Denaturation and the effects of temperature on hydrophobic-interaction and reversed-phase high-performance liquid chromatography of proteins : Bio-gel tsk-phenyl-5-pw column

Cytochrome c, myoglobin and lysozyme, as well as two synthetic peptides, TM-22 and TM-36, were used to examine denaturation of protein structure on a hydrophobic-interaction column, the Bio-Gel TSK-Phenyl -5-PW high-performance liquid chromatography column. The first three proteins were chosen because they have a monomeric structure while both synthetic peptides, which have the sequence Ac-(Lys-Leu-Glu-Ala-Leu-Glu-Gly)inn-Lys-amide where n  3 for TM-22 and n  5 for TM-36, are dimeric under solvent conditions used for hydrophobic-interaction chromatography. Only TM-36 is dimeric under reversed-phase conditions. Thus, denaturation of both tertiary and quaternary structure can be examined. The column was operated in both reversed-phase and hydrophobic-interaction modes. This, in combination with temperature variation between approximately 0-50°C, provided conditions where denaturing effects of the support could be examined. In reversed-phase mode, cytochrome c, myoglobin, and TM-22 were eluted in a denatured form throughout the temperature range. In contrast, lysozyme and TM-36 eluted primarily in their native conformation at low temperatures, but experienced partial or total denaturation at higher temperatures. Significantly, all of the polypeptides studied were denatured at room temperature by a conventional reversed-phase column, the Altex Ultrapore RPSC C-3 indicating that the Bio-Gel TSK-Phenyl-5-PW column is less denaturing. In the hydrophobic-interaction mode, the dimeric structure of TM-22 was totally disrupted at all temperatures, while TM-36, myoglobin, and cytochrome c underwent various degrees of partial denaturation as the temperature increased. Comparison of the temperatures at which the various polypeptides underwent denaturation on the column with their normal melting temperatures (where half the molecules are unfolded) demonstrated that the hydrophobic column itself, rather than the temperature, was primarily responsible for denaturation. Hence, even relatively “gentle” hydrophobic columns can promote denaturation of protein structure. Since the tertiary and quaternary structures of most proteins are stabilized by hydrophobic interactions, the possibility of denaturation must always be taken into consideration when a hydrophobic column is used.