ABSTRACT
The fluorescence of the single tryptophan (Trp69) of cutinase from Fusarium solani pisi, free in aqueous solution and adsorbed onto the surface of poly(methyl methacrylate) (PMMA) latex particles, was studied at pHs of 4.5 and 8.0. The monodisperse PMMA particles (d=106.0+/-0.1 nm) were coated with a quite compact monolayer of cutinase at both pH values. The Trp decay curve of the folded free cutinase in solution can only be fitted with a sum of four exponentials with lifetimes of 0.05, 0.3-0.4, 2-3, and 6-7 ns, irrespective of pH. The 50 ps lifetime is attributed to the population of Trp residues hydrogen bonded with the Ala32 and strongly quenched by a close disulfide bridge, while the other lifetimes are due to the non-hydrogen-bonded Trp rotamers. The 50 ps Trp lifetime component disappears by temperature melting and upon protein adsorption, owing to the disruption of the Trp-Ala hydrogen bond and the release of the Trp residue from the vicinity of the disulfide bridge. This shows that cutinase adsorption occurs by the region of the protein where the Trp is located, which agrees with the retention of cutinase enzymatic activity by adsorption at basic pH.
Subject(s)
Carboxylic Ester Hydrolases/chemistry , Nanoparticles/chemistry , Polymethyl Methacrylate/chemistry , Tryptophan/analysis , Tryptophan/chemistry , Absorption , Disulfides/chemistry , Fluorescence , Fusarium/enzymology , Models, Molecular , Protein Folding , Protein Structure, TertiaryABSTRACT
Thermosensitive poly(N-isopropylacrylamide)-based polymer particles were synthesised, and screened for the adsorption of human immunoglobulin G (hIgG). At pH 9 the adsorption on microgel particles was strongly affected by temperature, approximately 40 mg hIgG/g support (90% of initial hIgG) being adsorbed at 40 degrees C but only 10% of initial hIgG at 25 degrees C. At pH 5 the maximum adsorbed amount (20 mg hIgG/g support) was similar for both temperatures. The adsorption of hIgG on to charged poly(methyl methacrylate)/poly(N-isopropylacrylamide) core-shell latexes was negligible (5-10 mg hIgG/g support) at the same temperature and pH conditions. The lower adsorption of hIgG onto the core-shell particles is explained by steric interactions due to the small size of the shell.
Subject(s)
Acrylamides/pharmacokinetics , Immunoglobulin G/metabolism , Nanoparticles/chemistry , Polymers/chemistry , Acrylic Resins , Adsorption , Humans , Hydrogen-Ion Concentration , Immunoglobulin G/chemistry , Polymers/pharmacokinetics , Temperature , Time FactorsABSTRACT
Fluorescence, CD, and activity measurements were used to characterize the different conformational states of horseradish peroxidase A1 induced by thermal unfolding. Picosecond time-resolved fluorescence studies showed a three-exponential decay dominated by a picosecond lifetime component resulting from energy transfer from tryptophan to heme. Upon thermal unfolding a decrease in the preexponential factor of the picosecond lifetime and an increase in the quantum yield were observed approaching the characteristics observed for apoHRPA1. The fraction of heme-quenched fluorophore decreased to 0.4 after unfolding as shown by acrylamide quenching. A new unfolding pathway for HRPA1 was proposed and the effect of the low molecular weight solutes trehalose, sorbitol, and melezitose on this pathway was analyzed. Native HRPA1 unfolds with an intermediate between the native and the unfolded conformation. The unfolded conformation can refold to the native state or to a native-like conformation with no calcium ions upon cooling or can give an irreversible denatured state. The refolded conformation with no calcium ions was clearly identified in a second thermal scan in the presence of EDTA and shows secondary and tertiary structures, heme reincorporation in the cavity, and at least 59% of activity. Melezitose stabilized the refolded Ca2+-depleted protein and induced a more complex mechanism for heme disruption. The effect of sorbitol and trehalose were mainly characterized by an increase in the temperature of unfolding.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Protein Conformation , Circular Dichroism , Fluorescence Resonance Energy Transfer , Hot Temperature , Protein Denaturation , Spectrometry, Fluorescence , Time Factors , Tryptophan/chemistryABSTRACT
Os autores relatam um caso de comprometimento pulmonar por T. gondii, em homem de 57 anos. O diagnóstico foi realizado por meios sorológicos, após exaustiva avaliação clínico-radiológica. São discutidas as variantes clínicas de polimiosite apresentadas pelo paciente e o comprometimento do aparelho respiratório, tendo em vista as poucas citações desta entidade na literatura médica brasileira
Subject(s)
Humans , Male , Middle Aged , Leucovorin/therapeutic use , Lung Diseases, Parasitic/parasitology , Polymyositis/parasitology , Sulfadiazine/therapeutic use , Toxoplasmosis/diagnosis , Lung Diseases, Parasitic/diagnosis , Lung Diseases, Parasitic/drug therapy , Toxoplasmosis/drug therapyABSTRACT
Percepçöes, individualidade, linguagem, idéias, significado, cultura, escolha, moral e ética, todos existem em decorrência da evoluçäo e do funcionamento do sistema nervoso. Teme-se, por vezes, que a concepçäo da consciência como resultado de um processo biológico corresponda a uma ®profanaçäo do espírito humano¼, com conseqüente abandono do comportamento moral e ético. Na verdade, ao se investigar a consciência como fenômeno natural e näo místico, ampliam-se nossas possibilidades de entendê-la, com ganhos científicos, teóricos e sociais, além dos éticos e morais. Discute-se como a evoluçäo por seleçäo natural e a organizaçäo biológica do sistema nervoso permitem explicar as bases da individualidade, da intencionalidade, de representaçöes simbólicas e do significado. Fenômenos observados em pacientes com danos neurológicos reforçam a concepçäo de funcionamento modular do sistema nervoso; a consciência näo seria uma propriedade exclusiva de um módulo único do sistema nervoso, mas fruto do funcionamento sincrônico de diferentes módulos.
