| AUTHOR |
TITLE |
Kapila Ratnam,
Naomasa Shiraishi,
Wilbur H. Campbell,
Russ Hille |
Spectroscopic and Kinetic Characterization of the Recombinant Cytochrome c Reductase Fragment of Nitrate Reductase. IDENTIFICATION OF THE RATE-LIMITING CATALYTIC STEP
J. Biol. Chem. 1997 272: 2122-2128. |
Materials and Methods:
Rapid mixing experiments were performed using a Kinetic Instruments Inc. stopped-flow apparatus interfaced to an On-Line Instrument Systems (OLIS) model 3920Z data collection system. Enzyme samples, in a tonometer equipped with a three-way stopcock valve with a male Luer connector, were made anaerobic by alternately evacuating and flushing with oxygen-free argon on an anaerobic train. The tonometer was mounted on the stopped-flow apparatus and the enzyme rapidly mixed with anaerobic solutions of NADH. Kinetic transients obtained were analyzed and the rate constants determined using OLIS software. |
Hiroshi Yamazaki,
William W. Johnson,
Yune-Fang Ueng,
Tsutomu Shimada,
F. Peter Guengerich |
Lack of Electron Transfer from Cytochrome b5 in Stimulation of Catalytic Activities of Cytochrome P450 3A4. CHARACTERIZATION OF A RECONSTITUTED CYTOCHROME P450 3A4/NADPH-CYTOCHROME P450 REDUCTASE SYSTEM AND STUDIES WITH APO-CYTOCHROME b5
J. Biol. Chem. 1996 271: 27438-27444. |
Experimental Procedures:
Spectroscopy
UV-visible spectra were recorded using a Cary 14/OLIS spectrophotometer (On-Line Instrument Systems, Bogart, GA). Pre-steady-state absorbance measurements were made using an Applied Photosystems SX-17MV instrument (Applied Photophysics, Leatherhead, UK) operating at 37 °C. In most cases 90µl of an enzyme mixture were mixed with 10µl of a second solution (e.g. NADPH or O2-saturated buffer). In other situations, equal volumes (50µl) from the two syringes were mixed. |
Jong Hwa Kim,
Matthew G. Ryan,
Holger Knaut,
Russ Hille |
The Reductive Half-reaction of Xanthine Oxidase
J. Biol. Chem. 1996 271: 6771-6780. |
Materials and Methods:
Steady-state kinetic experiments were performed under O[(2)]-saturated conditions at 25 °C, using a Kinetic Instruments stopped-flow spectrometer interfaced to a PC-486 personal computer using software from On-Line Instrument Systems, Inc. (OLIS). |
Liuxin Huang,
Ronald J. Rohlfs,
Russ Hille |
The Reaction of Trimethylamine Dehydrogenase with Electron Transferring Flavoprotein
J. Biol. Chem. 1995 270: 23958-23965. |
Materials and Methods:
Kinetic Experiments
Kinetic experiments were carried out using a Kinetic Instrument Inc. stopped-flow apparatus equipped with an On Line Instruments Systems (OLIS) model 3920Z data collection system. Anaerobic TMADH was prepared as above in a tonometer equipped with a ground joint for the dithionite titration syringe, a side arm cuvette, and a three-way stopcock valve with a male Luer connector, and reduced by titration with sodium dithionite to the desired level. ETF was made anaerobic as above and transferred to a syringe fitted with a three-way valve so that anaerobic buffer could be added to the syringe to change the ETF concentration by serial dilution. The concentrations of ETF were at least 5 times larger than that of TMADH to ensure pseudo first-order conditions in the experiments described. Kinetic transients obtained after mixing reduced TMADH with ETF were monitored as transmittance voltage and collected by a high speed A/D converter, then converted to absorbance changes by OLIS software. |
Victor L. Davidson,
Limei Hsu Jones |
Complex Formation with Methylamine Dehydrogenase Affects the Pathway of Electron Transfer from Amicyanin to Cytochrome c-551i
J. Biol. Chem. 1995 270: 23941-23943. |
Experimental Procedures:
An On-Line Instruments (OLIS, Bogart, GA) RSM1000 stopped-flow rapid scanning spectrophotometer was used for kinetic measurements. All experiments were performed in 0.01 M potassium phosphate, pH 7.5, at 30 °C. Protein concentrations were determined from the previously reported extinction coefficients for each protein (Husain and Davidson, 1985, 1986; Husain et al., 1987). Non-linear curve fitting of data was performed using either OLIS software or Sigma Plot 5.0 (Jandel Scientific). |
Christopher H. Snyder,
Bernard L. Trumpower |
Ubiquinone at Center N Is Responsible for Triphasic Reduction of Cytochrome b in the Cytochrome bc1 Complex
J. Biol. Chem. 1999 274: 31209-31216. |
Experimental Procedures:
Kinetic Measurements
Kinetic measurements were performed at room temperature by rapid scanning stopped flow spectroscopy, using an OLIS Rapid Scanning Monochromator (On-Line Instrument Systems, Inc., Bogart, GA) equipped with a 1200 lines/mm grating blazed at 500 nm. This produced a spectrum of 75 nm width, centered at 555 nm, with a resolution of 0.4 nm. The dead time of the instrument was ~2 ms, and the end of this period was chosen as time 0, after which data were collected at 1000 scans/s. |
L. Chastine Bell-Parikh,
F. Peter Guengerich |
Kinetics of Cytochrome P450 2E1-Catalyzed Oxidation of Ethanol to Acetic Acid via Acetaldehyde
J. Biol. Chem. 1999 274: 23833-23840. |
Estimation of Binding Constants:
P450 2E1 (1.0µM) in 100 mM potassium phosphate buffer (pH 7.4, 2.0-ml total volume) was divided equally between two cuvettes, and a base-line spectrum was acquired from 340 to 480 nm using an OLIS-Aminco DW2a spectrophotometer (On-Line Instrument Systems, Bogart, GA). To the sample cuvette, 0.3 or 3.0µM 4-methylpyrazole was added, and the resulting "Type II" binding spectrum was recorded and designated as "100% binding." Aqueous potassium acetate (pH 7.4, 0-35 mM) or acetaldehyde (0-15 mM) was titrated into both the sample and reference cuvettes, and disappearance of the 4-methylpyrazole-induced Type II binding spectrum was quantified on the basis of the decrease in the absorbance difference ( A) between 424 and 389 nm A = A424 - A389). |
Gregory A. Hunter,
Gloria C. Ferreira |
Pre-steady-state Reaction of 5-Aminolevulinate Synthase. EVIDENCE FOR A RATE-DETERMINING PRODUCT RELEASE
J. Biol. Chem. 1999 274: 12222-12228. |
Stopped-flow Spectroscopy:
Rapid scanning stopped-flow kinetic measurements were conducted using a model RSM-1000 stopped-flow spectrophotometer equipped with a stopped-flow mixer with an optical path length of 1.5 mm (OLIS, Inc.). The dead time of this instrument is about 2 ms. Scans covering the wavelength range of 315-545 nm were collected at a rate of 1000 scans/s. For reactions longer than 3 s, the collected scans were averaged to yield either 62 or 31 scans/s to condense data files to a manageable size. An external water bath was used to maintain the temperature of the syringes (containing the reactants) and the stopped-flow cell compartment at 30 °C. The concentration of enzyme and ligands loaded into the syringes were always 2-fold greater than that reported in the figure legends, such that the reported concentrations represent the final concentrations present in the cell compartment after mixing. |
