Sym-dialkylsulfoxides (DASO), exhibiting both hydrophilic and hydrophobic character, are able to destabilize the native structure of proteins by weakening the hydrophobic interactions between non-polar residues as well by perturbing the structure of water surrounding the protein molecule. They showed effective cryoprotective effect on living system, more enhanced in the presence of diethylsulfoxide (DESO) than of dimethylsulfoxide (DMSO) or di-n-propylsulfoxide (DnPSO). The knowledge of the influence of DASO on the thermal denaturation and to the structural stability of protein is essential to understand their role in and we approached this problem by studying the thermal denaturation of lysozyme by means of Differential Scanning Calorimetry (DSC). MATERIALS AND METHODS: The lysozyme used in the present study is a salt-free, high purity from hen egg white Fluka product and used without further purification. DMSO is a low water content, high purity Fluka product; DESO and DnPSO were produced and purified according to the literature. The samples were made by dissolving the suitable amounts of the protein in sulfoxide ( 0-50 % w/w) stock solutions, containing 0,2 M NaCl and buffered at pH = 7.0 with phosphate buffer (≈ 10-3 M) and at pH = 5.0 with acetate buffer (≈ 10-3 M). DSC (Mettler Toledo TA-STAR 821e calorimeter) scans were performed at a heating rate of 1.0°C/min in the 5-90°C thermal range. The protein solutions were prepared at concentration of about 50 mg/cm3 and the effective amount present was confirmed by spectrophotometry at 280 nm. RESULTS: On heating, lysozyme solutions in the absence of DASO exhibit a endothermic DSC peak whose maximum temperature (Td) was found at 73.6 ± 0.1 °C, almost independent from the pH value (73.6 °C at pH = 5.0; 73.5 °C at pH = 7.0). Td dependence on both the DASO concentration and on the pH values is reported in Fig.1, showing a linear Td decrease in the presence of increasing DASO amounts. On the contrary the dependence of ΔHd on DASO concentration appear to be complex, showing an increase at lower and a lowering at higher DASO content. This behaviour can be presumable correlated to the complex behaviour of DASO on the sulfoxide-water systems. The thermal denaturation of lysozyme appears to be only partially reversible, and, in any case, it appeared to be significantly greater in the presence of DESO than of other sulfoxides. The lowering of Td at any concentration of DASO increased in correspondence of the hydrophobic character of the sulfoxide molecule. This result can be interpreted in terms of the hydrophobic interaction between the apolar part of the protein and the alkyl chains of the sulfoxides, thus relatively favouring the denaturated state. Moreover, as a consequence of the setting up of the hydrophobic interactions, the concentration of DASO molecules near to protein increases, with consequent modification of the water structure close the protein molecules. A similar result it was observed by studying the behaviour of lipidic bilayer in the water-DASO systems. The pH dependence on Td, high in the presence of low-hydrophobic DMSO and negligible in the presence of high-hydrophobic DnPSO, confirmed the noticeable role of the interaction between the alkyl chains and the apolar groups on the protein structure. It suggested that both the ‘water structure related’ interaction, due to the polar S=O group, as well the hydrophobic interaction are involved in the denaturation process. In particular, if both the effects occur in a similar extent, like in the presence of DESO, the reversibility of the lysozyme denaturation is enhanced. DESO acts as a ‘structure protecting substance’ against the irreversible thermal denaturation, and help to explain its particularly high cryoprotective effect found on the living systems .

DSC study of the Sym-Dialkylsulfoxides on the thermal denaturation of lisozyme solutions.

BONORA, SERGIO;TRINCHERO, ANDREA;
2004

Abstract

Sym-dialkylsulfoxides (DASO), exhibiting both hydrophilic and hydrophobic character, are able to destabilize the native structure of proteins by weakening the hydrophobic interactions between non-polar residues as well by perturbing the structure of water surrounding the protein molecule. They showed effective cryoprotective effect on living system, more enhanced in the presence of diethylsulfoxide (DESO) than of dimethylsulfoxide (DMSO) or di-n-propylsulfoxide (DnPSO). The knowledge of the influence of DASO on the thermal denaturation and to the structural stability of protein is essential to understand their role in and we approached this problem by studying the thermal denaturation of lysozyme by means of Differential Scanning Calorimetry (DSC). MATERIALS AND METHODS: The lysozyme used in the present study is a salt-free, high purity from hen egg white Fluka product and used without further purification. DMSO is a low water content, high purity Fluka product; DESO and DnPSO were produced and purified according to the literature. The samples were made by dissolving the suitable amounts of the protein in sulfoxide ( 0-50 % w/w) stock solutions, containing 0,2 M NaCl and buffered at pH = 7.0 with phosphate buffer (≈ 10-3 M) and at pH = 5.0 with acetate buffer (≈ 10-3 M). DSC (Mettler Toledo TA-STAR 821e calorimeter) scans were performed at a heating rate of 1.0°C/min in the 5-90°C thermal range. The protein solutions were prepared at concentration of about 50 mg/cm3 and the effective amount present was confirmed by spectrophotometry at 280 nm. RESULTS: On heating, lysozyme solutions in the absence of DASO exhibit a endothermic DSC peak whose maximum temperature (Td) was found at 73.6 ± 0.1 °C, almost independent from the pH value (73.6 °C at pH = 5.0; 73.5 °C at pH = 7.0). Td dependence on both the DASO concentration and on the pH values is reported in Fig.1, showing a linear Td decrease in the presence of increasing DASO amounts. On the contrary the dependence of ΔHd on DASO concentration appear to be complex, showing an increase at lower and a lowering at higher DASO content. This behaviour can be presumable correlated to the complex behaviour of DASO on the sulfoxide-water systems. The thermal denaturation of lysozyme appears to be only partially reversible, and, in any case, it appeared to be significantly greater in the presence of DESO than of other sulfoxides. The lowering of Td at any concentration of DASO increased in correspondence of the hydrophobic character of the sulfoxide molecule. This result can be interpreted in terms of the hydrophobic interaction between the apolar part of the protein and the alkyl chains of the sulfoxides, thus relatively favouring the denaturated state. Moreover, as a consequence of the setting up of the hydrophobic interactions, the concentration of DASO molecules near to protein increases, with consequent modification of the water structure close the protein molecules. A similar result it was observed by studying the behaviour of lipidic bilayer in the water-DASO systems. The pH dependence on Td, high in the presence of low-hydrophobic DMSO and negligible in the presence of high-hydrophobic DnPSO, confirmed the noticeable role of the interaction between the alkyl chains and the apolar groups on the protein structure. It suggested that both the ‘water structure related’ interaction, due to the polar S=O group, as well the hydrophobic interaction are involved in the denaturation process. In particular, if both the effects occur in a similar extent, like in the presence of DESO, the reversibility of the lysozyme denaturation is enhanced. DESO acts as a ‘structure protecting substance’ against the irreversible thermal denaturation, and help to explain its particularly high cryoprotective effect found on the living systems .
SIB 2004
8.13
8.13
S. Bonora; A. Trinchero; A. Torreggiani; A. Markarian
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/17677
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