Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength

Abstract

a b s t r a c t a r t i c l e i n f oDSC analysis has been used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Since the temperature at which protein unfolding occurs, T m , varies with different solution conditions, the effect on the melting temperature and the degree of refolding after thermal denaturation in low ionic strength sodium phosphate buffers (5-1000 mM) over a range of pH (5-9) in the presence/absence of disaccharides is examined. This study compares the enthalpies of unfolding during successive heating cycles to quantify reversibility following thermal denaturation. The disaccharides, trehalose and maltose were used to assess if the disaccharide induced increase in T m is reflected in the reversibility of thermally induced denaturation. There was extensive overlap between the T m values where non-reversible and reversible thermal denaturation occurred. Indeed, for pH 6, at the highest and lowest T m , no refolding was observed whereas refolding was observed for intermediate values, but with similar T m values having different proportions of refolded protein. We established a method to measure the degree of reversible unfolding following thermal denaturation and hence indirectly, the degree to which protein is lost to irreversible aggregation, and show that solution conditions which increase melt transition temperatures do not automatically confer an increase in reversibility. This type of analysis may prove useful in assessing the stability of proteins in both the biopharmaceutical and food industries.

Collagen from the Marine Sponges Axinella cannabina and Suberites carnosus: Isolation and Morphological, Biochemical, and Biophysical Characterization

“…In contrast to the previous transitions discussed, the examination of both the reversing and non-reversing components of this specific transition suggests that this process is, to a great extent, reversible. Taking into consideration that during the denaturation of small proteins (for instance lysozyme) [60] the reversible unfolding has the largest contribution, whereas the irreversible process still remains well detectable, we tentatively ascribe this low temperature transition to the denaturation of small molecular weight collagen species that are present in this sample.…”

Bigels formed via spinodal decomposition of unfolded protein

“…Gels composed of BSA only (at low ionic strength) were prepared over a range of protein concentrations (10-180 mg ml À1 ; f BSA = 0.007 to 0.133) and heated above the melt transition temperature (T m ) for BSA (determined using differential scanning calorimetry 24 ). BSA gels below a volume fraction f BSA = 0.059 (80 mg ml À1 ) are transparent and very weak with elastic moduli in the region of 1 kPa.…”

“…24 Samples were then heated at 80 1C for 1 hour in a water bath and cooled at room temperature for a further 1 hour. Gelatin samples were heated at 80 1C for 10 minutes in a water bath (to ensure that the gelatin was fully dissolved).…”

Thermoresponsive behavior of micellar aggregates from end-functionalized PnBA-b-PNIPAM-COOH block copolymers and their complexes with lysozyme

“…Finally it has to be noted that lysozyme is stable up to 70º C in similar solution conditions so we do not expect any alteration in its structure caused by temperature 37 .…”