Inositol Hexakisphosphate Induced Tertiary Conformational Transition In Horse Carbonmonoxyhemoglobin

The red blood cell of the domestic horse contains two haemoglobin types. The two haemoglobins were separated on a column of carboxymethylcellulose. The equilibrium constant, K equ , for the reaction of 5,5′‐dithiobis(2‐nitrobenzoate) — DTNB — with the CysF9[93]β sulfhydryl group of each haemoglobin was determined at 25°C as a function of pH. The reactivity of CysF9[93]β is affected by allosteric effectors such as the proton (H + ) and inositol hexakisphosphate (inositol‐P 6 ). Between pH 5.6 and 9.0 K equ decreased by about two to four orders of magnitude, demonstrating that H + is a heterotropic allosteric effector of haemoglobin with respect to its reaction with DTNB. Inositol‐P 6 also decreased K equ by about two to four orders of magnitude across the experimental pH range. CysF9[93]β exists in two tertiary conformations, r and t , in dynamic equilibrium. K rt , the equilibrium constant for the rt conformational transition, was determined for each of the two horse haemoglobins from an analysis of the pH dependence of K equ . The calculations from the pH dependence of K equ showed that the pK a values of the ionisable groups coupled to the DTNB reaction vary between 5.0 and 8.9. The equilibrium constants, K rt , for the r ↔ t tertiary structure transition, were 0.143 ± 0.05 and 0.446 ± 0.22 for the major and minor stripped horse haemoglobins respectively. In the presence of inositol‐P 6 , K rt for the major and minor were 2.219 ± 0.79 and 2.214 ± 0.83 respectively. The results show that inositol‐P 6 increases the relative population of the t tertiary conformation. So, it increases the affinity of CysF9[93]β by changing the relative distribution of two protein conformations. Support or Funding Information Covenant University, Ota. Nigeria.