| AUTHOR |
TITLE |
Ivana V. Yang,
H. Holden Thorp |
Kinetics of Metal-Mediated One-Electron Oxidation of Guanine in Polymeric DNA and in Oligonucleotides Containing Trinucleotide Repeat Sequences
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Experimental Section
Stopped-Flow Spectrophotometry. Kinetic experiments were carried out using an On Line Instrument Systems RSM-1000 stopped-flow spectrophotometer. The reactions were monitored spectrophotometrically from 350 to 580 nm for 2.0 s at 1000 scans/s for Ru(bpy)33+ and for 5 min at 21 scans/s for Fe(bpy)33+. Solutions were maintained at 25 ± 1 °C. M(bpy)33+ (M = Ru, Fe) and DNA were dissolved in ~10 mM H2SO4 (pH 2) and pH 8 phosphate buffer, respectively, and these solutions were then mixed to give a solution that was 50 mM in sodium phosphate, pH 7, with or without 800 mM NaCl after mixing. Concentrations of Ru(bpy)32+ and Fe(bpy)33+ in each run were obtained from Amin and Amax-Amin at 452 nm (ε = 14 600 M-1 cm-1) and at 524 nm (ε = 8400 M-1 cm-1), respectively. |
Erika E. Bullesbach,
Christian Schwabe |
The Relaxin Receptor-binding Site Geometry Suggests a Novel Gripping Mode of Interaction
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Experimental Procedures
Protein Determination-- Protein concentrations were measured by UV spectroscopy using an Olis Cary-15 spectrophotometer conversion (On-Line Instrument Systems, Inc.). Relaxin analogs (0.2-0.5 mg/ml) were dissolved in water. The specific absorption coefficient was calculated with 1.95 cm-2 mg-1 for B33 relaxin and relaxin analogs and 2.19 cm-2 mg-1 for B29 relaxin analogs. |
Ravi K. Nandigama,
Dale E. Edmondson |
Structure-Activity Relations in the Oxidation of Phenethylamine Analogues by Recombinant Human Liver Monoamine Oxidase A
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| Rapid-Scanning Stopped-Flow Kinetic Measurements. Time-dependent changes in absorption spectra upon mixing anaerobic solutions of MAO A and tyramine were obtained using an OLIS-RSM 1000 stopped-flow spectrophotometer (Olis, Inc., Bogart, GA) in the laboratory of W. David Wilson (Department of Chemistry, Georgia State University, Atlanta, GA). Kinetic and spectral data were analyzed using software provided by OLIS. |
Seun-Ah Yang,
Claude B. Klee |
Low Affinity Ca2+=Binding Sites of Calcineurin B Mediate Conformational Changes in Calcineurin A
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| OLIS offers a complete line of dual beam spectrophotometers configured for absorbance, fluorescence, and circular dichroism, with and without millisecond spectral acquisition rates; stopped-flow and titration accessories; and a full line of computer interfaces, electronic modernizations, and software for many of the best classic spectrophotometers of the 1960s and 70s. |
Shantanu Chowdhury,
Michael G. Thomas
Jorge C. Escalante-Semerena,
Ruma Banerjee |
The Coenzyme B12 Analog 5'- Deoxyadenosylcobinamide-GDP supports Catalysis by Methylmalonyl-CoA Mutase in the Absence of Trans-ligand Coordination
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Experimental Procedures
Equilibrium Binding Constants Measured by UV-visible Absorption Spectroscopy-- Binding of AdoCbl and AdoCbi-GDP to methylmalonyl-CoA mutase was followed spectrophotometrically using a Cary-118 spectrophotometer (Olis Instruments), in which the cuvette holder was maintained at 4 °C by a thermostatted water circulator. Methylmalonyl-CoA mutase (18.2 µM) in 150 µl of 50 mM potassium phosphate buffer, pH 7.5, was used as a blank. Spectra were recorded between 800 and 306 nm after each addition of AdoCbi-GDP (3-5-µl aliquots prepared in the same buffer), after incubation at 4 °C for 30 min. The final AdoCbi-GDP concentration used in these experiments was 43.1 µM. The change in absorbance at 460 nm at each concentration of AdoCbi-GDP was obtained by subtracting the spectrum of the same concentration of free AdoCbi-GDP in 50 mM potassium phosphate buffer, pH 7.5. Data were analyzed as described previously (26). A Kd of 4.9 µM obtained from the equilibrium fluorescence measurements was used to generate the best-fit line to the binding data obtained by absorption spectroscopy. |
Danilo S. Boskovic,
Sriram Krishnaswamy |
Exosite Binding Tethers the Macromolecular Substrate to the Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct Sites
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Experimental Procedures
Steady State Fluorescence Measurements-- ....Experiments with both substrates were also conducted using a customized rapid scanning fluorescence spectrophotometer (RSM-1000, On-Line Instrument Systems, Bogart, GA). Spectral-time data sets were obtained using the appropriate excitation wavelength for the probe studied and rapidly scanning the emission monochromator. Data collection times, time constants, and integration times were adjusted to permit the collection of 400 or 1000 spectra over 8-10 half-lives of the measured reaction. |
