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Thermodynamics of α-lactalbumin denaturation in hydrophobic-interaction chromatography and stationary phases comparison

1988; Elsevier BV; Volume: 458; Linguagem: Inglês

10.1016/s0021-9673(00)90556-9

1873-3778

Kálmán Benedek,

Chromatography in Natural Products

These studies present an evaluation of the role of surface hydrophobicity and temperature on the denaturation characteristics of α-lactalbumin under hydrophobic-interaction chromatography conditions, and a description of a model system to evaluate the role of chromatographic columns in the denaturation process of proteins. The chromatographic characteristics ofα-lactalbumin have been compared using commercially available poly(methyl); -(ethyl)- and -(propyl)aspartamide columns. These columns were designed for hydrophobic-interaction chromatography and represent a homologous series of stationary phases with increasing hydrophobic character. The elution profiles and retention times of α-lactalbumin have been also compared in different mobile phases and as a function of temperature. The chromatographic characteristics of the Beckman CAA-HIC column, another commercially available column for hydrophobic-interaction chromatography, have also been evaluated, permitting further insight into the effect of bonded phases on protein denaturation. The retention time-temperature curves obtained are sigmoidal, characteristic of the classical transition curves of protein denaturation. Thermodynamic parameters of the denaturation of α-lactalbumin are calculated from the retention data and the calculated transition temperatures and free energies of denaturation are used for comparison of the different columns. The model system and the calculated thermodynamic values represent a useful method for the evaluation of such columns, and can provide an estimate of the contribution of the stationary and mobile phases to the protein denaturation process.