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The regeneration of ATP from PEP and ADP was coupled to the oxidation of NADH and was observed as a decrease in absorbance at 380 nm (the maximal absorbance for NADH is at 340 nm, but 380 nm wavelength was used so that the signal remained within the linear range of the spectrophotometer for the duration of the experiment).
The change in absorbance at 408 nm (maximal absorbance for holomyoglobin) was plotted versus time and fit by a double-exponential equation to yield the first-order rate constants for heme dissociation using GraphPad Prism version 5.02 for Windows (GraphPad Software, La Jolla, CA).
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To allow direct comparison of the data, sample concentrations were adjusted by dilution so they had matching fluorophore maximal absorbances for bimane (typically Abs = 0.05, and absorbance maxima ranged from 386 to 397 nm depending on bimane location) and for BODIPY 507/545 at 507 nm.
Hence, to test the estimation of Km, the absorbance of uric acid was measured at 308 nm with the recombinant wildtype uricase of Bacillus fastidious A.T.C.C. 29604, but at 293 nm with the uricase from Asahi-Kasei, for the maximal absorbance of uric acid within the measurable range of the spectrophotometer.
Maximal absorbance of fast green is at 630 nm and 540 nm for Sirius red.
Quantum yield analysis for T4L N116X/N132B samples, and figures of these samples demonstrating how the maximal absorbance of the bimane peak shifts due to static complex formation between quencher and fluorophore.
The optimal reaction time was taken as the time corresponding to maximal absorbance of the samples.
The maximal absorbance of the unprotonated form of 4-nitroaniline was observed at 329 nm in CCl4.
The cell proliferation effect was calculated from the solvent control (0.1% ethanol -corrected absorbancethanol -corrected the percentabsorbanceimandabsorbancexpressedpositive control.
Triton X-100 (1%) and heat-inactivated complement were added to the wells to measure background or maximal absorbance of WST-1, respectively.
The supernatant was examined in an Ultrospec 2000 (Pharmarcia Biotech) spectrophotometer, and the optical density at 450 nm (maximal absorbance of lutein) was determined.
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