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TITLE |
Pereira, M. M.,
Carita, J. N.,
Teixeira, M. |
Membrane-Bound Electron Transfer Chain of the Thermohalophilic Bacterium Rhodothermus marinus: Characterization of the Iron-Sulfur Centers from the Dehydrogenases and Investigation of the High-Potential Iron-Sulfur Protein Function by in Vitro Reconstitut
Biochemistry; 1999; 38(4); 1276-1283. |
Experimental Procedures
Spectroscopic Techniques:
Visible spectra were obtained on a Beckman DU-70 or an Olis DW2 spectrophotometer, equipped with a temperature controller. For the thermal stability studies, visible spectra were recorded, after 30 min of incubation at each temperature, under anaerobic conditions. The stability was determined by following the absorption change at 410 nm. |
Pereira, M. M.,
Carita, J. N.,
Teixeira, M. |
Membrane-Bound Electron Transfer Chain of the Thermohalophilic Bacterium Rhodothermus marinus: A Novel Multihemic Cytochrome bc, a New Complex III
Biochemistry; 1999; 38(4); 1268-1275. |
Spectroscopic Techniques:
Electronic spectra were obtained on a Beckman DU-70 or on an OLIS DW2 spectrophotometer, at room and liquid nitrogen temperatures. |
Smithrud, D. B.,
Benkovic, P. A.,
Benkovic, S. J.,
Taylor, C. M.,
Yager, K. M.,
Witherington, J.,
Philips, B. W.,
Sprengeler, P. A.,
Smith, A. B., III,
Hirschmann, R. |
Investigations of an Antibody Ligase
Journal of the American Chemical Society; 1997; 119(2); 278-282. (Article) |
Steady-State Kinetics
The initial velocity of product formation was determined by measuring the change in absorbance of p-nitrophenol at 400 nM ( = 9600 cm-1·M-1) using a Cary (Olis-14) spectrophotometer. The appropriate amounts of substrates and antibody were mixed at 25 ˚C in 0.1 M 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.0, μ = 0.1 M NaCl, and containing 5% DMSO.
After correcting for the background rate of ester hydrolysis, the kcat and Km values were derived by fitting the data to the Michaelis-Menten equation. |
Thorsteinsson, M. V.,
Bevan, D. R.,
Potts, M.,
Dou, Y.,
Eich, R. F.,
Hargrove, M. S.,
Gibson, Q. H.,
Olson, J. S. |
A Cyanobacterial Hemoglobin with Unusual Ligand Binding Kinetics and Stability Properties
Biochemistry; 1999; 38(7); 2117-2126. |
Materials and Methods
Stopped-Flow Spectrophotometry. Association rates (k') for carbon monoxide (CO) and alkylisocyanide binding to cyanoglobin were determined by rapid mixing in a Gibson-Dionex stopped-flow apparatus equipped with an On-Line-Instruments System (OLIS) Model 3820 data collection system as described by Rohlfs et al. (29). In all the kinetic experiments, the concentration of protein was much less than that of ligand, so that the bimolecular binding processes behave as single pseudo-first-order reactions, with the observed rate constants proportional to the ligand concentration. |
Loeb, K. E.,
Zaleski, J. M.,
Hess, C. D.,
Hecht, S. M.,
Solomon, E. I. |
Spectroscopic Investigation of the Metal Ligation and Reactivity of the Ferrous Active Sites of Bleomycin and Bleomycin Derivatives
Journal of the American Chemical Society; 1998; 120(6); 1249-1259. |
Experimental Section
Charge-Transfer Region Absorption/MCD Instrumentation. Low-temperature UV/vis absorption spectra (5 K, 300-900 nm) were recorded on a Cary 17 UV/vis spectrophotometer equipped with a Janis Super Varitemp helium cryostat and interfaced to a Compaq 386 computer with OLIS software. Residual absorption due to buffer, glycerol, or glass scattering effects was subtracted from all of the spectra. |
Olesen, K.,
Veselov, A.,
Zhao, Y.,
Wang, Y.,
Danner, B.,
Scholes, C. P.,
Shapleigh, J. P. |
Spectroscopic, Kinetic, and Electrochemical Characterization of Heterologously Expressed Wild-Type and Mutant Forms of Copper-Containing Nitrite Reductase from Rhodobacter sphaeroides 2.4.3
Biochemistry; 1998; 37(17); 6086-6094. (Article) |
Methods and Materials
Activity Measurements
Oxidation of cyt c2+ was monitored in the Aminco-Morrow stopped-flow attachment coupled to a retrofitted DW-2 UV-vis spectrophotometer [the original by SLM Instruments, Urbana, IL, with retrofit by On Line Instrument Systems (OLIS), Inc., Jefferson, GA]. Kinetic data collection software and kinetic fitting software were provided by OLIS. Complementary steady-state activity measurements using nanomolar concentrations of enzyme and varying concentrations of nitrite and cytc2+ at the micromolar range and above were performed. |
Qu, K.,
Vaughn, J. L.,
Sienkiewicz, A.,
Scholes, C. P.,
Fetrow, J. S. |
Kinetics and Motional Dynamics of Spin-Labeled Yeast Iso-1-cytochrome c: 1. Stopped-Flow Electron Paramagnetic Resonance as a Probe for Protein Folding/Unfolding of the C-Terminal Helix Spin-Labeled at Cysteine 102
Biochemistry; 1997; 36(10); 2884-2897. (Article) |
Materials and Methods
Stopped-Flow UV-Vis.
Complementary stopped-flow measurements monitored by visible detection of the heme absorption were performed by an Aminco-Morrow stopped-flow attachment coupled to a retrofitted DW-2 UV-Vis spectrophotometer [the original by SLM Instruments (Urbana, IL), with retrofit by On Line Instrument Systems (OLIS) Inc. (Jefferson, GA)]. This stopped-flow device has a mixer with a 1970s design; we were warned about its potential for Schlieren mixing artifacts due to incomplete mixing of dilute buffer and concentrated GdnHCl (R. Hansen, Update Instrument, Inc., personal communication). We have observed such optical artifacts over a time of ~5 ms after mixing, and we therefore do not attempt to estimate with our optical stopped-flow system the optical absorption change during flow. |
Hui, H. L.,
Kavanaugh, J. S.,
Doyle, M. L.,
Wierzba, A.,
Rogers, P. H.,
Arnone, A.,
Holt, J. M.,
Ackers, G. K.,
Noble, R. W. |
Structural and Functional Properties of Human Hemoglobins Reassembled after Synthesis in Escherichia coli
Biochemistry; 1999; 38(3); 1040-1049. |
Experimental Procedures
Kinetics of CO Combination with Deoxygenated Hemoglobin by Stopped-Flow
These kinetics were measured with an OLIS U.S.A. (On Line Instrument Systems Inc., Bogart, GA) stopped-flow apparatus. The procedures were essentially those of Gibson as described by Doyle et al. Reactions were followed at 420 and 435 nm using a 1.7 cm path length cell. Concentrations of CO and hemoglobin (in heme equivalents) were 20 and 2 μM, respectively.
Kinetics of CO Recombination following Complete Photodissociation
Data acquisition and processing were carried out by an OLIS model 4000 data acquisition and instrument control system. Kinetic data were fitted to single or double exponential functions using successive integration or Levenberg-Marquardt fitting routines (8). |
Kwiatkowski, L. D.,
Hui, H. L.,
Wierzba, A.,
Noble, R. W.,
Walder, R. Y.,
Peterson, E. S.,
Sligar, S. G.,
Sanders, K. E |
Preparation and Kinetic Characterization of a Series of beta-W37 Variants of Human Hemoglobin A: Evidence for High-Affinity T Quaternary Structures
Biochemistry; 1998; 37(13); 4325-4335. (Article) |
Experimental Procedures
Kinetics of CO Combination with Deoxygenated Hemoglobin
These kinetics were measured with an OLIS (On line Instrument Systems Inc., Bogart, GA) U.S.A. stopped-flow apparatus which is similar to that described by Gibson and Milnes (1964). The procedures were essentially those of Gibson (1959) as described in Doyle et al. (1992). Reactions were followed at 420 and 435 nm using a 1.7 cm pathlength cell. Reactant concentrations after mixing were the same as in the complete photodissociation experiments.
Kinetic data from stopped-flow and flash photolysis experiments were obtained under four different experimental conditions. Measurements at 20 ˚C were carried out at pH 6 and 7 in 100 mM HCl plus bis-Tris buffer and at pH 8 in 100 mM HCl plus Tris buffer. Measurements were also carried out at 21.5 ˚C and pH 7.4 in a buffer containing 100 mM Tris-HCl, 100 mM NaCl, and 1 mM EDTA as used by Kiger et al. (1997) for measurements of equilibria of oxygen binding. |
Bauer, R.,
Danielsen, E.,
Hemmingsen, L., Sorensen, M. V.,
Ulstrup, J.,
Friis, E. P.,
Auld, D. S.,
Bjerrum, M. J. |
Structure and Dynamics of the Metal Site of Cadmium-Substituted Carboxypeptidase A in Solution and Crystalline States and under Steady-State Peptide Hydrolysis
Biochemistry; 1997; 36(38); 11514-11524. (Article) |
Materials and Methods
The effect of anions on the activity of the Cd-substituted enzyme toward the dipeptide Bz-Gly-L-Phe substrate was investigated by stopped-flow spectrophotometry using a High-Tech SF.53 instrument combined with the OLIS (Jefferson, GA) data acquisition and processing software. The software tool "Grapher" from Golden Software inc. was used to process the initial rates vs substrate concentration. |
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