Complex kinetics and residual structure in the thermal unfolding of yeast triosephosphate isomerase.

BACKGROUND: Saccharomyces cerevisiae triosephosphate isomerase (yTIM) is a dimeric protein that shows noncoincident unfolding and refolding transitions (hysteresis) in temperature scans, a phenomenon indicative of the slow forward and backward reactions of the native-unfolded process. Thermal unfolding scans suggest that no stable intermediates appear in the unfolding of yTIM. However, reported evidence points to the presence of residual structure in the denatured monomer at high temperature. RESULTS: Thermally denatured yTIM showed a clear trend towards the formation of aggregation-prone, ß-strand-like residual structure when pH decreased from 8.0 to 6.0, even though thermal unfolding profiles retained a simple monophasic appearance regardless of pH. However, kinetic studies performed over a relatively wide temperature range revealed a complex unfolding mechanism comprising up to three observable phases, with largely different time constants, each accompanied by changes in secondary structure. Besides, a simple sequential mechanism is unlikely to explain the observed variation of amplitudes and rate constants with temperature. This kinetic complexity is, however, not linked to the appearance of residual structure. Furthermore, the rate constant for the main unfolding phase shows small, rather unvarying values in the pH region where denatured yTIM gradually acquires a ß-strand-like conformation. It appears, therefore, that the residual structure has no influence on the kinetic stability of the native protein. However, the presence of residual structure is clearly associated with increased irreversibility. CONCLUSIONS: The slow temperature-induced unfolding of yeast TIM shows three kinetic phases. Rather than a simple sequential pathway, a complex mechanism involving off-pathway intermediates or even parallel pathways may be operating. ß-strand-type residual structure, which appears below pH 8.0, is likely to be associated with increased irreversible aggregation of the unfolded protein. However, this denatured form apparently accelerates the refolding process.

Physicochemical properties of collagen sheet from bovine femur.

PURPOSE: In the present study collagen sheets were prepared from the spongy part of the bovine femur and characterized from the physicochemical point of view. METHODS: The physicochemical properties of the collagen sheets were studied using scanning electronic microscopy (SEM) and thermogravimetrical analysis (TGA). RESULTS: The SEM studies showed that the collagen sheets are porous and exhibit a fibrous nature. The TGA study revealed that the collagen sheets are a two-phase system containing a protein and a mineral phase. In order to determine the collagen type present in the sample, the sheets were dissolved and studied by circular dichroism (CD) and electrophoresis techniques. The results obtained showed that the nature of the collagen is type I. CONCLUSIONS: The type I collagen sheets from bovine femur isolated in this study are proposed as a biomaterial for medical applications, for example, as an osteoinductive material or they could be used as a bone substitute.