Analysis of pH-dependent protein interactions with gel filtration medium

• Published in: Journal of Chromatography A 1992-02, Vol.591 (1), p.121-128
• Main Authors: Golovchenko, Nikolai P., Kataeva, Irina A., Akimenko, Vasily K.
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Interactions. Associations; Intermolecular phenomena; Molecular biophysics
• ISSN: 0021-9673
• DOI: 10.1016/0021-9673(92)80229-N

A prepacked Superose 12 HR 10 30 column was used to study the effects of elution ionic strength and pH on the chromatographic behaviour of a strong hydrophobic Clostridium thermocellum endoglucanase (1) and two weak hydrophobic proteins, Clostridium thermocellum endoglucanase C and egg white lysozyme. Ion-exclusion or ion-exchange interactions between weakly hydrophobic proteins and the gel matrix observed at low ionic strength, depending on whether the pH of the elution buffer was higher or lower than the p I values of the proteins. These interactions were due to the presence of negatively charged groups on the surface of Superose and could be eliminated at any pH by adding electrolyte at a concentration determined by its chemical identity. The optimum results were observed with sodium sulphate at a concentration of 100 m M. The chromatographic behaviour of strong hydrophobic endoglucanase (1) on a Superose column as a function of pH was much more complex because of two interplaying effects, electrostatic and hydrophobic. Ideal size-exclusion chromatography could be achieved only in a narrow range of the conditions: first, the mobile phase must contain a weak salting-out electrolyte such as NaCl, and second, the mobile phase pH must be high enough that hydrophobic interactions between the solute and support are balanced by their electrostatic repulsion. At pH>p I, the retardation of endoglucanase (1) gradually increased with decreasing pH as a result of lowering of repulsive electrostatic interactions whether or not the buffer ionic strength was high. At pH < p I a drastic increase in the capacity factor k′ was observed owing to the additivity of hydrophobic and ion-exchange effects. Overall, the chromatographic behaviour of endoglucanase (1) on a Superose column could be adequately described in terms of the theory of potential barrier chromatography. The explanation presented could obviously be valid for the behaviour of any protein on any gel matrix, as its mechanism was described using lumped physical notions such as hydrophobic and electrostatic interactions, charges and potentials. Virtually all currently known gel filtration media are more or less hydrophobic and are weak cation exchangers.