AUTHOR

TITLE

TEXT

Manon Couture, Tapan Kanti Das, H. Caroline Lee, Jack Peisach, Denis L. Rousseau, Beatrice A. Wittenberg, Jonathan B. Wittenberg, and Michel Guertin

Chlamydomonas Chloroplast Ferrous Hemoglobin. HEME POCKET STRUCTURE AND REACTIONS WITH LIGANDS J. Biol. Chem. 1999 274: 6898-6910.

Experimental Procedures

Ligand Reaction Rates-- Reaction rates were measured using a Hi-Tech model 61 (Salisbury, UK) stopped-flow apparatus interfaced to an OLIS Data Acquisition/Computation System (On Line Instruments Systems, Bogart, GA). Rates were computed using the OLIS system.

Stephen M. G. Duff, Jonathan B. Wittenberg, and Robert D. Hill

Expression, Purification, and Properties of Recombinant Barley (Hordeum sp.) Hemoglobin. OPTICAL SPECTRA AND REACTIONS WITH GASEOUS LIGANDS J. Biol. Chem. 1997 272: 16746-16752.

Experimental Procedures

Ligand Reaction Rates

These were measured using an Hi-Tech model SF 61 (Salisbury, UK) stopped-flow apparatus interfaced to an OLIS Data Acquisition/Computation System (On-Line Instrument Systems, Bogart, GA). Rates were computed using either the Olis system or Origins 4.1 (Microcal Software Northampton, MA). Most kinetic experiments used Hb purified by chromatography on Mono Q HR only, omitting the final hydrophobic interaction chromatography step.

Gang Wu, Chunhong Wei, Richard J. Kulmacz, Yoichi Osawa, and Ah-lim Tsai

A Mechanistic Study of Self-inactivation of the Peroxidase Activity in Prostaglandin H Synthase-1

J. Biol. Chem. 1999 274: 9231-9237.

Experimental Procedures
Kinetics of PGHS Heme Spectral Changes Induced by Peroxide-- Measurements were performed on the Bio-Sequential stopped-flow instrument in the kinetic scan-spectral reconstruction mode or on an Olis RSM-1000 rapid-scan stopped-flow instrument (On-Line Instrument Systems, Inc., Bogart, GA). The former approach was used to acquire data over shorter wavelength ranges (typically, 390-430 nm in 2-nm increments) and at lower enzyme concentrations. Kinetic scan mode data obtained at serial wavelengths were first processed by the singular value decomposition (SVD) method  and then fitted to kinetic models using the Glint analysis package to deconvolute spectral intermediates. The Olis rapid-scan instrument was used principally to obtain spectral data in the visible region, where a more concentrated sample is required. Similar SVD analysis and global fitting routines were used to analyze data obtained with the Olis instrument to resolve dominant spectral intermediates and to determine the associated reaction rate constants. A set of published kinetic data and a mechanistic model for the PGHS peroxide reaction containing three spectral species  was used to validate the reliability of the SVD/global fitting software supplied with the Applied Photophysics and Olis instruments and achieved an excellent agreement. 

Young-Lan Hyun, Zhenyu Zhu, and Victor L. Davidson

Gated and Ungated Electron Transfer Reactions from Aromatic Amine Dehydrogenase to Azurin

J. Biol. Chem. 1999 274: 29081-29086.

Experimental Procedures
Transient kinetic experiments were performed using an On-Line Instrument Systems (OLIS, Bogart GA) RSM1000 rapid-scanning stopped-flow spectrophotometer. The experimental procedures for the rapid mixing experiments were as described previously for the reactions of the O-quinol AADH with azurin. For the reactions of the N-quinol, it was necessary to use anaerobic conditions. This is because the N-quinol AADH exhibited significant reactivity with O2, and during the incubation times before mixing some conversion of N-quinol to N-semiquinone occurred. Anaerobicity was achieved by deaerating buffers and including in the buffers a mixture of glucose oxidase (1 unit/ml), glucose (30 mM), and catalase (24 units/ml). Unless otherwise indicated, reactions were performed in 0.25 M potassium phosphate, pH 7.5. N-Quinol AADH was prepared by the addition of 1 M equivalent of tyramine per TTQ. O-Quinol AADH was prepared by titration with sodium dithionite. Kinetic data collected in the rapid-scanning mode were reduced by factor analysis using Global Fit, the singular value decomposition algorithm provided by OLIS. Singular value decomposition-reduced data were then globally fit by a robust version of the Levenberg and Marquardt (non-linear method of least squares using the fitting routines of the Global Fit software.

Kapila Ratnam, Naomasa Shiraishi, Wilbur H. Campbell, and Russ Hille

Spectroscopic and Kinetic Characterization of the Recombinant Wild-type and C242S Mutant of the Cytochrome b Reductase Fragment of Nitrate Reductase

J. Biol. Chem. 1995 270: 24067-24072.

Materials and Methods

Ultraviolet/visible spectra were recorded using a Hewlett Packard 8452A single-beam diode array spectrophotometer; visible/near-infrared spectra were recorded with an On-Line Instrument Systems (OLIS) modernized Cary-14.

Rapid mixing experiments were carried out using a Kinetic Instruments Inc. stopped-flow apparatus interfaced with an OLIS model 3920Z data collection system.

The kinetic transients obtained after mixing were monitored as transmittance voltages collected by a high speed A/D converter and converted to absorbance changes, and the rate constants were determined by OLIS Inc. software.

Mei-Huei Jang, Jaswir Basran, Nigel S. Scrutton, and Russ Hille

The Reaction of Trimethylamine Dehydrogenase with Trimethylamine

J. Biol. Chem. 1999 274: 13147-13154.

Experimental Procedures

Pre-steady-state 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.

Kinetic transients were monitored as transmittance voltages collected by a high speed A/D converter and converted to absorbance changes by OLIS software.

Takashi Yonetani, Antonio Tsuneshige, Yuxiang Zhou, and Xuesi Chen

Electron Paramagnetic Resonance and Oxygen Binding Studies of -Nitrosyl Hemoglobin. A NOVEL OXYGEN CARRIER HAVING NO-ASSISTED ALLOSTERIC FUNCTIONS

J. Biol. Chem. 1998 273: 20323-20333.

Experimental Procedures

Oxygen Equilibrium Measurements-- Oxygen equilibrium curves were measured by an improved version of Imai's automatic method (19) with the following modifications. Absorbance was monitored using a computer-controlled Olis -Cary 118 spectrophotometer (Olis, Bogart, GA).

Stefano Bettati, Laura D. Kwiatkowski, Jeffrey S. Kavanaugh, Andrea Mozzarelli, Arthur Arnone, Gian Luigi Rossi, and Robert W. Noble

Structure and Oxygen Affinity of Crystalline des-His-146 Human Hemoglobin in the T State

J. Biol. Chem. 1997 272: 33077-33084.

Both stopped flow studies and flash photolysis measurements were carried out at pH 6, 7, and 8. The buffers were prepared by titrating an amount of HCl equal to 0.1 M chloride with solid bis-Tris base (for pH 6 and 7) and solid Tris base (for pH 8). Inositol hexaphosphate, IHP (Sigma), was obtained in the sodium form. The pH of the stock solution (0.2 M) was adjusted to pH 5.6 with the protonated form of Amberlite IR-120 resin. IHP was added to give a final concentration of 0.1 mM. Reactions were followed at 420 and 435 nm, and the temperature of the sample maintained at 20 ？. Data collection and processing was by an Olis model 4000 data acquisition and instrument control system as already described (31). Rigorous error analysis for the fitted rate constants is difficult. Because of the large numbers of data points obtained in each kinetic experiment, the estimated errors of the fits are very small. However, reproducibility between and among experiments indicates a more reasonable estimate of error to be ?0% for the rate constants and ?5% for the fractional contributions of the kinetic components of the reaction. 

James T. Hazzard, Shanthi Govindaraj, Thomas L. Poulos, and Gordon Tollin

Electron Transfer between the FMN and Heme Domains of Cytochrome P450BM-3. EFFECTS OF SUBSTRATE AND CO

J. Biol. Chem. 1997 272: 7922-7926.

Materials and Methods

Difference spectra were collected with an OLIS-modified Cary 15 spectrophotometer using the fully oxidized BM3_t as a reference spectrum.

Transient kinetic data were collected using a Tektronix TDS 410A digitizing oscilloscope and analyzed on a PC using KINFIT (OLIS).

Paul S. Kingma and Neil Osheroff

Spontaneous DNA Damage Stimulates Topoisomerase II-mediated DNA Cleavage

J. Biol. Chem. 1997 272: 7488-7493.

Experimental Procedures
Spectroscopic Determination of Oligonucleotide Melting Temperatures 
Ultraviolet absorbance measurements were recorded using a modified Cary-14/OLIS spectrophotometer (On-Line Instrument Systems). Reaction temperatures were controlled by a jacketed cell holder connected to a water bath. Oligonucleotide melting was determined by monitoring changes in A260 as a function of temperature over a range of 10-65 ？. Melting curves were derivatized incrementally and smoothed with the software provided by the manufacturer. The melting temperature was defined as the temperature at which the first order derivative of the melting curve reached its maximum. 

Wei Jiang and David D. Hackney

Monomeric Kinesin Head Domains Hydrolyze Multiple ATP Molecules before Release from a Microtubule

J. Biol. Chem. 1997 272: 5616-5621.

Materials and Methods

Stopped flow turbidity measurements were performed with an OLIS stopped flow spectrophotometer at 320 nm and 1.6 cm path length.