ABSTRACT
The reductase domains of neuronal NOS, endothelial NOS and two constitutive nitric oxide synthase (cNOS) share higher sequence similarity (>60%). In order to evaluate the role of ferredoxinNADP+ reductase (FNR) module in adjusting NOS catalytic activities, chimeras were by interchanging the FNRlike module between endothelial NOS and neuronal NOS in the present study. The assays of steadystate enzymatic activities for cytochrome c and ferricyanide reduction, NO synthesis and NADPH oxidation were performed spectrophotometrically. The two NOS FNR modules transferred their ferricyanide reductase character to the chimera enzymes. Results showed that the FNR module was important in adjusting electrons flow through the reductase domain and out of the FMN module. Results indicated that the FNR module was critical in controlling the electron transfer capacities of the FMN module.
Subject(s)
Biocatalysis , Ferredoxin-NADP Reductase/metabolism , Nitric Oxide Synthase/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Cytochromes c/metabolism , Ferricyanides/metabolism , Flavins/metabolism , Heme/metabolism , Kinetics , NADP/metabolism , Nitric Oxide/biosynthesis , Oxidation-Reduction , Oxygenases/metabolism , Rats , Recombinant Fusion Proteins/isolation & purification , Spectrum Analysis , Time FactorsABSTRACT
The interactions between Trypsin and bifendate (DDB) or analogs (I, II and III) were investigated by fluorescence, UV-visible absorption, resonance light scattering, synchronous fluorescence and 3D spectroscopy under mimic physiological conditions. The results revealed that DDB and analogs caused the fluorescence quenching of Trypsin by the formation of DDB/I/II/III-Trypsin complex. The quenching and energy transfer mechanisms were discussed. The binding constants and thermodynamic parameters at three different temperatures were obtained. The hydrophobic interaction was the predominant intermolecular forces to stabilize the complex. Results showed that DDB was the stronger quencher and bound to Trypsin with higher affinity than other three analogs.
Subject(s)
Biphenyl Compounds/metabolism , Spectrometry, Fluorescence/methods , Trypsin/metabolism , Biphenyl Compounds/chemistry , Energy Transfer , Hydrogen-Ion Concentration , Kinetics , Light , Protein Binding , Protein Conformation , Scattering, Radiation , Spectrophotometry, Ultraviolet , TemperatureABSTRACT
The interaction between ovalbumin (OVA) and three purine alkaloids (caffeine, theophylline and diprophylline) was investigated by the aid of intrinsic and synchronous fluorescence, ultraviolet-vis absorbance, resonance light-scattering spectra and three-dimensional fluorescence spectra techniques. Results showed that the formation of complexes gave rise to the fluorescence quenching of OVA by caffeine, theophylline, and diprophylline. Static quenching was confirmed to results in the fluorescence quenching. The binding site number n, apparent binding constant KA and corresponding thermodynamic parameters were measured at different temperatures. The binding process was spontaneous molecular interaction procedures in which both enthalpy and Gibbs free energy decreased. Van der Waals forces and hydrogen bond played a major role in stabilizing the complex. The comparison between caffeine, theophylline, and diprophylline was made, and thermodynamic results showed that diprophylline was the strongest quencher and bound to OVA with the highest affinity among three compounds. The influence of molecular structure on the binding aspects was reported.
Subject(s)
Caffeine/chemistry , Dyphylline/chemistry , Ovalbumin/chemistry , Theophylline/chemistry , Binding Sites , Fluorescence , Hydrogen Bonding , Molecular Structure , Protein Binding , Spectrometry, Fluorescence , ThermodynamicsABSTRACT
The interactions of bifendate (DDB) or analogs (Bicyclol, I, II and III) with catalase are analyzed by spectrophotometric methods. The fluorescence spectra results show the intrinsic fluorescence of catalase is strongly quenched by DDB or analogs with a static quenching procedure. The binding constants are obtained at three temperatures. The thermodynamics parameters (ΔH, ΔS, ΔG) indicate the hydrophobic and electrostatic interactions play a major role in the interaction. The results of synchronous fluorescence, UV-vis absorption and three-dimensional fluorescence spectra demonstrate that the microenvironments of Trp residue of catalase are disturbed by the analogs. Thermodynamic results showed that DDB is the strongest quencher and bind to catalase with the highest affinity among five compounds.