Contrasting effect of crowder and tertiary isomerizations on ligand binding properties of two rabbit hemoglobin derivatives

Abstract

The need to explore the effect of molecular crowder on the tertiary structure of protein and determine how it affects the
function of such protein was the motivation for this work. Reaction of 5,5’dithobis(2-nitrobenzoate) (DTNB), a reagent
which reacts reversibly with hemoglobin sulfhydryl groups were carried out in dilute solution and in up to 100 mg/cm3
Ficoll 70. The reversible binding of DTNB to hemoglobin sulfhydryl group had previously shown to be linked to some
ionizable groups which are linked to the reversible binding of oxygen with hemoglobin. Reaction of rabbit oxy and
carbonmonoxy hemoglobin with DTNB was studied in the range of 5.8 ≤ pH ≤ 8.8. The data were fitted to a model which
takes into account the ionization of the ionizable groups on the reversible binding of DTNB with the hemoglobin.
Whereas, the affinity of carbonmonoxy hemoglobin for DTNB is reduced in the presence of Ficoll 70 (Fic), the affinity of
oxyhemoglobin for DTNB is enhanced in the presence of the crowding agent. This finding is an indication that crowding
may have possible physiological consequence of enhancing the binding of physiologically import ligand to hemoglobin.