6] Determining subunit dissociation constants in natural and recombinant proteins

Abstract

Publisher Summary Subunit dissociation in proteins depends on the strength of the interfaces between the subunits that vary considerably for different proteins. The extent, to which such dissociations can be measured—that is, to tetramers, dimers, or monomers—depends on the resolution of the separation procedure and the sensitivity of the detection system. This chapter describes gel-filtration procedure. Two aspects of determining subunit dissociation constants by FPLC (fast protein liquid chromatography) on Superose-12 are emphasized. In the procedure described in this chapter, the tetramer to dimer dissociation for hemoglobin is measured. Mutant hemoglobins, either natural variants or recombinant proteins with amino acid substitutions in the α 1 β 2 subunit interface where tetramerdimer dissociation occurs, are especially prone to changes in their oxygen binding affinity. With the advent of recombinant expression systems that produce hemoglobin mutations at any position in either α or β chains, the availability of a rapid and simple method to estimate tetramer dissociation constants on small amounts of protein would be advantageous.