Analysis of pH-dependent protein interactions with gel filtration medium.
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 were observed at low ionic strength, depending on whether the pH of the elution buffer was higher or lower than the pI 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 mM. 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 greater than pI, 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 less than pI a drastic increase in the capacity factor k' was observed owing to the additivity of hydrophobic and ion-exchange effects.(ABSTRACT TRUNCATED AT 250 WORDS)
Golovchenko
N P
NP
Department of Anaerobic Processes, Institute of Biochemistry and Physiology of Microorganisms, Puschino, Moscow Region.
Kataeva
I A
IA
Akimenko
V K
VK
D002480:Cellulase / Q000737:chemistry; D002850:Chromatography, Gel / Q000295:instrumentation*; D003013:Clostridium / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry; D009994:Osmolar Concentration; D011506:Proteins / Q000737:chemistry*
D011506:Proteins; D009113:Muramidase; D002480:Cellulase