Ligand-dependent reactivity of the CysB5[23] b sulfhydryl group of the major haemoglobin of chicken

Chicken haemoglobin contains eight reactive sulfhydryl groups per (tetramer) molecule, as determined by Boyer titration with p-chloromercury(II)benzoic acid. However, only four of these sulfhydryls are reactive towards 5,5@-dithiobis(2-nitrobenzoic acid) (DTNB). They are at the F9[93] and B5[23] positions of each of the two b subunits in the molecule. The time course of the DTNB reaction is biphasic. With oxyhaemoglobin, k the apparent second-order rate constant of the fast phase, increases app, monotonically with pH, the simple proÐle resembling the titration curve of a diprotic acid; the pH-dependence of k for the app slow phase is bowl-shaped. With carbonmonoxyhaemoglobin and aquomethaemoglobin, k for the fast phase is bowl-shaped app whilst k for the slow phase increases monotonically with pH. Quantitative analyses of the simple proÐles show that the app reactivity of the sulfhydryl group to which they may be attributed is subject to the inÑuence of two ionizable groups on the molecule, with mean pK values of 6.4^0.1 and ca. 8.4^0.3. These values are assigned to HisHC3[146]b and CysF9[93]b, a pKa respectively. Quantitative analyses of the bowl-shaped proÐles show that the reactivity of the sulfhydryl group to which they may be attributed is subject to the inÑuence of two ionizable groups on the protein, with mean pK of 6.85^0.05 and 8.3^0.2. as These values are assigned to HisG19[117]b and CysB5[23]b, respectively. It is highly signiÐcant that the CysB5[23]b sulfhydryl groups of carbonmonoxy- and aquomet-haemoglobin react ca. 100 times faster than that of oxyhaemoglobin. By contrast, the di†erence in the reactivities of the CysF9[93]b sulfhydryls of the three haemoglobin derivatives is no more than four-fold. This indicates that, in chicken haemoglobin, changes in the haem ligand give rise to structural changes in the neighbourhood of the CysB5[23]b sulfhydryl which are far more signiÐcant than those in the neighbourhood of the CysF9[93]b sulfhydryl

Desensitization is a property of the cholinergic binding region of the nicotinic acetylcholine receptor, not of the receptor-integral ion channel

The reversible acctylchollne esternse inhibitor (-}.physostiilmine (¢serine) is th© prolotypc of a new class of nie~tinlc ucetylcholinc receptor (nAChR) activating liga,ds: it induces cation fluxes into nAChR.rich membrane vesicl~s from 7~r#eda marmoeata cle~:tric tissue even under condl. lions of antalionist blocked :tcctylcholin~ binding sil~s (Okonjo, Kuhlm~mn. Maelicke. Neuron, in press). This su~tlest's that escrine exerts it~ than. nel.activating proi'~rty via binding sites at the nAChR separate from those of tile natural transmitter. We now report thllt eserine e'-m activate the channel wen when the receptor has t~en preincub~ttcd (des©nsitiz©d) with elevated concentrations of acetylcholi~e, Titus the confornudional state Of the receptor corresponding to de~nsitixation is confined to the transmitter bindinB rclli0n, leaving the ch=tr'4nel fully activatable ,- albeit only from other than the tr~msmitter bindin~ site(s)

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

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