Understanding the Effects of Polydispersity on Protein Conjugation

Abstract

The level of polydispersity or aggregation in a protein population is an important factor to consider when performing conjugation or labeling reactions. In such reactions, the more aggregated portions of a polydispersed protein population have a tendency to be less labeled—or have a lower degree of labeling (DOL)—than the more highly labeled, less aggregated portions of the population. The mechanism by which this differential labeling occurs is thought to work as follows: When conjugating a protein to a detection mechanism or other conjugate of interest, the reaction, as it occurs in solution, can be visualized as taking place in a vast, empty space. In such a scenario, each particle in the reaction is very dilute relative to the total volume of the reaction. Therefore, each protein particle in the reaction has a similar, average number of chemical interactions with the conjugate molecules in the reaction, regardless of the protein particle’s size or number of subunits. The number of molecular interactions a particle will have during the conjugation reaction is very unlikely to be affected by the number of subunits it contains; conversely, however, the number of subunits will have a considerable effect on the DOL calculated for a given set of the dispersed population. If we assume the monomeric portion of a dispersed protein population receives an average of one label during conjugation, then we would also assume that the more aggregated portion of the population will receive the same average number of labels and have the same number of labels per particle. However, because the two sets of particles (monomeric and aggregated) do not contain the same number of subunits, the degree of labeling will differ quite significantly. This is because the amount of protein present is inversely related the degree of labeling; as the protein concentration increases, the degree of labeling decreases. The study described in this thesis seeks to demonstrate that the different polydispersed populations present in a conjugate solution will have significant differences in their DOL with the smaller, less aggregated particles in the conjugate having a DOL than larger, more aggregated particles. To do this, two special batches of fluorescent protein conjugates were prepared, one labeled with Alexa Fluor 594 and the other with Alexa Fluor 488. Both of these conjugates were fractionated by size and the fractions of interest were promptly assayed to determine their DOL and to compare the level of labeling in each of the polydispersed populations in the conjugate sample. The data collected for both prepared conjugates clearly showed that there were considerable differences in DOL and a clear trend indicating that the DOL of each species in a polydispersed conjugate population decreased as the level of aggregation, or number of protein subunits per protein, increased.