title: Sorbitol prevents the self-aggregation of unfolded lysozyme leading to and up to 13 degrees C stabilisation of the folded form.
abstract: We present a calorimetric investigation of stabilisation of hen egg-white lysozyme with sorbitol in the pH range 3.8-10.5. Differential scanning calorimetry and steady-state fluorescence were used to determine the denaturation temperatures of lysozyme as a function of sorbitol concentration. The fluorescence data were collected in the presence of 2M urea to lower the melting point of the protein to an observable range of the instrument. The effect of sorbitol on the activation energy of unfolding was investigated by scanrate studies. The effect of sorbitol lysozyme interaction was investigated using isothermal titration calorimetry. The titration experiments were performed with folded as well as unfolded lysozyme to investigate in more detail the nature of the interaction. The data obtained in those experiments show a remarkable stabilisation effect of sorbitol. We observed a 4.0 degrees C increase in the Tm for 1 M sorbitol in the pH range 3.8-8.5 by scanning calorimetry. The effect increases dramatically at pH 9.5 where we observe a 9.5 degrees C stabilisation. An increase in the sorbitol concentration to 2 M stabilises lysozyme by 11.3-13.4 degrees C in the pH range 9.5-10.5. In the absence of urea, no significant effects of sorbitol were observed on the activation energy for unfolding for lysozyme at pH 4.5. This indicates together with the results from the titration experiments that sorbitol may stabilise the folded form of lysozyme by destabilising the unfolded form of lysozyme. At pH values at and above lysozyme's pI (approximately 9.3), the unfolding of the protein is accompanied with a substantial amount of self-aggregation seen in the calorimetry experiments in the ratio of DeltaH(cal)/DeltaH(vH). In the presence of sorbitol, the self-aggregation was counterbalanced by higher sorbitol concentrations. These results strongly suggest a negative influence of sorbitol on the unfolded form of lysozyme and thereby stabilising the native form.
authors.affiliation: The Biostructure and Protein Engineering Group, Department of Life Science, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark. steffen.petersen@nanobio.aau.dk
authors.forename: Steffen B
authors.initials: SB
authors.lastname: Petersen
authors.forename: Virpi
authors.initials: V
authors.lastname: Jonson
authors.forename: Peter
authors.initials: P
authors.lastname: Fojan
authors.forename: Reinhard
authors.initials: R
authors.lastname: Wimmer
authors.forename: Shona
authors.initials: S
authors.lastname: Pedersen
mesh_terms: D002152:Calorimetry, Differential Scanning / Q000379:methods; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D013012:Sorbitol / Q000737:chemistry*
chemical_list: D013012:Sorbitol; C421864:hen egg lysozyme; D009113:Muramidase