PubMed · 9255801
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M D Vancetto, J M de Oliveira, S M Prado, F Fratelli, H G Higashi. 1997. Tetanus toxoid purification: a case study.. https://doi.org/10.1038/nbt0897-807
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Analyses of unfiltered and filtered (< 0.45 micron and < 0.10 micron) groundwater samples from 15 selected wells in crystalline bedrock aquifers of the Oslo area, Norway, have been studied for 62 chemical elements. While concentrations of almost all elements vary over several orders of magnitude between the individual wells, the discrepancy between filtered and unfiltered samples from the same well are rather small, not exceeding one order of magnitude. Many elements show no influence of filtration at all, while one element (Sn) suggests that filtration may actually introduce contamination to the samples. Correlation between unfiltered and filtered samples is high for most elements. The study shows that: (1) even unfiltered samples will satisfactorily reflect general water chemistry as long as drinking water (i.e. by definition rather 'clean' water, with low particulates) is collected; (2) filtered samples do not necessarily reflect 'true' solution chemistry (an elusive concept); and (3) the differences between samples filtered at < 0.45 micron and < 0.10 micron are so minimal for most elements, that the additional effort invested in ultra-filtration may not be justified for bedrock groundwater samples.
A general method is presented here for the determination of the Km, kcat, and kcat/Km of fluorescence resonance energy transfer (FRET) substrates using a fluorescence plate reader. A simple empirical method for correcting for the inner filter effect is shown to enable accurate and undistorted measurements of these very important kinetic parameters. Inner filter effect corrected rates of hydrolysis of a FRET peptide substrate by hepatitis C virus (HCV) NS3 protease at various substrate concentrations enabled measurement of a Km value of 4.4 +/- 0.3 microM and kcat/Km value of 96,500 +/- 5800 M-1 s-1. These values are very close to the HPLC-determined Km value of 4.6 +/- 0.7 microM and kcat/Km value of 92,600 +/- 14,000 M-1 s-1. We demonstrate that the inner filter effect correction of microtiter plate reader velocities enables rapid measurement of Ki and Ki' values and kinetic inhibition mechanisms for HCV NS3 protease inhibitors.
Alpha-Acetolactate-deficient Lactococcus lactis ssp. lactis biovar. diacelylactis are utilised in several industrial processes for producing diacetyl and alpha-acetolactate. They can be selected by screening after random mutagenesis. We improved a previously described screening method [Monnet, C., Schmitt, P., Diviès, C., 1997. Appl. Environ. Microbiol. 63, 793-795], which makes it possible to screen up to 1000 colonies per agar plate, whereas the previous method allowed to screen only 60 colonies per agar plate. The new screening method facilitates selection of alpha-acetolactate-deficient mutants.