Liver Glutamate dehydrogenase (GDH) is a homohexamer of 336 KDa. [1]. Due to the reversible oxidative-reductive properties of GDH, it is a prime candidate for incorporation into a biocatalyst [2]. We report the use of agarose isoelectric focusing (IEF) as a tool for the determination of protein-polyelectrolyte binding and calculate the stoichiometry of protein-polyelectrolyte complex formation [3]. The degree of protein-polyelectrolyte interaction was determined by the amount of protein migrating to its normal isoelectric point. The dilution profiles of all polyelectrolytes examined were dramatically different, indicating unique mechanisms of interaction. Of the 7 polyelectrolytes examined, only 3, Dextran sulphate, DEAE Dextran and Gafquat HS100 bound GDH as determined by IEF. These bound at considerably lower concentrations than expected (0.12µM-0.02mM). The stoichiometry of protein to polyelectrolyte complex formation was determined as 45:1. Electrophoretic analysis can be used as a preliminary screening process for determining the stoichiometry of protein-polyelectrolyte binding. We aim to determine whether polyelectrolytes used at these considerably reduced concentrations do indeed confer stability to the protein in question. The decreased polyelectrolyte concentration required to retain enzyme activity will significantly reduce the cost of stabilising proteins in all possible applications. This work was funded by European Union Brite EuRam project PRO.M.O.FIL.M., project no: BE97-4511.

[1] Peterson, P. E., Pierce, J. & Smith, T.J. (1997). J. Struct. Biol. 120, 73-77.

[2] Yao, T., Suzuki, S., Nakahara, T., Nishino, H. (1998). Talanta. 45, 917-923.

[3] Katakis, I., Ye, L. & Heller, A. (1994). J. Am. Chem. Soc. 116, 3617-3618.