Reversible reaction of 5,5′-dithiobis(2-nitrobenzoate) with the CysF9[93]β sulfhydryl groups of the hemoglobins of the domestic cat: Variation of the equilibrium and reverse rate constants with pH

Tertiary conformational transition in sheep hemoglobins induced by reaction with 5,5´-dithiobis(2-nitrobenzoate) and by binding of inositol hexakisphosphate

“…2 and 4 of [9]). This abolition arose because iodoacetamide bound to the sulphydryl irreversibly, thereby shifting the r ⇌ t tertiary equilibrium entirely to [10][11][12][13][14]. If iodoacetamide binds irreversibly to the CysF9[93]β site, thereby shifting the r ⇌ t equilibrium completely to the right and making it impossible to detect [9], it follows that, since DTNB reacts with CysF9[93]β reversibly, it cannot shift the r ⇌ t equilibrium completely to the right but can only influence its relative position.…”

“…3 and 4 confirm this expectation: inositol-P 6 lowers the values of k R by about an order of magnitude throughout the pH range 5.6 to 9. Using a pQ TNB value of 5.27 [11], we quantitatively analyzed the data presented in these figures with Eq. (4).…”

“…This abolition arises because iodoacetamide binds irreversibly to the CysF9[93]β sulphydryl and shifts the r ⇌ t equilibrium completely to the right, in favor of the t conformation [9]. By contrast, the reaction of this sulphydryl group with 5,5´-dithiobis(2-nitrobenzoate) -DTNB -is a reversible process [10][11][12][13][14] and therefore does not abolish the transition.…”

Organic phosphate binding inhibits high pH t-isomerization of the ß-chain in straw-coloured fruit bat (Eidolon helvum) haemoglobin

“…In mammalian haemoglobins, the reaction of 5,5'-dithiobis(2nitrobenzoate) (DTNB) with CysF9[93]â sulfhydryl group has been used as indicator of tertiary and the quaternary structure. [1][2][3] This reaction occurs reversibly. Therefore, the r↔t transition which occurs at the tertiary level is also in dynamic equilibrium when DTNB is bound to the CysF9[93]â sulfhydryl group.…”

“…7 Previous work has demonstrated that the most striking functional changes in haemoglobin occur when allosteric effectors get fully bound to oxy-haemoglobin, the consequences of which include a dramatic decrease in the oxygen affinity, an enhanced Bohr effect, and almost disappearance of oxygen cooperativity, emulating a Root effect-like behaviour. [9][10] ValNA1 [1]â, HisNA2 [2]â, LysEF6[82]â and HisH21 [143]â have been implicated in the binding of 2,3-BPG to haemoglobin. 9 The same residues have been reported to bind inositol-P 6 , an organic phosphate, which like 2,3-BPG, has a high density of negative charge.…”