REACTIVITIES OF THE SULPHYDRYL GROUPS OF DOG HEMOGLOBIN

Dog hemoglobin has four sulphydryl groups at positions olfl (G18) and p93 (F9), all of which are titratable with mercurials. Only two of these, however, react with non-mercurial sulphydryl reagents. Kinetic results indicate that the reacting site might be the p”” (F9). An examination of the environment of the alI1 (G18) shows that this sulphydryl must be unreactive towards non-mercurials because of the presence near it of several interacting groups. These are the carboxyl group of Glu 27u, which is only 4.5 A away; the carbonyl of Val 1070; and the hydroxyl of Tyr24a. There is also a strong interaction with the carboxyl of Glu 116~ which, though 12 A away, is separated from the (Y”’ (G18) not by water but by protein, a low dielectric constant medium. All these interactions would considerably raise the pK of the Cys llla thiol. Therefore reaction with non-mercurial sulphydryl reagents via nucleophilic attack by the thiol anion becomes impossible. The effect of inositol hexaphosphate on the kinetics of the sulphydryl group reaction was investigated. Inositol hexaphosphate slows down the reaction by a factor of three for a 10 M excess of inositol hexaphosphate per hemoglobin tetramer and makes about 25% of the sulphydryl contents of the 0’” and CX”’ sites unavailable for reaction by any sulphydryl reagent

Effect of Organic Phosphates on the Sulfhydryl Reactivities of Oxyhemoglobins A and S*

The /393 sulfhydryl groups of oxyhemoglobins A and S display a difference in reactivity with 5,5’-dithiobis- 2-nitrobenzoic acid. It is concluded that this difference arises from differences in tertiary structure in the vicinity of the p”“ site. Organic phosphatesd ecrease the pg3s ulfhydryl reactivity. We have used this effect to measure the organic phosphate binding constants. Hemoglobin S binds organic phosphates very weakly compared to hemoglobin A. This result indicates that the structure at the organic phosphate binding site is different in the two oxyhemoglobins and may be the result of differences in the structure of the NHa-terminal ends of the p chains

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

Role of the Relaxation of the Iron(ll1) Ion Spin States Equilibrium in the Kinetics of Ligand Binding to Methaemoglobin

Temperature-jump experiments of the reaction of the thiocyanate ion with human aquomethaemoglobin have been performed in the presence of a 10-fold excess of inositol hexakisphosphate (inositolP,). Two kinetic phases corresponding to the a and /3 subunits were observed. Kinetic parameters of the reaction were evaluated from the reciprocal relaxation times on the basis of a fast relaxation of the iron(iii) ion spin states equilibrium before binding of the ligand. The association, ki,, and dissociation, k-i,, rate constants determined were: k,, = 225 dm3 mol-' s-', k-aL = 1.52 s-', k,, = 2430 dm3 mol-' s-', k-pL = 6.51 s-' at 27"C, pH 6.44. There was good agreement between the equilibrium constant of the ligand binding step determined by static methods (Kequ = 204 & 11 dm3 mol-') and that evaluated from kinetic data [(KaLKpL)1'2 = 235 & 12 dm3 mol-'1. The value ksJkaL = 11 obtained ensured proper separation of the two kinetic phases. Analyses of the subunit relaxation amplitudes, a€,, , showed that inositolP, perturbed the absorption spectrum of the /3 subunits. This suggests that in the presence of the organic phosphate, methaemoglobin behaves as a protein with independent binding sites rather than as an allosteric molecule. The kinetic and relaxation amplitude spectral characteristics of the subunits, in the presence of inositolP, have demonstrated that the kinetic dynamics are effectively decoupled in a stable tetramer

Reversible reaction of 5,5V-dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: Acetylation of NH3 + terminal group of the h chain transforms the complex pH dependence of the forward apparent second order rate constant to a simple form

We demonstrate kinetically that the reaction of 5,5V-dithiobis(2-nitrobenzoate) with the CysF9[93]h sulfhydryl group of domestic cat hemoglobins is a reversible process. In the major hemoglobin, in which the NH3 + terminal group of GlyNA1[1]h is free, kf, the apparent forward second order rate constant, has a complex pH dependence profile. In the minor hemoglobin, the NH3 + terminal group of SerNA1[1]h is acetylated, and the pH dependence profile of kf is simple. These results support the proposal that the positively charged groups at the organic phosphate binding site are electrostatically linked to CysF9[93]h. Quantitative analyses of the complex profiles enabled us to estimate pKas of 7.47T0.3; 6.53T0.03 and 8.49T0.3 for GlyNA1[1]h, HisH21[143]h and other histidines within 2 nm of the sulfhydryl, and CysF9[93]h, respectively, of the major hemoglobin. Analyses of the simple profiles gave pKas of 6.33T0.17 and 8.54T0.5 for HisH21[143]h and other histidines within a distance of 2 nm of the sulfhydryl, and CysF9[93]h of the minor hemoglobin, respectively

Relaxation amplitude analysis of thiocyanate and formate binding to human aquomethemoglobin A

The kinetics of the reaction of thiocyanate and formate ions with aquomethemoglobin can be adequately accounted for by a scheme in which the ligand-binding step in both the alpha and beta subunits is preceded by a fast transition of the iron atom from high to low spin (Okonjo, K.O. (1980) Eur. J. Biochem. 105, 329-334). Amplitude expressions derived from this scheme are used to analyse the relaxation amplitude data for alpha and beta subunits within the methemoglobin tetramer. The mean of the reaction enthalpies for ligand binding by the subunits within the tetramer is in good agreement with the reaction enthalpy for ligand binding by the methemoglobin tetramer obtained from a Van't Hoff plot of equilibrium titration data

Subunit iron spin heterogeneity in human aquomethemoglobin A

On the basis of a reaction scheme in which the ligand binding steps are preceded by fast iron spin transitions (Okonjo, K.O. (1980) Eur. J. Biochem. 105, 329-334; Iwuoha, E.I. and Okonjo, K.O. (1985) Biochim. Biophys. Acta 829, 327-334), the spin equilibrium constants of methemoglobin subunits are calculated from kinetic and equilibrium binding parameters with azide ion as ligand. The results demonstrate the existence of thermodynamic spin heterogeneity within the tetramer

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)

A second pathway of activation of the Torpedo acetylcholine receptor channel

We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine (D-eserine) with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligandinduced ion flux into nAChR-rich membrane vesicles and of equilibrium binding. We find that (—)physostigmine induces cation flux (and also binds to the receptor) even in the presence of saturating concentrations of antagonists of acetylcholine, such as D-tubocurarine, a-bungarotoxin or antibody WF6, The direct action on the acetylcholine receptor is not affected by removal of the methylcarbamate function from the drug and thus is not due to carbamylation of the receptor. Antibodies FKl and benzoquinonium antagonize channel activation (and binding) of eserine, suggesting that the eserine binding site(s) is separate from, but adjacent to, the acetylcholine binding site at the receptor. In addition to the channel activating site(s) with an affinity of binding in the 50 nM range, there exists a further class of low-affinity (K^ ~ mM) sites from which eserine acts as a direct blocker of the acetylcholine-activatcd channel. Our results suggest the existence of a second pathway of activation of the nAChR channel

Transition of hemoglobin between two tertiary conformations: The transition constant differs significantly for the major and minor hemoglobins of the Japanese quail (Cortunix cortunix japonica)

We demonstrate that 5,5′-dithiobis(2-nitrobenzoate) – DTNB – reacts with only CysF9[93]β and CysB5[23]β among the multiple sulfhydryl groups of the major and minor hemoglobins of the Japanese quail (Cortunix cortunix japonica). Kequ, the equilibrium constant for the reaction, does not differ very significantly between the two hemoglobins. It decreases 430-fold between pH≈5.6 and pH≈9: from a mean of 7±1 to a mean of 0.016±0.003. Quantitative analyses of the Kequ data based on published X-ray and temperature-jump evidence for a tertiary structure transition in liganded hemoglobin enable the calculation of Krt, the equilibrium constant for the r←→t tertiary structure transition. Krt differs significantly between the two hemoglobins: 0.744±0.04 for the major, 0.401±0.01 for the minor hemoglobin. The mean pKas of the two groups whose ionizations are coupled to the DTNB reaction are about the same as previously reported for mammalian hemoglobins