Discontinuous assay method

The need to use a discontinuous assay method does not automatically mean that the HPLC method is the procedure of choice. For HPLC to be suitable, it must be possible to separate the components, and some method for detection and quantitation must be available. Next, neither the ingredients in the reaction mixture nor those used to terminate the reaction should produce problems for the separation and detection. Finally, the enzyme itself should be considered. 

The discontinuous method measures activity by separating the product from the substrate. Assays characteristic of this group usually require two steps, since separation often does not include detection. Thus, first, the substrate and the product are separated, and usually the amount of product formed is measured. Assays that use radiochemical substrates are included in this group, since radiochemical detectors are unable to differentiate between the radiolabel of the substrate and that of the product. Examples of enzymes whose assay methods fall into this category are legion, and these approaches characterized by a separation step. 

In addition, unlike many other discontinuous assays that focus on only one of the components of the reaction, the HPLC assay offers the potential to monitor several. For example, consider adenosine kinase, the enzyme that uses two substrates and forms two products according to the reaction Ado + ATP —> AMP + ADP. Since HPLC can readily separate all four compounds (see Fig. 1.4), and all four compounds can be detected at 2S4 nm, it is apparent that with the HPLC method, the level of each component can be monitored during the course of the reaction, providing a complete analysis of each time point. ... 

Not all assays require a separation step, and this fact may be used to develop a classification scheme for assay methods. Assays that require no separation have been grouped under the heading continuous assay methods, while discontinuous methods incorporate those that do. 

The elution method involves scraping off the separated zones of samples and standards and elution of the substances from the layer material with a strong, volatile solvent. The eluates are concentrated and analyzed by use of a sensitive spectrometric method, gas or liquid column chromatography, or electroanalysis. Scraping and elution must be performed manually because the only commercial automatic micropreparative elution instrument has been discontinued by its manufacturer. The elution method is tedious and time-consuming and prone to errors caused by the incorrect choice of the sizes of the areas to scrape, incomplete collection of sorbent, and incomplete or inconsistent elution recovery of the analyte from the sorbent. However, the elution method is being rather widely used (e.g., some assay methods for pharmaceuticals and drugs in the USP Pharmacopoeia). 

Boeker, E. A. (1987). Analytical methods for fitting integrated rate equations. A discontinuous assay. Biochem. J. 245, 67-74. 

Sphaeroplasts were prepared by slight modifications to published methods [12,13]. Lysis of sphaeroplasts was effected by a combination of osmotic lysis and gentle mechanical disruption [14]. Discontinuous sucrose-density gradients were constructed and fractions were then assayed for protein, PG and marker enzymes for different organelles. 

Assays. Protein concentrations were measured by the method of Bradford (18) and the various contractile protein ATPase activities by tRe method of Martin and Doty (19). Gel electrophoresis was carried out by the method of Ames (20) on 1.5 ran polyacrylamide slabs using the discontinuous SDS buffer system of Laemnli (21). Dried gels were scanned at 550 nm for densiometry measurements. 

The method of Kato and Nakai (27) for determining protein surface hydrophobicity was adapted for evaluating procyanidin binding to BSA and Gl. The procedure is based on the fact that the fluorescence quantum yield of cis-parinaric acid increases 40-fold when cis-parinaric acid enters a hydrophobic environment from a hydrophilic environment. The digestion of BSA by trypsin in the presence of procyanidin dimer, procyanidin trimer and black bean procyanidin polymer was evaluated by discontinuous sodium dodecyl sulfate (SDS) slab gel electrophoresis and a picryl sulfonic acid (TNBS) assay (28). 

Classical low values for the mammalian enzyme that have appeared in the literature are the result of enzyme inactivation by hydrogen peroxide when measurements were carried out with peroxide levels in excess of 10 mM over time scales of 10 minutes or longer. The rapid sampling/titration method of Bonnichsen overcame the inactivation problem and permitted a satisfactory correlation of the overall catalytic measurements and Chance s observations on the intermediate complex (compound 1). Eventually, the introduction of the UV detector/spectrophotometer and the consequent assay based upon the UV absorbance of peroxide (35) further simplified the process by eliminating the discontinuous titrimetric assay. 

The pharmacokinetics of FLU and its metabolite 7-OH FLU in sheep tissue was studied using the HPLC method with fluorescence detection (207). Tissue samples were extracted with ethyl acetate. After drying, phosphate buffer (pH 7.8) and hexane were added, and aqueous (lower) phase was injected into an HPLC system. Extraction recovery was 75% for FLU and 60% for 7-OH FLU. The limit of detection was 1 and 4 ytrg/kg for both compounds. The elimination of OXO in eggs (albumen, yolk) was described using an HPLC assay with fluorimetric detection. The limits of quantitation were 5 tg/kg in albumen and yolk. Of the overall oxolinic acid detected in eggs, 95% was concentrated in the albumen. Detectable residues persisted for 9 and 7 days, respectively, in albumen and yolk after the treatment was discontinued (208). Albumen sample was homogenized with water and hydrochloric acid and extracted with ethyl acetate. The supernatant was evaporated and the rest dissolved in mobile phase. Extraction recovery was 65.2%, RSD of 5.3, for a concentration of 10 ig/kg. 

The methods in use for the assay of enzymatic activities may be divided into three groups. These will be referred to as the continuous, coupled, and discontinuous methods (see Table 1.1). 

Colorimetric assay of ATP hydrolysis, fluorimetric assay of proton pumping and measurement of ATP synthesis by a discontinuous luciferin/luciferase method have been described elsewhere (2). 

Future trends in the separation area will include translation of all these methods to microchip format, which promises to lead the next revolution in chemical analysis. MEKC and isotachophoresis, a CE separation technique in a discontinuous buffer system, have already been adapted to microchips and applied to assay herbicides, biogenic amines, and ions. Micro-channels on a chip-like structure are likely to be exploited more frequently in CE after further development of nanotechnology because it results in extremely rapid separations that consume only picoliter sample volumes and introduce the possibility of merging sample preparation and analysis in a single device. 

The initial steps of solubilization, DEAE chromatography and gel filtration were slight modifications of reported procedures [19]. The pooled fractions from a Sephacryl S-200 (Pharmacia) column were applied to a Mono Q HR5/5 FPLC column (Pharmacia) and eluted in a 20 ml gradient from 0-350 mM NaCl in 0.25 M sucrose-20 mM Tris-HCl pH 7.3. Fractions of 1 ml were collected and assayed for binding of NAA-1-[ C] (61 mCi/mmol, Amersham) by one of three methods [20]. The most active fractions were pooled, desalted and lyophilized. This preparation (approx. 50% receptor) was used either for monoclonal antibody production or was fully purified by native PAGE in a neutral pH discontinuous system [3]. The gel was briefly electroblotted (5 min, 10 mA) to nitrocellulose and the small fraction of transferred proteins visualized by rapid staining [8]. This blot was then used to locate precisely the bulk protein bands remaining in the gel. 

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