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Kinetics and Equilibrium of the Reaction of Ellman's Reagent with Domestic Horse(Equus caballus) Haemoglobin

Omotosho, O. E. (2014) Kinetics and Equilibrium of the Reaction of Ellman's Reagent with Domestic Horse(Equus caballus) Haemoglobin. PhD thesis, Covenant University, Ota.

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Abstract

CysF9[93]β exists in two tertiary conformations, r and t, which are in dynamic equilibrium. The reactivity of the CysF9[93]β sulphydryl group and the oxygen affinity of haemoglobin (Hb) are affected by protons (H+) and organic phosphates such as inositol hexakisphosphate (inositol-P6). This study was aimed at determining the effects of inositol-P6 and pH on the relative populations of the two conformations. The major and minor haemoglobins in horse haemolysate were separated using a column of Whatman’s carboxymethylcellulose (CMC 52). Equilibrium studies of the reaction of CysF9[93]β with Ellman’s reagent, 5,5'-dithiobis(2-nitrobenzoate) (DTNB), equilibrated for six hours in the presence and absence of inositol-P6 were carried out at 25°C. 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. These experiments were carried out on the oxy, carbonmonoxy and aquomet derivatives of each haemoglobin in the pH range of 5.6 to 9.0. The number of sulphydryl groups in haemoglobin was determined by titrations with p-hydroxymercury(II)benzoate (pMB) and DTNB. The effects of these relative populations on the equilibrium and the kinetics of the reaction of CysF9[93]β of horse haemoglobin with Ellman’s reagent were determined. The pseudo-first order kinetics, with the [DTNB] in excess of the Hb concentration, were studied in the presence and absence of inositol-P6. Values of the observed rate constant, kobs, were plotted against [DTNB] to obtain the apparent second order forward rate constant, kF. 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 oxy- and aquomet- Hb. Theoretical calculations from the pH dependence of Kequ showed that the pKa values of the ionisable groups coupled to the DTNB reaction vary between 5.0 and 8.9. The equilibrium constants, Krt, 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-P6, Krt for the major and minor were 2.219 ± 0.79 and 2.214 ± 0.83 respectively. Theoretical calculations from the pH dependence of kF showed that the pKa values of the ionisable groups coupled to the DTNB reaction vary between 5.0 and 8.9. The plot of kobs against [DTNB] was linear at each pH, with a non-negligible positive intercept. This is an indication that the reaction of CysF9[93]β of horse haemoglobins with DTNB is reversible. In the presence of inositol-P6, values of kF increased across the pH range. Under the same experimental conditions, the binding of inositol-P6 to horse haemoglobin shifted the tertiary conformation in favour of the t state; the minor Hb has a higher affinity for DTNB than the major Hb except for aquomet with inositol-P6.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Haemoglobin, Equus caballus, Ellman’s reagent, kinetics, equilibrium
Subjects: Q Science > QD Chemistry
Divisions: Faculty of Engineering, Science and Mathematics > School of Chemistry
Depositing User: Mrs Patricia Nwokealisi
Date Deposited: 23 Jan 2015 15:50
Last Modified: 23 Jan 2015 15:50
URI: http://eprints.covenantuniversity.edu.ng/id/eprint/3383

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