RESUMO
The fluorescence and synchronous fluorescence spectra were employed to study the binding between sodium azide (NaN3) and horseradish peroxidase (HRP), which was affected by the molecular conformation and the microenvironment of the fluorescence residues. The mechanism of the fluorescence quenching of HRP by NaN3 was discussed and the binding constant and the number of binding sites of non-covalent bond between them were calculated.
Assuntos
Fluorescência , Peroxidase do Rábano Silvestre/química , Azida Sódica/química , Espectrometria de Fluorescência/métodos , Algoritmos , Sítios de Ligação , Peroxidase do Rábano Silvestre/metabolismo , Cinética , Ligação Proteica , Projetos de Pesquisa , Azida Sódica/metabolismoRESUMO
Comparing the fluorescence spectra of tobacco peroxidase I (TOP I) solution and the solutions titrated by iodine anions, the number of binding locus and the binding constant of iodine anion to TOP I were calculated by using the modified Stern-Volmer equation. The mechanism of the quenching of fluorescence spectra and the distribution of tryptophane residues in the TOP I molecule were discussed.
Assuntos
Nicotiana/enzimologia , Peroxidase/química , Proteínas de Plantas/química , Espectrometria de Fluorescência/métodos , Algoritmos , Ânions/química , Concentração de Íons de Hidrogênio , Iodo/química , Cinética , Peroxidase/metabolismo , Proteínas de Plantas/metabolismo , Ligação Proteica , Projetos de Pesquisa , TemperaturaRESUMO
The ultraviolet/visible spectra of TOP I were studied. It was proved that TOP I is an acid enzyme containing it hemochrome as an agon. When the pH reduced, the Soret absorption in UV-Vis region exhibited a blue shift, when pH increased exhibited a red shift. The result of the influence of carbamide, the denaturant, on the spectra showed that TOP I may have a unfolded structural change in the solution, which made the peptide chain completely extend. After adding Fe(III), Fe(II), Cu(II), Zn(II), Co(II), Ni(II) and Sn(II) to the apo-TOP I, the UV-Vis spectra changed except for Fe(III), which almost did not change at all. The strongest characteristic absorption peak of the Soret showed a blue shift to different extent and it became weak gradually. The situation of alpha-strip and beta-strip remained unchanged while the relative strength decreased.
Assuntos
Nicotiana/enzimologia , Peroxidases/química , Espectrofotometria Ultravioleta/métodos , Dicroísmo Circular/métodos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/fisiologia , Conformação Proteica , Espectrofotometria Infravermelho/métodos , Análise Espectral RamanRESUMO
Tobacco peroxidase (TOPI) has been purified by ammonium sulfate fractionation and column chromatography, involving ion exchange on DE-52 cellulose, gelfiltration on Sephadex G-75 and ion exchange on DEAE-sephadex A-50. The specific activity of peroxidase purified was 4,826 U/mg. It has been demonstrated by SDS-PAGE as a single band. Its molecular weight (matrix assisted laser desorption/ionization time of flight mass spectra) and isoelectric point (pI) have been determined to be 21,888.5 and 3.5 respectively. The native enzyme is one of a haemachrome-containing acidic protein and has its soret maximum at 402 nm and alpha and beta bands at 636 nm and 498 nm, respectively. Its characteristic absorption spectra and fluorescence spectra changed in different pH and denaturant. It has its own characteristic absorption spectra and fluorescence spectra.
