Tertiary Conformational Transition In Horse Haemoglobin Induced By Inositol Hexakisphosphate
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Abstract
Description
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, Kequ, 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-P6). Between pH 5.6 and 9.0 Kequ 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-P6 also decreased
Kequ 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.
Krt, the equilibrium constant for the r t conformational transition, was
determined for each of the two horse haemoglobins from an analysis of the pH
dependence of Kequ. The 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 fast and slow
stripped horse haemoglobins respectively. In the presence of inositol-P6, Krt for
the fast and slow were 2.219 ± 0.79 and 2.214 ± 0.83 respectively. 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.
Keywords
QC Physics, QD Chemistry, QH301 Biology