Retention mechanism of lactate dehydrogenase in anion-exchange chromatography

The anion-exchange retention mechanism of lactate dehydrogenase (LDH) isoenzymes was examined under conditions where the muscle (M) subunit was not retained by the sorbent matrix. Using the stoichiometric displacement model of retention to quantitate the number (Zn) of ionic groups on the protein that interact with the sorbent matrix, it was determined that Zn for the LDH isoenzymes increases incrementally as the number of heart (H) subunits is increased up to a total of three H subunits. Both the MH3 and H4 isoenzymes have the same Zn. As the subunits in this tetrameric enzyme are arranged in a tetrahedral structure, these data indicate that steric limitations prevent any more than three subunits from interacting simultaneously with the sorbent surface. The reason why the MH3 and H4 isoenzymes could be separated is apparently that there are multiple equivalent faces in the H4 isoenzyme, and this both increases the probability and rate of adsorption and decreases the probability and rate of desorption for the H4 relative to the MH3 isoenzyme. It was concluded from these studies that the three-dimensional structure of a biopolymer will determine those surface residues which are in a position to interact with a sorbent surface and that multiple subunits or domains of a protein may act cooperatively over considerable distances in biopolymer-surface interactions.