PubMed · 5967612
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G Thieme. A versatile device for microscopic spectrofluorometry.. https://doi.org/10.1111/j.1748-1716.1966.tb03337.x
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An enzymatically optimized, miniaturized (20 microl) fluorimetric assay of galactose-1-phosphate-uridyltransferase using dried blood spots for newborn screening is presented. The Beutler reaction principle has been adapted to the microtiter plate technology and acetone/methanol was used for complete deproteinization. A special ultramicro multiwell screening plate resistant to organic solvents has been developed and employed. The assay is simple, sensitive and inexpensive, due to small reagent volumes and the low prices of ultramicro screening plates. The reaction is linear with galactose-1-phosphate-uridyltransferase activity up to 120 min of incubation time. It shows low imprecision and good correlation to a quantitative validation test. For standardization the use of plate means or medians of activity or fluorescence values is proposed. Individual blank measurement prevents false negative assessments.
The proton translocating ATP synthase is conceived as a rotatory molecular engine. ATP hydrolysis by its headpiece, CF1, drives the rotation of subunit gamma relative to the hexagonally arranged large subunits, (alphabeta)3. We investigated transition states of the rotatory drive by polarized confocal fluorometry (POCOF) as applied to single molecules of engineered, immobilized and load-free spinach-CF1. We found that the hydrolysis of ATP caused the stepped and sequential progression of subunit gamma through three discrete angular positions, with the transition states of gamma being too shortlived for detection. We also observed the stepped motion of epsilon, whereas delta was immobile as (alphabeta)3.
The simultaneous determination of nafcillin and methicillin was accomplished by constant wavelength synchronous fluorimetry, in combination with partial least-squares multivariate calibration. The total luminescence information of the compounds was used to optimize the spectral data set to perform the calibration. A calibration set of standard samples was designed by combination of a factorial design, with three levels for each factor, and a central composite design. A comparative study of the results obtained using the excitation, the emission, the constant wavelength synchronous and the constant energy synchronous spectral data as analytical signals was performed. The data, set composed of the constant wavelength synchronous spectra, was selected as the analytical signal. The method was successfully applied to the determination of nafcillin and methicillin in mixtures.