| AUTHOR |
TITLE |
Lydakis-Simantiris, N.,
Betts, S. D.,
Yocum, C. F. |
Leucine 245 Is a Critical Residue for Folding and Function of the Manganese Stabilizing Protein of Photosystem II
Biochemistry; 1999; 38(47); 15528-15535. |
Materials and Methods
CD and Optical Spectroscopy.
The spectra were then normalized to the spectrum of the most concentrated sample. Optical spectroscopy was carried out at room temperature on an OLIS-modified Cary-17 instrument. All samples were in 10 mM KH2PO4, pH 6.0, except for unfolded wild-type MSP which was transferred by dialysis to 3 M urea, 50 mM MES, pH 6.0, 50 mM NaCl. |
Hanna, I. H.,
Roberts, E. S.,
Hollenberg, P. F. |
Molecular Basis for the Differences in Lidocaine Binding and Regioselectivity of Oxidation by Cytochromes P450 2B1 and 2B2
Biochemistry; 1998; 37(1); 311-318. (Article) |
Experimental Procedures
Spectral Binding Studies. Binding spectra were recorded on an Aminco DW2 spectrophotometer equipped with the OLIS operating system (On-Line Instruments Bogart, GA) essentially as described (20). The various P450s were suspended to a final concentration of 0.5-1.0 nmol/mL in a 50 mM Tris-HCl buffer, pH 7.4, containing 20% glycerol and 150 mM KCl and placed into two cuvettes (sample and reference). LIDO was added sequentially to the sample cuvette either in a methanolic solution or dissolved in water,3 while the reference cuvette received an equal volume of the vehicle solvent. Difference spectra were recorded from 360 to 450 nm after each addition. Dissociation constants were determined using nonlinear fits of the average absorbance differences between 388 and 420 nm at various concentrations of LIDO. |
Kent, U. M.,
Hanna, I. H.,
Szklarz, G. D.,
Vaz, A.D.N.,
Halpert, J. R.,
Bend, J. R.,
Hollenberg, P. F. |
Significance of Glycine 478 in the Metabolism of N-Benzyl-1-aminobenzotriazole to Reactive Intermediates by Cytochrome P450 2B1
Biochemistry; 1997; 36(39); 11707-11716. (Article) |
Experimental Procedures.
Spectral Determinations. P450, either 1 nmol of r2B1 or 1 nmol of r2B1 G478A, was incubated with 100 µg of DLPC for 30 min at 4 °C. The reconstituted proteins were diluted to 0.5 µM with 50 mM potassium phosphate (pH 7.4). The samples were divided and placed in the reference and sample cuvettes and scanned from 350 to 500 nm before and after each addition of substrate on a DW 2 UV/visible spectrophotometer (SLM Aminco, Urbana, IL) equipped with an OLIS spectroscopy operating system (On-Line Instrument Systems, Inc., Bogart, GA). To the sample cuvette were added 1 µL aliquots of BBT (0-580 µM) or n-octylamine (0-14 µM) dissolved in DMSO while the reference cuvette received DMSO only. Spectral binding constants were calculated by linear regression after the inverse of the absorbance change versus the inverse of the BBT concentration was plotted. |
Zhu, Z.,
Davidson, V. L. |
Identification of a New Reaction Intermediate in the Oxidation of Methylamine Dehydrogenase by Amicyanin
Biochemistry; 1999; 38(15); 4862-4867. |
Experimental Procedures.
An On-Line Instrument Systems (OLIS) RSM 1000 rapid-scanning stopped-flow spectrophotometer was used for transient kinetic experiments. Unless otherwise indicated, reactions were initiated by mixing 4 M MADH with 40 M amicyanin at 25 °C under the indicated buffer conditions. This concentration of amicyanin was sufficient to maintain pseudo-first-order reaction conditions and saturate the reaction. Raising the amicyanin concentration to 200 M yielded identical results. Data were fitted either by global kinetic and factor analysis using the OLIS software, as described previously (5), or by analysis of the change in absorbance with time at selected single wavelengths as indicated. |
Bishop, G. R.,
Davidson, V. L. |
Electron Transfer from the Aminosemiquinone Reaction Intermediate of Methylamine Dehydrogenase to Amicyanin
Biochemistry; 1998; 37(31); 11026-11032. (Article) |
Experimental Procedures
Rapid Kinetic Measurements. Rapid kinetic measurements were made using an On-Line Instrument Systems (OLIS) RSM 1000 stopped-flow spectrophotometer. All reactions were monitored over a range of wavelengths between 330 and 490 nm where each of the three redox forms of TTQ exhibits a visible absorbance maximum. The visible absorbance maximum of amicyanin is at 595 nm, and its extinction coefficient is much smaller than that of MADH. Therefore, redox-linked changes in amicyanin do not interfere with the analysis of the absorbance changes of oxidized, semiquinone, or reduced MADH. In mixing experiments, MADH was the limiting reactant with a concentration of 1-4 µM. All experiments were performed under saturating pseudo-first-order conditions with the amicyanin concentration in at least 20-fold excess of that of MADH. |
Davidson, V. L.,
Jones, L. H.,
Zhu, Z. |
Site-Directed Mutagenesis of Phe 97 to Glu in Amicyanin Alters the Electronic Coupling for Interprotein Electron Transfer from Quinol Methylamine Dehydrogenase
Biochemistry; 1998; 37(20); 7371-7377. (Article) |
Experimental Procedures
An On-Line Instrument Systems (OLIS) RSM1000 stopped-flow spectrophotometer was used for transient kinetic experiments. For each set of mixing experiments, the concentration of MADH was fixed at 2 µM and the amicyanin concentration was varied. The amicyanin concentration was always in at least 10-fold excess of MADH to maintain pseudo-first-order reaction conditions. Values of kobs were determined from global fits of the raw data or analysis of the change in absorbance at 443 nm. |
Bishop, G. R.,
Davidson, V. L. |
Catalytic Role of Monovalent Cations in the Mechanism of Proton Transfer Which Gates an Interprotein Electron Transfer Reaction
Biochemistry; 1997; 36(44); 13586-13592. (Article) |
Experimental Procedures
Kinetic measurements were made using an On-Line Instrument Systems (OLIS) RSM1000 stopped-flow spectrophotometer. MADH was the limiting reactant with its concentration fixed between 1 and 4 mM. Pseudo-first-order conditions were maintained so that the concentration of amicyanin was much greater than that of MADH and well above the Kd for complex formation at each pH and salt concentration. Reactions were monitored between 330 and 490 nm where each of the redox forms of TTQ exhibits a different visible absorbance maximum. The absorbance maximum of amicyanin is at 595 nm, and because of its low extinction coefficient relative to that of MADH, redox-linked changes in amicyanin absorbance do not contribute significantly to the absorbance changes of oxidized, semiquinone, and reduced MADH (Davidson et al., 1995). |
Davidson, V. L.,
Jones, L. H., \
Graichen, M. E.,
Mathews, F. S.,
Hosler, J. P. |
Factors Which Stabilize the Methylamine Dehydrogenase-Amicyanin Electron Transfer Protein Complex Revealed by Site-Directed Mutagenesis
Biochemistry; 1997; 36(42); 12733-12738. (Article) |
Experimental Procedures
An On-Line Instrument Systems (OLIS) RSM1000 stopped-flow spectrophotometer was used for transient kinetic experiments. Reactions were monitored at 443 nm, which is an isosbestic point for the semiquinone and oxidized forms of MADH and is where MADH exhibits a of 26 200 M-1 cm-1 on conversion from reduced to semiquinone (Husain et al., 1987). |
Falzon, L.,
Davidson, V. L. |
Intramolecular Electron Transfer in Trimethylamine Dehydrogenase: A Thermodynamic Analysis
Biochemistry; 1996; 35(37); 12111-12118. (Article) |
| Kinetic Analysis. Stopped-flow experiments were performed essentially as previously described (Falzon & Davidson, 1996) using an On-Line Instrument Systems (OLIS, Bogart, GA) RSM 1000 stopped-flow spectrophotometer. Data were collected and analyzed using OLIS software on an IBM compatible 486 personal computer. We have observed that the reduced forms of TMADH are quite stable against reoxidation by air, and, therefore, anaerobic conditions were not necessary for these experiments. TMADH (2-4 µM) was mixed with various concentrations of trimethylamine (50 µM to 4 mM) in 0.1 or 0.01 M potassium phosphate buffer, pH 7.5, at different temperatures. The rate of intramolecular ET (monitored by the change in absorbance at 365 nm) was slow enough under these conditions to be measured by the stopped-flow technique. Under these conditions the varied substrate was in excess of the concentration of the fixed reactant, oxidized TMADH. |
Bishop, G. R.,
Brooks, H. B.,
Davidson, V. L. |
Evidence for a Tryptophan Tryptophylquinone Aminosemiquinone Intermediate in the Physiologic Reaction between Methylamine Dehydrogenase and Amicyanin
Biochemistry; 1996; 35(27); 8948-8954. (Article) |
Experimental Procedures
Rapid kinetic measurements were made using an On-Line Instrument Systems (OLIS) RSM1000 stopped-flow spectrophotometer. All reactions were monitored over a range between 300 and 500 nm where each of the three redox forms of TTQ exhibits a visible absorbance maximum. |
