Tertiary conformational transition constant of guinea pig haemoglobin determined from the reaction of 5,5′-dithiobis (2-nitrobenzoate) with CysF9[93]β and CysH3[125]β

Abstract

We have determined Kequ, the equilibrium constant for the reaction of 5,5′-dithiobis(2-nitrobenzoate) — DTNB — with the CysF9*93+β and CysH3*125+β sulphydryl groups of various derivatives of guinea pig haemoglobin at 25oC. In the pH range 5.6 to 9, Kequ decreases almost 50-fold: from a mean of 3.45 ± 0.2 to a mean of 0.073 ± 0.01. Quantitative analyses of the pH dependence profiles of Kequ enable the determination of Krt, the equilibrium constant for the rt tertiary conformational transition of haemoglobin. The t isomer population is 53.9 (± 2)%. In the r conformation the pKas of the amino acid residues whose ionisations are coupled to the reaction of DTNB with the sulphydryl groups are 5.74 ± 0.02 — for a combination of HisNA2*2+β and HisH21*143+β) — and 7.74 ± 0.2 for ValNA1*1+β); in the t conformation they are 5.88 ± 0.05 and 8.23 ± 0.1, respectively.
Keywords --- guinea pig haemoglobin; sulphydryl groups; 5,5′-dithiobis(2-nitrobenzoate); equilibrium constants; tertiary conformational transition.