Further Assays

1 Enzymatic Method for Determining Enantiomeric Excess (EMDee) 

A somewhat different approach to determining the enantiopurity of a sample is based on the idea that an appropriate enzyme selectively processes one enantiomer, giving rise to a UV/visible signal [17]. An example concerns determination of the enantiopurity of chiral secondary alcohols, the (S) enantiomer being oxidized selectively by the alcohol dehydrogenase from Thermoanaerobium sp. The rate of this process can be monitored by a UV/visible plate reader due to the formation of NADPH (absorption at 340 nm), which relates to the quantity of the (S) enandomer present in the mixture. About 4800 ee determinadons are possible per day, accuracy amoundng to 10%. Although the screen was not specifically developed to evaluate chiral alcohols produced by an enzymadc process, it is conceivable that this could be possible after an appropriate extraction process. 

Conventional gas chromatography (GC) based on the use of chiral stationary phases can handle only a few dozen ee determinations per day. In some instances GC can be modified so that, in optimal situations, about 700 exact ee and E determinations are possible per day [29]. Such meclium-throughputmay suffice in certain applications. The example concerns the lipase-catalyzed kinetic resolution of the chiral alcohol (R)- and (S)-18 with formation of the acylated forms (R)- and (S )-19. Thousands of mutants of the lipase from Pseudomonas aeruginosa were created by error-prone PCR for use as catalysts in the model reaction and were then screened for enantioselectivity [29]. 

Using a stationary phase based on a /3-cyclodextrin derivative (2,3-di-0-ethyl-6-O-te/T-bu ty klimethy Isily 1-/3-CD), complete separation of (R)- and (S)-18 (but not of (R)/(S)-19) is possible within 3.9 min. Because the configuration comprises two simultaneously operating GC units, about 700 exact ee determinations of (R)f(S)-lS are possible per day. Moreover, the corresponding values for the conversion and the selectivity factor E (or s) are likewise automatically provided in microtiter-format, which means that the ee of (R)/(S)-19 is also accessible. Of course, every 

The advantage of fluorescence-based assays is their high sensitivity. It is therefore perhaps surprising that few such systems have been developed for evaluating the enantioselectivity of enzyme-catalyzed reactions. Fluorescence as a detection method is used in an enzyme-coupled assay [26] (see Section 9.3.4.3) and in the capillary array electrophoresis [25] (see Section 9.3.6.5). If several substrates need to be screened simultaneously, fluorescence-based substrate arrays as enzyme fingerprinting tools can be used, although enantioselectivity still needs to be addressed [26e], 

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A further assay method using a G-6-P, G-6-PDH, NADP, MHb, MHbR system in which MHb was prepared from human hemolyzates as... 

Table 27.2 illustrates urea determination with the use of sensors, manufactured as described in point 3 of Table 27.1. Initial concentrations of aqueous NiS04 solution and chloroform NaDDC solution were varied. The optimum results (minimum mean square deviation and maximum correlation between the introduced and determined concentrations) are observed for the case when the initial concentration of NiS04 exceeds four times the initial chloroform NaDDC concentration. For further assays, the sensor was used meeting the conditions described in point 3 of Table 27.1 and in point 3 of Table 27.2. Solutions contained 0.04 g NaDDC in 2 ml of chloroform and 0.2 g NiS04 in 2 ml of H20.

Thus, some environmental scientists suggest that tests be used that measure toxicity rather than individual contaminants. Toxic samples could then be further assayed for specific contaminants if necessary to identify point sources and/or water treatment procedures. Relatively rapid, affordable and dependable assays would be a boon for developing country communities, in the same way as earlier rapid tests were for fecal contamination. The latter have proven to be usable in a sustainable manner in developing country communities, empowering them to monitor water safety and to act appropriately when necessary. 

Filter paper activity, which describes the overall cellulolytic activity of an enzyme preparation, was determined by the method of Mandels et al. (16). A 1 x 6 cm strip of Whatman no.l filter paper (Hillsboro, OR), which equals 50 mg of cellulose, served as the substrate and was added to the sample solution containing 0.5 mL of appropriate diluted enzyme (supernatant of culture broth) and 1.0 mL of 0.05 M citrate buffer (pH 4.8). After 60 min of incubation at 50°C, the hydrolysis was terminated by the addition of 3 mL of DNS solution, and the mixture was further assayed for reducing sugar content by the DNS method. One international filter paper unit (FPU) was defined as the amount of enzyme that releases 1 pmol of glucose/min under the assay conditions. Activities were reported as FPU/milliliter. 

The demand for enzyme assays that not only monitor overall activity but also en-antioselectivity stimulated the development of further assay systems that are still, however, in a rather experimental state with respect to high-throughput enzyme screening applications. These methods include assays based on electron spin resonance spectroscopy (ESR) [91], nuclear magnetic resonance (NMR) [92,93], IR-thermography [94] or electrospray ionization spectrometry (ESI-MS) [95]. 

Various classes of drugs can be identified from complex mixtures by this method. UV-Vis spectra can help to recognize different structural groups then, FTIR can further identify the compounds. All the spectra can be included in UV-Vis or IR spectral libraries and can be used as references for further assays. 

First, the antibody specificity as to what epitope locations are identified needs to be delineated. Epitopes located on the stable part of the cTnl molecule should be a priority. Further, assays need to clarify whether different cTnl forms (i.e., binary versus ternary complex) are recognized in an equimolar fashion by the antibodies used in the assay. Specific relative responses need to be described for the following cTnl forms free cTnl, the I-C binary complex, the T-I-C ternary complex, along with oxidized, reduced, and phos-phorylated iso forms of the three cTnl forms. Further, the effects of different anticoagulants on binding of cTnl need to be addressed. ... 

The PR8, PR9 and PR 10 populations, which contained mutants with higher rates of H2 evolution than that of WT (see above), were subsequently submitted to the MZ selective pressure following deactivation by 5% 02. The surviving populations (less than 3% of the initial cell density), MZ12, MZ13 and MZ14, were resuspended in liquid medium and plated to yield single colonies. Two hundred and forty clones from each of the three populations were screened as above, and selected clones were further assayed to determine their 02 tolerance relative to WT cells. 

The situation has since turned aroimd, at least in some ways, as efforts continue by both the government and the pharmaceutieal companies to further assay plant sources — and whatever else comes to mind. 

NaCl and lysed using a FastPrep FP120 cell disrupter (Qbiogene, USA). After centrifugation at 4 °C, the supernatants were stored at -70 °C for further assays. 

The addition of a conunercial fertilizer with salts of nitrogen, phosphorous and potassium (10 10 10) was evaluated. The vinasse appears to have adequate amounts of these components for microbial development, since a decline in their radial growth was observed when these salts were added to the media (Table 2), even when reduced (1/5) doses were evaluated (data not shown). As a control, fertilizer was replaced with the NPK salts from A. nidulans complete medium. Thus in further assays, vinasse medium was standardized as vinasse supplemented with two percent molasses (w v). 

It is customary to monitor the elution of proteins by virtue of an increase in absorbance of the eluate, either at 280nm or, for greater sensitivity, at 2l4nm. Some caution is advised here, as many organic compounds absorb light at these wavelengths, and an absorbance peak at 280nm cannot be assumed to reflect a peak of proteins. Further assays are required to confirm the presence of protein and the complexity of the mixture. 

Further assays were conducted in order to try and ascertain the mechanism of protection by 44, 43, and 9, with the hope of shedding light on the resnlts obtained in the TBARS assay. 

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