KINETIC AND EQUILIBRIUM STUDIES OF THE REACTION OF 5,5'-DITHIOBIS(2-NITROBENZOATE) WITH HAEMOGLOBINS OF DOG AND DONKEY

Abstract

The CysF9[93]β sulphydryl group is an indicator for tertiary and quaternary structure change in haemoglobin. Allosteric effectors such as proton and inositol hexakisphosphate (inositol-P6) influence its reactivity. This work was undertaken to study the effects of inositol-P6 and pH on the kinetics and equilibrium of the reaction of CysF9[93]β of dog (Canis familiaris) and donkey (Equus asinus) haemoglobins with 5,5'-dithiobis(2-nitrobenzoate) (DTNB).

The number of sulphydryl groups in haemoglobin was determined by titrations with p-hydroxymercury(II)benzoate (pMB) and DTNB. The pseudo-first order kinetics of the reaction of haemoglobin with DTNB were studied at 25oC, with and without inositol-P6. Values of observed rate constant (kobs) were plotted against [DTNB] to obtain the apparent second order forward rate constant, kF. Equilibrium studies of the DTNB reaction were carried out at 25°C, with and without inositol-P6. An equation was derived for the determination of the equilibrium constant of the reaction, Kequ, within a series of equilibria. Kinetics and equilibrium experiments were carried out on the oxy, carbonmonoxy and aquomet derivatives of the haemoglobins in the pH range 5.6 to 9.0.

The number of sulphydryl groups in dog haemoglobin reacting with DTNB and pMB were 2 and 4 per tetramer respectively, while those of donkey haemoglobin gave two sulphydryl groups per haemoglobin tetramer for both reagents. The plot of kobs against [DTNB] was linear at each pH, with a non-negligible positive intercept, indicating that the reaction of CysF9[93]β of dog and donkey haemoglobins with DTNB is a reversible process. The slope of each plot (kF) varied with pH, giving different profiles for the three haemoglobin derivatives. Inositol-P6 had a significant effect on kF for the two haemoglobins. The Kequ showed strong pH dependences for all derivatives of the two haemoglobins. Inositol-P6 had only a minor effect on the affinity of dog haemoglobin for DTNB. By contrast, it increased the affinity of donkey haemoglobin by two orders of magnitude. These results could not be accounted for by changes in the tertiary conformation transition constant, Krt caused by inositol-P6. Inositol-P6 had little effect on Krt in the case of dog haemoglobin: Krt for stripped dog haemoglobin was calculated as 0.75 ± 0.13; in the presence of inositol-P6 Krt became 0.70 ± 0.20. Krt for stripped donkey haemoglobin increased from 0.46 ± 0.02 to 0.83 ± 0.20 in the presence of inositol-P6. This indicated that changes in tertiary structure govern the affinity of haemoglobin for DTNB.