Transition of haemoglobin between two tertiary conformations: Inositol hexakisphosphate increases the transition constant and the affinity of sheep haemoglobin for 5,5′-dithiobis(2-nitrobenzoate)

• Published in: Biochimica et biophysica acta 2009-03, Vol.1794 (3), p.398-409
• Main Authors: Okonjo, Kehinde Onwochei, Adeogun, Idowu, Babalola, J. Oyebamiji
• Format: Article
• Language: English
• Subjects: 5,5′-dithiobis(2-nitrobenzoate) affinity; Amino Acid Sequence; Animals; Carboxyhemoglobin - chemistry; CysF9β sulphydryl; Cysteine - chemistry; Dithionitrobenzoic Acid - chemistry; Haemoglobin tertiary conformation; Hemoglobins - chemistry; Hemoglobins - drug effects; Hydrogen-Ion Concentration; Inositol hexakisphosphate; Kinetics; Phytic Acid - pharmacology; Protein Conformation; Protein Structure, Tertiary; Sheep; Transition constant
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2008.11.006

The equilibrium constant ( K equ) for the reaction of 5,5′-dithiobis(2-nitrobenzoate) — DTNB — with the CysF9[93]β sulphydryl group of the haemoglobins of the sheep decreases by about two orders of magnitude between pH ≈ 5.6 and 9.2: from a mean of 7.2 ± 1 to a mean of 0.044 ± 0.01. Calculations from the pH dependence of K equ show that in the r ⇌ t tertiary conformational transition of haemoglobin the t isomer population is 50.7 and 61.8% for the major and minor haemoglobins, respectively. In the presence of inositol hexakisphosphate (inositol-P 6), K equ increases for both haemoglobins by about an order of magnitude through most of the pH range. The t isomer population also increases to 82.1 and 79.6% for the major and minor haemoglobins, respectively. These results indicate that inositol-P 6 increases the affinity of the sulphydryl for DTNB by increasing the population of the t isomer. It is highly probable that a minimum four-state model that includes the r ⇌ t transition is required for a full understanding of haemoglobin function.