Effect of anionic surfactant on the reaction of 2,2(1)-dithiobispyridine with bovine oxyhemoglobin as a function of temperature.

The reaction of bovine oxyhemoglobin with 2,2(1)-dithiobispyridine in the absence and presence of sodium n-dodecyl sulphate has been studied as a function of pH and temperature. The quantitative analysis of the pH dependence of the apparent second order rate constants shows that two ionizable groups are linked to the reaction in the native form. These are His HC3 (146) beta and Cys F9 (93) beta. Their pKa values are 6.5 and 8.5. In the denatured form, only one ionizable group is the modulating factor. This group is Cys F9 (93) beta. Its pKa is 8.35. The activation parameters for the reaction of the denatured form of bovine oxyhemoglobin with 2,2(1)-dithiobispyridine are lower than those of the native form. The reason is that the sulphydryl group is more exposed in the denatured form that in the native form. In addition, there is an increase in the rupturing of salt bridges and hydrogen bonds between the CO and NH peptide backbone. There is also a decrease in van der Waals interactions in the denatured form compared to the native form.

Hemoglobins with multiple reactive sulphydryl groups: the reaction of pigeon hemoglobin with 5,5'-dithiobis (2-nitrobenzoic acid).

Pigeon hemoglobin has eight reactive sulphydryl groups per (tetramer) molecule, as determined by Boyer titration with p-chloromercuribenzoate. However, only four of these are titratable with 5,5'-dithiobis(2-nitrobenzoate) under the same experimental conditions. The time course of the reaction of pigeon hemoglobin with 5,5'-dithiobis(2-nitrobenzoate) is biphasic. In the pH range 6-9, the fast phase is between one and two orders of magnitude faster than the slow phase. For the fast phase, kapp, the apparent second-order rate constant, increases monotonously with pH. Quantitative analysis reveals that the reaction of the sulphydryl group responsible for this phase is coupled to the ionization of two groups with pKa values of 6.15 +/- 0.1 and 8.5 +/- 0.1. These pKa values are assigned to HisHC3(146) beta and to the CysF9(93) beta sulphydryl group, respectively. For the slow phase the kapp vs. pH profiles are bowl-shaped. Analysis reveals that the reaction of the sulphydryl group to which this phase may be attributed is coupled to the ionization of two groups with mean pKa values of 6.53 +/- 0.1 and 8.25 +/- 0.1. Examination of the structure of hemoglobin allows us to assign these values to HisG19(117) beta and CysB5(23) beta, respectively. The CysB5(23) beta sulphydryl is in the region of the molecule where amino acid substitutions have been found to give rise to significant changes in the oxygen affinity of hemoglobin [Huang et al. (1990), Biochemistry 29, 7020-7023].

Hemoglobins with multiple reactive sulphydryl groups: the reaction of dog hemoglobin with 5,5'-dithiobis (2-nitrobenzoate).

Dog hemoglobin has four sulphydryl groups per (tetramer) molecule located at the G18(111)alpha and F9(93)beta positions. The two sulphydryls at the G18(111)alpha positions are unreactive toward nonmercurial sulphydryl reagents, but those at the F9(93)beta positions are reactive toward these reagents. We have studied the kinetics of the reaction of dog hemoglobin with 5,5'-dithiobis (2-nitrobenzoic acid) as a function of pH. At all pH values studied, the reaction is kinetically monophasic. Quantitative analysis of the pH dependence of the apparent second-order rate constant shows that two ionizable groups are linked to the reaction of the sulphydryl group. Their pKa values are 5.57 and 9.0. These values are assigned to HisHC3(146)beta and to the CysF9(93)beta sulphydryl. We find that dog carbonmonoxyhemoglobin is significantly--almost an order of magnitude--less reactive than the aquomet, azidomet, and oxy derivatives. This result may be due to a greater tendency (at acid pH) for the salt bridge between HisHC3(146)beta and AspFG1(94)beta to form in the carbonmonoxy than in the other derivatives. Formation of this salt bridge is known to hinder access to the CysF9(93)beta sulphydryl [Perutz, M. F. (1970), Nature 228, 734-739].

Biochemical characterization of a novel channel-activating site on nicotinic acetylcholine receptors.

We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine and several structurally related compounds with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligand-induced ion flux into nAChR-rich membrane vesicles, direct binding studies and photoaffinity labeling. (-)Physostigmine acts as a channel-activating ligand at low concentrations and as a direct channel blocker at elevated concentrations. Channel activation is not inhibited by desensitizing concentrations of ACh or ACh-competitive ligands (including alpha-bungarotoxin and D-tubocurarine) but is inhibited by antibody FK1 and several other compounds. From photoaffinity labeling using tritiated physostigmine and mapping of the epitope for the Phy-competitive antibody FK1, the binding site for physostigmine is located within the alpha-subunit of the Torpedo nAChR and is distinct from the acetylcholine binding site. Our data suggest a second pathway of nAChR channel activation that may function physiologically as an allosteric control of receptor activity.

Ionizable groups linked to the reaction of 2,2'-dithiobispyridine with hemoglobin.

The pH-dependence of the second-order rate-constant for the reaction of 2,2'-dithiobispyridine with the CysF9(93) beta sulphydryl group of hemoglobin in the R quaternary structure is analyzed in terms of a tentative model based on the observation that this sulphydryl exists as a mixture of two tertiary conformations in dynamic equilibrium. For the four aquomethemoglobins studied (human A and S, dog and rabbit), the equation derived from this model gives a better fit than a simpler equation based on the assumption of only one tertiary conformation. For the corresponding carbonmonoxyhemoglobins the simpler equation gives a better fit. The dog and rabbit oxy and azidomet data are better fitted by the model equation, whereas the data for the corresponding human A and S derivatives are better fitted by the simpler equation. From the analysis pKa values of 6.1 and 8.7 are obtained for the ionization of groups coupled to the presumed conformational transition. The pKa of 6.1 is assigned to HisHC3(146) beta; the pKa of 8.7 is assigned to the CysF9(93) beta sulphydryl group in its external conformation. It is estimated that the pKa of this sulphydryl may be as high as 12.9 in its internal conformation.

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 ligand-induced 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, alpha-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 FK1 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 microM range, there exists a further class of low-affinity (Kd approximately mM) sites from which eserine acts as a direct blocker of the acetylcholine-activated channel. Our results suggest the existence of a second pathway of activation of the nAChR channel.

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

The reversible acetylcholine esterase inhibitor (-)-physostigmine (eserine) is the prototype of a new class of nicotinic acetylcholine receptor (nAChR) activating ligands: it induces cation fluxes into nAChR-rich membrane vesicles from Torpedo marmorata electric tissue even under conditions of antagonist blocked acetylcholine binding sites (Okonjo, Kuhlmann, Maelicke, Neuron, in press). This suggests that eserine exerts its channel-activating property via binding sites at the nAChR separate from those of the natural transmitter. We now report that eserine can activate the channel even when the receptor has been preincubated (desensitized) with elevated concentrations of acetylcholine. Thus the conformational state of the receptor corresponding to desensitization is confined to the transmitter binding region, leaving the channel fully activatable-albeit only from other than the transmitter binding site(s).

Temperature-jump studies on hemoglobin. Kinetic evidence for a non-quaternary isomerization process in deoxy- and carbonmonoxyhemoglobin.

Temperature jumps on mixtures of hemoglobin and pH indicators give rise to relaxation signals in the microsecond range. The pH and concentration dependences of the reciprocal relaxation time, 1/tau, may be rationalized on the basis of a reaction scheme in which a slow isomerization process in the protein moiety is coupled to a rapid co-operative ionization of two protons. At 11 degrees C the rate constants of the isomerization are kr = 4.2(+/- 1.8) x 10(4) s-1 and kf = 1.3(+/- 0.1) x 10(4) s-1 in deoxyhemoglobin; in carbonmonoxyhemoglobin they are kr = 3.9(+/- 1.3) x 10(4) s-1 and kf = 5.3(+/- 1.8) x 10(3) s-1. The pKa values of the coupled ionizing groups are 5.3 in deoxy- and 6.0 in carbonmonoxyhemoglobin. Modification of the CysF9(93) beta sulfhydryl group with iodoacetamide abolishes the pH dependence of 1/tau, suggesting that this sulfhydryl is involved in the isomerization process. Consideration of the X-ray structure of oxyhemoglobin allows a structural interpretation of the results. It is concluded that the isomerization may be important for the physiological function of hemoglobin, but that it is not identical with the quaternary structure transition.

The uptake of protons by heme-linked ionizable groups on azide binding to methemoglobin.

When azide ion reacts with methemoglobin in unbuffered solution the pH of the solution increases. This phenomenon is associated with increases in the pK values of heme-linked ionizable groups on the protein which give rise to an uptake of protons from solution. We have determined as a functional of pH the proton uptake, delta h+, on azide binding to methemoglobin at 20 degrees C. Data for methemoglobins A (human), guinea pig and pigeon are fitted to a theoretical expression based on the electrostatic effect of these sets of heme-linked ionizable groups on the binding of the ligand. From these fits the pK values of heme-linked ionizable groups are obtained for liganded and unliganded methemoglobins. In unliganded methemoglobin pK1, which is associated with carboxylic acid groups, ranges between 4.0 and 5.5 for the three methemoglobins; pK2, which is associated with histidines and terminal amino groups, ranges from 6.2 to 6.7. In liganded methemoglobin pK1 lies between 5.8 and 6.3 and pK2 varies from 8.1 to 8.5. The pH dependences of the apparent equilibrium constants for azide binding to the three methemoglobins at 20 degrees C are well accounted for with the pK values calculated from the variation of delta h+ with pH.

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.

The effect of heme-linked ionizable groups on cyanide binding to methemoglobin.

The pH dependence of the kinetics of the binding of cyanide ion to methemoglobins A and S and to guinea pig and pigeon methemoglobins appears to be not directly correlated with the net charges on the proteins. The kinetics can, however, be adequately explained in terms of three sets of heme-linked ionizable groups with pK1 ranging between 4.9 and 5.3, pK2 between 6.2 and 7.9, and pK3 between 8.0 and 8.5 at 20 degrees C. pK1 is assigned to carboxylic acid groups, pK2 to histidines and terminal amino groups, and pK3 to the acid-alkaline methemoglobin transition. Kinetic second order rate constants have also been determined for the binding of cyanide ion by the four sets of methemoglobin species present in solution. The pKi values and the rate constants of methemoglobin S are strikingly different from those of methemoglobin A. This result is explained in terms of different electrostatic contributions to the free energy of heme linkage arising from differences in the environments of ionizable groups at the surfaces of the two molecules.

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.