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