(240b) Further Study of the Effect of Electrostatic Properties in Binary Protein Ultrafiltration

Our research group has been working continuously on the study of the effect of electrostatic properties in binary protein ultrafiltrations using three model binary protein systems, bovine serum albumin (BSA, MW 67 kD, pI 4.7)/hen egg lysozyme (HEL, MW 14 kD, pI 11.0), BSA/cytochrome c (CytC, MW 15 kD, pI 10.5) and BSA/alpha-lactalbumin (alpha-LA, MW 15 kD, pI 5.2). HEL and CytC were selected because of their similarity in size and electrostatic properties while alpha-LA was selected because of its significant difference in isoelectric point. Ultrafiltration experiments using two different 30,000-MWCO membranes, polyestersulfone (PES, negatively charged) and composite regenerated cellulose (CRC, relatively neutral charged) membranes, were studied at three different pH values, pH 4.7, pH 7 and pH 10.

Previously our group presented the comparison of the ultrafiltration performances of two of those systems, BSA/HEL and BSA/CytC. HEL and CytC are similar in size and charge properties, so it was expected that the ultrafiltration of those two systems would show similar fractionation performances. However our research showed that the fractionation performances of BSA/CytC system were consistently better than those of the BSA-HEL system in almost all experimental conditions studied. On the average base, the selectivity of CtyC/BSA is 2 to 3 times of that of HEL/BSA and the yield of CytC is around 1.25 times of that of HEL. Post-experimental tests of apparent zeta-potential and resistance associated with fouling had suggested that membrane fouling should not be the dominating cause of the significant differences observed from those two systems. The results suggested that more detailed protein charge characteristics such as the charge density or distribution of amino acids of the protein molecule might be the true cause to those phenomena.

The ultrafiltration of BSA/alpha-LA system showed significantly different performances than the other two binary protein systems. This was expected since the pI of alpha-LA is 5.2, which is far different from the pI of HEL and CytC, but is close to that of BSA. Those differences in ultrafiltration performances include different pH dependence and different sieving and selectivity profiles. What especially drew attention was the phenomenon of reverse selectivity observed in several cases, which means that the selectivity of alpha-LA over BSA was less than one. In other words, the selectivity of BSA over alpha-LA was achieved. This may be caused by the severe interactions between alpha-LA and the membrane.

Based on the results obtained from the study of those three model binary protein systems under various ultrafiltration conditions, our group are currently working on the development of a more complete model on the effect of electrostatic properties in binary protein ultrafiltrations which include the more detailed study of protein conformation, protein charge density, protein-protein interactions and protein-membrane interactions. The findings will be discussed.