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Polyethyleneimine-Citrate and Phosphate Systems. Potential Applications in Protein Separation. Alejandra Manzur, Darío Spelzini, Diana Romanini, Beatriz Farruggia and Guillermo Picó Bioseparation Lab. Physical Chemistry Department. Faculty of Biochemical and Pharmaceutical Sciences. University of Rosario, FonCyT, CIUNR and CONICET. Suipacha 570 (S2002RLK) Rosario. ARGENTINA. E-mail: gpico@fbioyf.unr.edu.ar Charged polymers are widely distributed in nature; they have also been obtained synthetically as random linear molecules. They precipitate in solution in a pH range and have been used for protein purification by means of ionic exchange. These polyelectrolytes interact with small ions of opposite charge to form complexes which can be precipitated in a pH range (DpH) where they behave like ampholytes. The aqueous solution behaviour of polyethyleneimine (PEI) (a cationic synthetic polymer, pKa 9.6) in the citrate and phosphate (Pi) presence was investigated by turbidimetry. The variation of the absorbance at 420 nm with pH, polymer concentration and ionic strength was explored. The complex formation was also assayed as a potential application to isolate proteins [1]. It has been found that in the citrate or phosphate presence, PEI forms insoluble complexes in the pH interval between 4 to 8 where they behave like ampholytes. Both complexes do not show exactly the same pH value of precipitation because the PEI-anion stechiometry is different. Phosphate and citrate have three pKa but with different values, which makes their acid basic parameters to be slightly different. The isoelectric pHs were 5.5 and 6.2 for phosphate and citrate respectively. Citrate showed a better precipitation effect than phosphate. The precipitate was reversibly dissolved in NaCl (for concentrations higher than 0.5 M). The formation of the complex between PEI and Pi or citrate can be explained considering the acid base equilibrium that takes place in a mixture of PEI with these salts. At pHs bellow 8, all PEI amine groups are protonated, since its pKa is 9.6. Therefore a coulombic interaction between the particles of opposite charge is carried out. The formed complex may have a negative or positive electrical net charge, depending on the PEI/anion ratio and the pH of the medium [2]. The phase diagram showed that the isoelectric pH of the complex was independent of the PEI/anion ratio. The complex was formed at pH 5.5 in the presence of Bovine serum albumin (pI 4.8), lysozyme (pI 11.2) and pepsin (pI 2.5). The latter was completely precipitated with the polymer-anion complex, while albumin and lysozyme were not. The insoluble complex was dissolved in NaCl 2.5 M, the pepsin recovery was 98% (in citrate) and 87 % (in Pi), while 5 % of albumin and 0% of lysozyme were recovered in both systems. Studies of pepsin thermal stability (by differential scanning calorimetry) showed that polyethyleneimine presence increased the enzyme denaturation temperature. Circular dichroism spectra of pepsin revealed no loss of secondary and tertiary enzyme structure by polyethyeneimine. Pepsin enzymatic activity did not vary within 24 hs. in presence of PEI. These results open up the possibility to use this method to isolate acidic enzymes from their natural source. Experiments using other pure proteins and their natural source are in progress in our laboratory. ________________ [1] U. Dissing, B. Matiasson. J Biotecnol., 52 (1996) 1-10. [2]C.S. Patrickios et al. Langmuir, 15 (1998) 1613-1620

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