RESUMEN
OBJECTIVES: The purpose of this study was to evaluate how evaporation affects the shelf life of a one-bottle universal adhesive. METHODS: Three different versions of Scotchbond Universal (SBU, 3M ESPE, Seefeld, Germany) were prepared using a weight-loss technique. SBU0 was left open to the air until maximal weight loss was obtained, whereas SBU50 was left open until 50% of evaporation occurred. In contrast, SBU100 was kept closed and was assumed to contain the maximum concentration of all ingredients. The degree of conversion (DC) was determined by using Fourier transform infrared spectroscopy on different substrates (on dentin or glass plate and mixed with dentin powder); ultimate microtensile strength and microtensile bond strength to dentin were measured as well. RESULTS: DC of the 100% solvent-containing adhesive (SBU100) was higher than that of the 50% (SBU50) and 0% (SBU0) solvent-containing adhesives for all substrates. DC of the adhesive applied onto glass and dehydrated dentin was higher than that applied onto dentin. Even though the ultimate microtensile strength of SBU0 was much higher than that of SBU50 and SBU100, its bond strength to dentin was significantly lower. CONCLUSIONS: Evaporation of adhesive ingredients may jeopardize the shelf life of a one-bottle universal system by reducing the degree of conversion and impairing bond strength. However, negative effects only became evident after more than 50% evaporation.
Asunto(s)
Adhesivos , Resistencia a la TracciónRESUMEN
Pressure unfolding-refolding and the subsequent aggregation of human serum albumin (HSA) was investigated by high-pressure Fourier transform infrared measurements. HSA is completely unfolded at 1 GPa pressure, but the unfolding is not cooperative. Hydrogen-deuterium exchange experiments suggest that a molten globule-like conformation is adopted above 0.4 GPa. An intermediate was formed after decompression, which differs from the native state only slightly in terms of the secondary structure, but this intermediate is more stable against the temperature-induced gel formation than the pressure-untreated native protein. This observation can be explained by assuming that the pressure unfolded-refolded protein is in a misfolded state, which is more stable than the native one.
Asunto(s)
Pliegue de Proteína , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Calefacción , Humanos , Presión , Desnaturalización Proteica , Renaturación de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Refolding of hen egg white lysozyme after pressure unfolding was measured by FTIR spectroscopy. The high-pressure treatment was found to be useful for unfolding/refolding studies because pressure acts against aggregation, and therefore no irreversible aggregation takes place during the pressure treatment. After the release of the pressure, folding intermediate structures were found which were formed during the decompression of the lysozyme. These were aggregation prone when heated, as indicated by their lower stability against aggregation. The intermediates were only formed if the protein was unfolded, subdenaturing pressures could not populate these intermediates. We introduced the notion of a superfunnel to describe the free energy landscape of interacting polypeptide chains. This can explain the propensity of folding intermediates to aggregate. A possible Gibbs-free energy landscape for lysozyme was constructed for the whole pressure-temperature plane.
Asunto(s)
Muramidasa/química , Unión Proteica , Animales , Cristalografía por Rayos X , Presión , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier , TemperaturaRESUMEN
The pressure stability of horseradish peroxidase isoenzyme C and the identification of possible stabilizing factors are presented. The effect of heme substitution, removal of Ca(2+), binding of a small substrate molecule (benzohydroxamic acid), and reduction of the disulfide bonds on the pressure stability were investigated by FTIR spectroscopy. HRP was found to be extremely stable under high pressure with an unfolding midpoint of 12.0 +/- 0.1 kbar. While substitution of the heme for metal-free mesoporphyrin did not change the unfolding pressure, Ca(2+) removal and substrate binding reduced the midpoint of the unfolding by 2.0 and 1.2 kbar, respectively. The apoprotein showed a transition as high as 10.4 kbar. However, the amount of folded structure present at the atmospheric pressure was considerably lower than that in all the other forms of HRP. Reduction of the disulfide bonds led to the least pressure stable form, with an unfolding midpoint at 9.5 kbar. This, however, is still well above the average pressure stability of proteins. The high-pressure stability and the analysis of the pressure-induced spectral changes indicate that the protein has a rigid core, which is responsible for the high stability, while there are regions with less stability and more conformational mobility.
Asunto(s)
Calcio/química , Disulfuros/química , Hemo/química , Peroxidasa de Rábano Silvestre/química , Amidas , Apoenzimas/química , Sitios de Unión , Estabilidad de Enzimas , Ácidos Hidroxámicos/química , Ligandos , Metales/química , Oxidación-Reducción , Porfirinas/química , Presión , Unión Proteica , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
The structure of the wheat gamma 46 gliadin was investigated, in aqueous solutions, under high pressure or temperature by the use of ultraviolet and fluorescence spectroscopic techniques. We found that high pressure (above 400 MPa) induces a change in the protein conformation that results in a decrease of the polarity of the environment of aromatic amino acids. This new conformation was able to bind the hydrophobic probe, 8-anilino-1-naphtalene-sulfonic acid (ANS), indicating an increase in the gliadin surface hydrophobicity. Thermodynamic parameters of this conformational change were measured and infrared spectroscopy studies were used to probe the potential secondary structure modifications. The high stability of gamma 46 gliadin could be related to its elastic character, as the observed changes were always found to be reversible.
Asunto(s)
Gliadina/química , Triticum/química , Naftalenosulfonatos de Anilina/química , Colorantes Fluorescentes/química , Presión , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de Fourier , TemperaturaRESUMEN
The pressure denaturation of trypsin from bovine pancreas was investigated by fluorescence spectroscopy in the pressure range 0. 1-700 MPa and by FTIR spectroscopy up to 1000 MPa. The tryptophan fluorescence measurements indicated that at pH 3.0 and 0 degrees C the pressure denaturation of trypsin is reversible but with a large hysteresis in the renaturation profile. The standard volume changes upon denaturation and renaturation are -78 mL.mol-1 and +73 mL.mol-1, respectively. However, the free energy calculated from the data in the compression and decompression directions are quite different in absolute values with + 36.6 kJ.mol-1 for the denaturation and -5 kJ. mol-1 for the renaturation. For the pressure denaturation at pH 7.3 the tryptophan fluorescence measurement and enzymatic activity assays indicated that the pressure denaturation of trypsin is irreversible. Interestingly, the study on 8-anilinonaphthalene-1-sulfonate (ANS) binding to trypsin under pressure leads to the opposite conclusion that the denaturation is reversible. FTIR spectroscopy was used to follow the changes in secondary structure. The pressure stability data found by fluorescence measurements are confirmed but the denaturation was irreversible at low and high pH in the FTIR investigation. These findings confirm that the trypsin molecule has two domains: one is related to the enzyme active site and the tryptophan residues; the other is related to the ANS binding. This is in agreement with the study on urea unfolding of trypsin and the knowledge of the molecular structure of trypsin.