in the non linear region on mixing, before any reaction and the plug has been pumped to the flow-through cuvette.

The maximum dilution is 67.7 times for the tubing configuration investigated, having a minimum sample volumetric flow rate and maximum diluent and assay mixture volumetric flow rates. This is extended to 101.0 times by using the extra pump. The predicted initial optical densities are thus 0.835 and 0.557. In contrast it would be 0.018 for the conventional ADH assay (section 2.1.2.3). It appears that if the optical density increase of 0 .0 2 /s is scaled for the decrease in dilution the linear measurement region is exceeded before several successive readings are made. Evaluation from the minimum time interval is not preferred; subsequent readings would be available as well. In practice ADH assays at such high concentrations were inconsistent with the rate of reaction fluctuating at lower values than expected. This may be the result of product inhibition, the sample presence not being negligible compared to the assay reagents or the mixing being determining which will be discussed.

Therefore to imitate the conventional assay it is necessary to add a small quantity of a prediluted sample stream to the assay mixture stream the remainder going to waste, utilising four pumps and two valves as previously. In each case the minimum volumetric flow rate is mixed with the maximum. This achieves a substantial dilution. The assay may be mimicked more closely by relaxing the upper limit on the tube bore, for example the diluent to 4.0 mm (80 ml/min) and the assay mixture to 4.5 mm (98 ml/min). It has been shown that for a high throughput in narrow internal diameter tubing the flow rate is rapid and mixing coils should not be required.

The effect of the reagent concentration for a constant overall sample fraction is considered to see whether it may be manipulated to adjust the range of a run. The

reaction rate initially increases with reagent concentration according to the Michaelis- Menten equation.

[5]

^ ^max [ g ] + [ 4 . 5 ]

where V is the rate of catalysis, the maximum, [S] is the substrate concentration and Km is the Michaelis constant. It is based on a model in which a discrete enzyme- substrate complex is an intermediate in the catalysis. The substrate binds to a specific region of the enzyme termed the active site. Where the substrate concentration is low it becomes negligible compared to the Michaelis constant and thus is proportional to the reaction rate from equation 4.5. At high concentration it is much greater than the Michaelis constant and the reaction rate equals the maximum. However the reaction rate then decreases with reagent concentration, the overall profile appearing to be bell-shaped. The reduction may be caused by reversion of the product to its original form or inhibition of the active sites by the substrate. For instance the rate of reaction was observed to be 0 following a five-fold increase, the ethanol concentration being 17.5% by volume. Limits may also be set by diffusion such as the frequency of encounter between the enzyme and substrate. The reaction rate therefore may not be greatly increased by adjusting the reagent concentration.

The optical system of the stopped-flow analyser is required to have minimal measurement error and an excellent sensitivity for the on-line monitoring of optical density. This involves focusing high intensity light at 340 nm on the cuvette with a maximum detection range by the photodiode. The reaction rate measured is a function of the pathlength, see equation 2.2. It may be varied such as by substimting for a flow-through cuvette of 1 mm pathlength. An optical system fitted with a beam splitter (or a second complete optical assembly) is another possibility, though was not incorporated. The redissolved sample would be analysed in the cell having the lower pathlength, the supernatant sample in the cell having the higher value. Consequently the magnitude between the optical densities is reduced.