Inositol Hexakisphosphate Increases The Transition Constant And The Affinity Of Horse Aquomethemoglobin for 5,5′‐dithiobis(2‐nitrobenzoate)

Abstract

CysF9[93]β exists in two tertiary conformations, r and t, which are in dynamic equilibrium. The equilibrium constant, Kequ, for the reaction of 5,5′‐dithiobis(2‐nitrobenzoate) — DTNB — with the CysF9[93]β sulfhydryl group of each hemoglobin 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‐P6). The major and minor haemoglobins in horse haemolysate were separated using a column of Whatman's carboxymethylcellulose (CMC 52). The absorbance of the product of DTNB reaction, 5‐thio‐2‐nitrobenzoate, at different concentrations of DTNB and 50 μmol (haem) dm−3 was read at 412 nm. The absorbance was then substituted into an appropriate equation derived for the determination of the equilibrium constant, Kequ, for the reaction. Kequ decreased the orders of magnitude between pH 5.6 and 9.0 in the absence and presence of inositol‐P6. Inositol‐P6 increased the affinity of the major and minor Hb for DTNB but decreased the affinity of the minor aquomet‐Hb. The results show that inositol‐P6 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.Study objectiveThe study also seeks to find the equilibrium between the r and t isomers in the reaction of DTNB with CysF9[93]β sulphydryl group of the horse aquomethemoglobins. The relative populations of these two conformations from the pH dependence of equilibrium and kinetic parameters and the effect of inositol‐P6 on these relative populations were determined.MethodsPreparation and separation of horse hemoglobin had been described in Omotosho et al., 2017. Preparation and determination of aquomethemoglobin concentration: Aquomethemoglobin was prepared by the addition of a two‐fold excess of potassium ferricyanide, K3Fe(CN)6, to oxyhemoglobin of a known concentration. An approximately 1 mol dm−3 K3Fe(CN)6 solution was prepared by dissolving 0.33 g of the reagent in 1 cm3 of distilled water. The volume of the K3Fe(CN)6, v cm3, added to V cm3 of hemoglobin to give a two‐fold excess is related to the concentration of the hemoglobin solution.ResultsDomestic horse contains two hemoglobin types: A, the fast hemoglobin, and B, the slow hemoglobin. The two haemoglobins have identical α chains. Horse hemoglobin consists of two electrophoretically distinct components. From Figs. 1 and 2 it is seen that inositol‐P6 increases the affinity of CysF9[93]β of each haemoglobin for DTNB by about three orders of magnitude throughout the pH range 5.6 to 9.2.ConclusionFigs. 1 and 2 report the dependence of Kequ, the equilibrium constant of the reaction of DTNB with CysF9[93]β, on pH. The values of Kequ decrease by about two orders of magnitude for slow aquomet hemoglobin over the range 5.6 ≥ pH ≤ 9.0 whereas there in the fast hemoglobin, the values of Kequ increases by about six orders of magnitude and there is an increase in the affinity for the aquomet derivative. This shows that there is more insight on the nature of the tertiary structure transition in both T and R state hemoglobin.Support or Funding InformationWe appreciate Covenant University Centre for Research, Innovation and Development for the financial support.