Propidium Binding Research Articles

A flexible cation binding site in the multidrug major facilitator superfamily transporter LmrP is associated with variable proton coupling.

The multidrug major facilitator superfamily transporter LmrP from Lactococcus lactis mediates protonmotive-force dependent efflux of amphiphilic ligands from the cell. We compared the role of membrane-embedded carboxylates in transport and binding of divalent cationic propidium and monovalent cationic ethidium. D235N, E327Q, and D142N replacements each resulted in loss of electrogenicity in the propidium efflux reaction, pointing to electrogenic 3H(+)/propidium(2+) antiport. During ethidium efflux, single D142N and D235N replacements resulted in apparent loss of electrogenicity, whereas the E327Q substitution did not affect the energetics, consistent with electrogenic 2H(+)/ethidium(+) antiport. Different roles of carboxylates were also observed in fluorescence anisotropy-based ligand-binding assays. Whereas D235 and E327 were both involved in propidium binding, the loss of one of these carboxylates could be compensated for by the other in ethidium binding. The D142N replacement did not affect the binding of either ligand. These data point to the presence of a dedicated proton binding site containing D142, and a flexible proton/ligand binding site containing D235 and E327, the contributions to proton and ligand binding of which depend on the chemical structure of the bound ligand. Our findings provide the first evidence that multidrug transport by secondary-active transporters can be associated with variable ion coupling.

New multipotent tetracyclic tacrines with neuroprotective activity

The synthesis and the biological evaluation (neuroprotection, voltage dependent calcium channel blockade, AChE/BuChE inhibitory activity and propidium binding) of new multipotent tetracyclic tacrine analogues ( 5– 13) are described. Compounds 7, 8 and 11 showed a significant neuroprotective effect on neuroblastoma cells subjected to Ca 2+ overload or free radical induced toxicity. These compounds are modest AChE inhibitors [the best inhibitor ( 11) is 50-fold less potent than tacrine], but proved to be very selective, as for most of them no BuChE inhibition was observed. In addition, the propidium displacement experiments showed that these compounds bind AChE to the peripheral anionic site (PAS) of AChE and, consequently, are potential agents that can prevent the aggregation of β-amyloid. Overall, compound 8 is a modest and selective AChE inhibitor, but an efficient neuroprotective agent against 70 mM K + and 60 μM H 2O 2. Based on these results, some of these molecules can be considered as lead candidates for the further development of anti-Alzheimer drugs.

A computational study of the binding of propidium to the peripheral anionic site of human acetylcholinesterase.

Combined docking and molecular dynamics (MD) simulations were carried out in order to investigate the binding mode of propidium at the human acetylcholinesterase (HuAChE) peripheral site. Two different docking protocols followed by cluster analyses were performed, allowing the identification of five high-populated and low-energy configuration families. To dynamically explore the behavior of the ligand at the peripheral HuAChE binding site, six complexes (five low-energy and one high-energy) were submitted to 2.5 ns of MD simulations. The representative propidium/HuAChE binding modes were chosen on the basis of both the docking energy score and the dynamic stability of the complexes throughout the MD simulations. The most stable poses of propidium at HuAChE PAS were similar to those experimentally determined with the murine enzyme. We therefore suggest that the present modeling protocol might be used in the dynamic investigation of the interactions of a small-molecule inhibitor with a surface-like binding site of a protein. Finally, because of the biological relevance of the target studied here, the present results can be of interest for the rational design of molecules potentially useful in the treatment of the Alzheimer's disease.

Thioflavin T Is a Fluorescent Probe of the Acetylcholinesterase Peripheral Site That Reveals Conformational Interactions between the Peripheral and Acylation Sites

Three-dimensional structures of acetylcholinesterase (AChE) reveal a narrow and deep active site gorge with two sites of ligand binding, an acylation site at the base of the gorge, and a peripheral site near the gorge entrance. Recent studies have shown that the peripheral site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, but the question of whether the peripheral site makes other contributions to the catalytic process remains open. A possible role for ligand binding to the peripheral site that has long been considered is the initiation of a conformational change that is transmitted allosterically to the acylation site to alter catalysis. However, evidence for conformational interactions between these sites has been difficult to obtain. Here we report that thioflavin T, a fluorophore widely used to detect amyloid structure in proteins, binds selectively to the AChE peripheral site with an equilibrium dissociation constant of 1.0 microm. The fluorescence of the bound thioflavin T is increased more than 1000-fold over that of unbound thioflavin T, the greatest enhancement of fluorescence for the binding of a fluorophore to AChE yet observed. Furthermore, when the acylation site ligands edrophonium or m-(N, N,N-trimethylammonio)trifluoroacetophenone form ternary complexes with AChE and thioflavin T, the fluorescence is quenched by factors of 2.7-4.2. The observation of this partial quenching of thioflavin T fluorescence is a major advance in the study of AChE for two reasons. First, it allows thioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site initiates a change in the local AChE conformation at the peripheral site that quenches the fluorescence of bound thioflavin T. The data provide strong evidence in support of a conformational interaction between the two AChE sites.

Organophosphorylation of Acetylcholinesterase in the Presence of Peripheral Site Ligands: DISTINCT EFFECTS OF PROPIDIUM AND FASCICULIN

Structural analysis of acetylcholinesterase (AChE) has revealed two sites of ligand interaction in the active site gorge: an acylation site at the base of the gorge and a peripheral site at its mouth. A goal of our studies is to understand how ligand binding to the peripheral site alters the reactivity of substrates and organophosphates at the acylation site. Kinetic rate constants were determined for the phosphorylation of AChE by two fluorogenic organophosphates, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide (DEPQ) and 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC), by monitoring release of the fluorescent leaving group. Rate constants obtained with human erythrocyte AChE were in good agreement with those obtained for recombinant human AChE produced from a high level Drosophila S2 cell expression system. First-order rate constants kOP were 1,600 +/- 300 min-1 for DEPQ and 150 +/- 11 min-1 for EMPC, and second-order rate constants kOP/KOP were 193 +/- 13 microM-1 min-1 for DEPQ and 0.7-1.0 +/- 0.1 microM-1 min-1 for EMPC. Binding of the small ligand propidium to the AChE peripheral site decreased kOP/KOP by factors of 2-20 for these organophosphates. Such modest inhibitory effects are consistent with our recently proposed steric blockade model (Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216). Moreover, the binding of propidium resulted in a clear increase in kOP for EMPC, suggesting that molecular or electronic strain caused by the proximity of propidium to EMPC in the ternary complex may promote phosphorylation. In contrast, the binding of the polypeptide neurotoxin fasciculin to the peripheral site of AChE dramatically decreased phosphorylation rate constants. Values of kOP/KOP were decreased by factors of 10(3) to 10(5), and kOP was decreased by factors of 300-4,000. Such pronounced inhibition suggested a conformational change in the acylation site induced by fasciculin binding. As a note of caution to other investigators, measurements of phosphorylation of the fasciculin-AChE complex by AChE inactivation gave misleading rate constants because a small fraction of the AChE was resistant to inhibition by fasciculin.

Different effects of two peripheral anionic site-binding ligands on acetylcholinesterase active-site gorge topography revealed by electron paramagnetic resonance

Both propidium and monoclonal antibody (mAb) 25B 1 bind to the peripheral anionic site region of fetal bovine serum acetylcholinesterase (FBS AChE). Using electron paramagnetic resonance (EPR) with spin-labelled organophosphate specifically bound to the AChE active-site serine, we studied the effects of both ligands on the topography of the AChE active-site gorge. After incubation of FBS AChE with Fab fragments of mAb 25B1, freedom of motion of our spin label became more restricted, suggesting closing of the gorge. Stabilization against heat denaturation was also observed. No alterations in the freedom of motion or protection against heat denaturation could be detected after propidium binding. Our results demonstrate that two ligands binding to the peripheral anionic site region of AChE have different effects, suggesting a complex structure for this region of the molecule that allows various types of interactions with different ligands. We also demonstrate that EPR is a suitable tool for studying microtopographical alterations at the active sites of cholinesterases.

Thermodynamic investigation of the association of ethidium, propidium and bis-ethidium to DNA hairpins

We have used a combination of calorimetric and spectroscopic techniques to investigate the association of the bis-intercalator ethidium homodimer (bis-ethidium) to short DNA hairpins with sequences: d(GCGCT 5GCGC) and d(CGCGT 5CGCG). The helix-coil transition of each hairpin, investigated by UV and calorimetric melting protocol, takes place in monomolecular two-state transitions with characteristic enthalpies of ∼37 kcal mol −1 for disrupting the four dG-dC base pairs of the hairpin stems. Deconvolution of the bis-ethidium-hairpin calorimetric titration curves indicate that each hairpin contains two distinct binding sites for the ligand: a high affinity site in the stem ( K b ∼10 7) that accommodates one bis-ethidium molecule and a lower affinity site ( K b ∼10 6) located probably at the loop that accommodates two bis-ethidium molecules. The overall stoichiometries of three ligands per hairpin are in agreement with those obtained in continuous variation experiments using visible spectroscopy. The interaction of bis-ethidium for each type of sites results in enthalpy driven reactions, with average binding enthalpies, ΔH b, of −13.1 and −12.1 kcal mol −1 for the stem and loop sites, respectively. Comparison to the thermodynamic profiles of ethidium and propidium binding reveals that the bis-ethidium binding to the stem site of each hairpin has a more favorable free energy term of −1.4 kcal mol −1 and more favorable enthalpy of −4.2 kcal mol −1. These suggest that only one phenanthridine ring of bis-ethidium intercalates in the stem, while the second planar ring is exposed to solvent or weakly associated to the surface of DNA. The K bs for bis-ethidium binding to the loop sites are about two orders of magnitude larger than the mono-intercalators and correspond to a more favorable free energy of −2.0 kcal mol −1. The enthalpies of binding are also more favorable for bis-ethidium by −2 to −4 kcal mol −1. Overall, the magnitude of K b for each ligand, and for each site, is in qualitative agreement with the electrostatic contribution from the actual number of positive charges of the ligand. The increased favorable enthalpic contributions of bis-ethidium are consistent with larger hydrophobic contributions, while the increase in unfavorable entropy contributions are consistent with the higher ordering of bis-ethidium in the stem and loop sites.

Thermodynamics and Kinetics of Propidium Binding to Poly(A)·Poly(U) in Aqueous Solution

Abstract The propidium binding to poly(A)·poly(U) has been investigated thermodynamically and kinetically in buffers containing different concentrations of NaCl at various temperatures. Free propidium had an absorption maximum at 493 nm, and this shifted to about 530 nm for propidium fully bound to poly(A)·poly(U) in all solutions. The association constant for the reaction was 1.42×106 mol−1 dm3 at 2.02×10−2mol dm−3 of Na+concentration, and about 26 times larger than that at 1.02 mol dm−3 Na+. The dependence of the association constants indicated the enthalpies for the reaction at the low and high Na+ concentrations were 39.8 and −8.3 kJ mol−1, respectively. The entropies were 251 and 63 J K−1 mol−1, respectively. These thermodynamical results suggested that the association reaction of propidium to poly(A)·poly(U) may play different behaviors at the low and high salt concentrations. Kinetic results by a micro stopped-flow method indicated that the propidium binding to poly(A)·poly(U) was a single step at the low Na+ concentration, while at the high Na+concentration it consisted of two steps which contained the slow unimolecular process after the binding step.

Temperature dependence of enthalpy changes for ethidium and propidium binding to DNA: Effect of alkylamine chains

Calorimetric titrations have been performed on the binding of ethidium and propidium to calf thymus DNA at temperatures in the 15-60 degrees C range. Enthalpy changes (delta HB) derived from these experiments performed with the new Omega reaction calorimeter have a precision of +/- 0.10 kcal/mol or less at all temperatures. For ethidium (a monocation), delta HB varies little with temperature, and the heat capacity change (delta CP) for the binding reaction derived from these parameters is 10 cal/deg/mol. In contrast, delta HB changes from -6.5 to -8.1 kcal/mol for DNA binding of propidium (a dication due to a charged amine group at the end of an alkyl chain attached to the phenanthridine ring nitrogen), and delta CP is -57 cal/deg/mol. At 21 degrees C a plot of delta HB vs mole ratio is curved downward for propidium in the 0.08-0.25 range, whereas the same plot at 45 degrees C is a straight line from 0.05 to 0.15 and sharply downward thereafter. Similar plots for ethidium follow the latter pattern between 25 and 50 degrees C. These observations and our analyses of delta HB and delta SB are consistent with the hypothesis that the location in the DNA complex and the rotational motion of the alkylamine chain change substantially over the temperature range in this study. Only near 50 degrees C is delta HB equal for the binding of these two cations to DNA, and caution must be used in analyses of enthalpic effects when the temperature dependence for delta HB is not available.

Interaction of an organophosphate with a peripheral site on acetylcholinesterase

O-Ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (MPT) is an active site directed inhibitor of acetylcholinesterase (AChE). Inhibition of the Electrophorus electricus (G4) enzyme follows classical second-order kinetics. However, inhibition of total mouse skeletal muscle AChE and inhibition of the individual molecular forms from muscle, including the monomeric species, do not proceed as simple irreversible bimolecular reactions. Similarly, complex inhibition kinetics are observed for the purified enzyme from Torpedo californica. AChE can be cross-linked with glutaraldehyde into a semisolid matrix. Under these conditions the abnormal concentration dependence for MPT inhibition is accentuated, and a range of MPT concentrations can be found where inhibition of polymerized AChE is far less than that observed at lower concentrations. Inhibition in certain concentration ranges is partially reversible after removal of all unbound ligand. Thus, there are two different modes of organophosphorus inhibition by MPT: the classical irreversible phosphorylation of the active site and a reversible interaction at a site peripheral to the active center. Propidium, a well-studied peripheral site ligand, can prevent the later interaction. Hence, the second site of MPT interaction with AChE may overlap or be linked to the peripheral anionic site of AChE characterized by the binding of propidium and other peripheral site inhibitors.

Enthalpy and entropy changes for the intercalation of small molecules to DNA. II. Ethidium and propidium fluoride.

AbstractEnthalpy changes (ΔHB) for the binding of ethidium (a monocation) and propidium (a dication) to calf thymus DNA have been determined calorimetrically in piperazine‐N, N′‐bis(2‐ethanesulfonic acid) buffer with the fluoride ion as the counterion. Heats of dilution for the fluoride salts of ethidium and propidium were substantially less than the corresponding values found for other halide salts of these cations. At a Na+ ion concentrations of 0.019, ΔHB = −8.3 and −7.9 ± 0.3 kcal mol−1 for ethidium and propidium, respectively. For these two cations, just as was observed for the naphthalene monoimide (monocation) and diimide (dication) [H. P. Hopkins, K. A. Stevenson, and W. D. Wilson, (1986) J. Sol. Chem. 15, 563–579], ΔHB is within the same experimental error for both cations. Apparently, charge–charge interactions in DNA–cation complexes produce only small changes in the enthalpy for the system. In the concentration range 0.019–0.207, the ΔHB values for propidium did not depend appreciably on the Na+ ion concentration, and a similar pattern was shown to exist for ethidium. When these results were combined with ΔGB values for the binding of these cations to DNA, we found the variation of ΔSB with Na+ ion concentration to be remarkably close to the predictions of modern polyelectrolyte theory, i.e., propidium binding to DNA causes approximately twice as many Na+ ions to be released into the bulk solution as does the binding of ethidium. The much stronger binding of propidium, relative to ethidium, at low ionic strengths is thus seen to be primarily due to entropic effects.

Sequence-dependent cooperative interactions in A/T-containing oligo- and polydeoxyribonucleotides.

To evaluate the length and sequence dependence of the unusual interaction properties observed for nonalternating A/T sequences in deoxyribonucleic acid (DNA) [Wilson, W. D., Wang, Y. H., Krishnamoorthy, C. R., & Smith, J. C. (1985) Biochemistry 24, 3991-3999], we have synthesized the oligomers d(A-T)6, dA10 X dT10, and d(A6-T6) and evaluated their interaction with the intercalator propidium. Propidium visible spectral shifts on adding all three oligomers are quite similar. Low-temperature spectrophotometric binding measurements indicate that d(A-T)6 has a significantly larger binding constant for propidium than dA10.dT10, as with the analogous alternating and nonalternating DNA polymers. The oligomer dA10.dT10 displays positive cooperativity in its propidium binding isotherm, and its binding constant increases with increasing temperature while d(A-T)6 does not display positive cooperativity, and its binding constant decreases with temperature, again as with the analogous polymers. van't Hoff plots indicate that the propidium binding enthalpies are approximately -9 and +6 kcal/mol for the alternating and nonalternating DNA samples, respectively. The mixed-sequence self-complementary oligomer d(A6-T6) has an unusual low-temperature binding isotherm which suggests a single strong binding site and a larger number of weaker binding sites which bind propidium cooperatively. A van't Hoff plot indicates that the cooperative sites d(A-T)6 have binding constants and binding enthalpies similar to dA10.dT10. Similar rate constants are observed in the sodium dodecyl sulfate driven dissociation reaction of propidium from d(A-T)6 and d(A6-T6), but the association reaction of propidium is significantly slower with d(A6-T6) than with d(A-T)6.(ABSTRACT TRUNCATED AT 250 WORDS)

Intercalators as probes of DNA conformation: Propidium binding to alternating and non-alternating polymers containing guanine

Propidium iodide is used as a structural probe for alternating and non-alternating DNA polymers containing guanine and the results are compared to experiments with poly[d(A-T)2], poly(dA . dT) and random DNA sequences. Viscometric titrations indicate that propidium binds to all polymers and to DNA by intercalation. The binding constant and binding site size are quite similar for all alternating polymers, non-alternating polymers containing guanine and natural DNA. Poly(dA . dT) is unusual with a lower binding constant and positive cooperativity in its propidium binding isotherms. Poly(dA . dT) and poly(dG . dC) have similar salt effects but quite different temperature effects in propidium binding equilibria. Polymers and natural DNA have similar rate constants in their SDS driven dissociation reactions. The association rate constants are similar for the alternating polymers and poly(dG . dC) but are significantly reduced for poly(dA . dT). These results suggest that natural DNA, the alternating polymers, and non-alternating polymers containing guanine convert to an intercalated conformation with bound propidium in a very similar manner.

Mechanism of intercalation: ion effects on the equilibrium and kinetic constants for the interaction of propidium and ethidium with DNA.

AbstractThe effects of sodium ion concentration on the binding isotherms and association and dissociation reaction rates for the interaction of the closely related intercalating dication, propidium, and the monocation, ethidium, with DNA have been determined by spectrophotometric binding and stopped‐flow kinetics methods. The binding of propidium to DNA is best described by a neighbor‐exclusion binding isotherm (two base pairs per binding site) with negative ligand cooperatively on binding. The cooperativity parameter is fairly independent of salt concentration, while the log of the observed equilibrium binding constant varies with −log [Na+], with a slope of two for the propidium–DNA interaction. These effects of the sodium ion on the equilibrium binding of propidium with DNA are similar to those previously described for the dication, quinacrine [Wilson, W.D. & Lopp, I.G. (1979) Biopolymers, 18, 3025–3041]. Ethidium behaves, as a function of salt, as a monocation binding to DNA with neighbor exclusion and without ligand cooperativity. Equations are derived for two limiting kinetics models for intercalation involving binding of the intercalator to a preequilibrium, open state of DNA (model I) or binding form a preequilibrium externally associated state of the intercalator with DNA (model II). Model II gives the best fit to all of the kinetic results if it is assumed that the initial external interaction of the intercalator with DNA is similar to the exchange of free and condensed simple counterions. Intercalation then occurs from this state following an opening transition of the base pairs of the double helix. This model predicts a larger effect of salt concentration on the association than on the dissociation reaction, and that is what is experimentally observed. The intercalation conformational change makes a significant contribution to the ionic effects for both the equilibrium binding and the kinetic constants. The dissociation results and the association reaction results under pseudo‐first‐order conditions could be fit with single exponential curves under the conditions of our experiments.

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA.

AbstractAt low temperature and low salt concentration, both imino proton and 31p‐nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow‐exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring‐current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow‐ and fast‐exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor‐exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor‐exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in 31P‐nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately −2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at −3.9 to −4.0 ppm relative to −4.35 in uncomplexed DNA. The simplest neighbor‐binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation‐induced unwinding of the double helix over several base pairs and/or a DNA sequence‐ and conformation‐dependent heterogeneity in intercalation‐induced chemical shifts and resulting exchange rates.

Circular dichroism studies of acetylcholine sterase conformation. Comparison of the 11 S and 5.6 S species and the differences induced by inhibitory ligands

Circular dichroism studies were carried out in the vacuum ultraviolet region for 11 S and 5.6 S species of acetylcholinesterase from Torpedo. As the 5.6 S acetylcholinesterase forms larger oligomers in the absence of detergent, the CD spectrum was measured both with and without detergent. Secondary structure analysis of the CD spectrum for 11 S acetylcholinesterase shows 33% α-helix, 23% β-sheet (14% antiparallel and 9% parallel), 17% turns and 26% other structure. Binding of edrophonium to the active site of 11 S acetylcholinesterase increases α-helix, while binding of propidium to the peripheral site increases β-sheet. The β-sheet content is slightly higher for 5.6 S than 11 S acetylcholinesterase in water. When the detergent is added to 5.6 S acetylcholinesterase, the 190 nm and 220 nm bands become less intense, although the analyses of the two spectra are similar. No significant change is observed for the 5.6 S form in either solvent on binding ligands. The prediction of both parallel and antiparallel β-sheet suggests that at least one domain in these multidomain proteins belongs to the α / β tertiary structural type.

Equilibrium and kinetic studies of the interaction of site-specific ligands with acetylcholinesterase from Electrophorus electricus.

The interactions of edrophonium chloride, gallamine triiodide, and propidium diiodide with affinity-purified acetylcholinesterase from Electrophorus electricus have been examined under conditions of low ionic strength (0.001 M Tris, pH 8.0) using kinetic and fluorescence titration techniques. Edrophonium is a competitive inhibitor of the steady-state hydrolysis of acetylthiocholine, with an inhibition constant, Kcomp, of 1.2 × 10−8 M. Double reciprocal plots in the presence of either gallamine or propidium are nonlinear. Similarly, the pre-steady-state carbamoylation of the enzyme by 7-(dimethylcarbamoyloxy)-N-methyl quinolinium iodide is competitively inhibited by edrophonium, whereas the intercepts of the double reciprocal plots of pseudo-first-order rate constant of carbamoylation versus substrate concentration are displaced downwards in the presence of gallamine or propidium. These results, and those of equilibrium binding studies utilizing the fluorescence properties of bound propidium, suggest that gallamine and propidium compete for a peripheral class of anionic sites on the enzyme, whereas edrophonium binds to the anionic subsite of the catalytic site. The characteristics of propidium binding to the eel enzyme differ from those previously observed with enzyme isolated from Torpedo californica. Whereas the tetrameric Torpedo enzyme possesses four binding sites of equal affinity for propidium, the eel enzyme appears to have two classes of propidium binding site. One set of approximately two sites per tetramer is characterized by a dissociation constant of approximately 2–5 × 10−8 M; a second set of two sites bind propidium with a dissociation constant of 4 × 10−6 M. Possible reasons for these differences are discussed.

Propidium as a probe of acetylcholine receptor binding sites

The fluorescence of propidium was enhanced in the presence of acetylcholine receptor (AcChR) purified from Torpedo californica electroplax membranes. This fluorescence enhancement has been exploited to titrate the AcChR and to search for the effects of other cholinergic ligands on propidium binding by equilibrium and stopped-flow kinetic techniques. Propidium bound to half of the α-bungarotoxin binding sites on the AcChR with a K d = 1.8 × 10 −6, M and various bis-quaternary cholinergic analogs acted as competitors of propidium binding, the dissociation constant for these cholinergic ligands being obtained by competitive binding treatment of the data. Carbamylcholine did not displace propidium at concentrations corresponding to its known dissociation constant, but it affected the fluorescence yield of bound propidium in the proposed ternary complex. The site for propidium binding is likely to be a cationic binding site near one of the specific cholinergic neurotransmitter binding sites. The stopped-flow results allow calculation of the rates of dissociation ( k −1 = 0.2 s −1) of propidium from AcChR-propidium complexes and estimation, to a first approximation, of the association rate constant value ( k 1 = 1 × 10 5, M −1 S −1).

Interaction of fluorescence probes with acetylcholinesterase. The site and specificity of propidium binding.

A bis-quaternary fluorescence probe, propidium diiodide, has been found to exhibit a tenfold enhancement of fluorescence when bound to acetylcholinesterase from Torpedo california. The complex is characterized by a high affinity, KD = 3.0 times 10-7 M, and 1:1 stoichiometry with the 82,000 molecular weight subunit of acetylcholinesterase. A wide variety of other quaternary ammonium ligands such as decamethonium, gallamine, d-tubocurarine, tetraethylammonium, and tetramethylammonium will completely dissociate propidium from the enzyme as will monovalent and divalent inorganic cations. The competitive dissociation does not show cooperative behavior or a distinct, requirement for occupation of multiple sites of different affinity to produce displacement. While a directly competitive relationship can be illustrated macroscopically, the various quaternary ligands show a different susceptibility toward inorganic cation displacement. The affinity of propidium relative to gallamine increases with ionic strength. This finding indicates that there is not complete equivalence in the negative subsites to which quaternary groups bind. Although edrophoniumwill also displace propidium from the enzyme, the dissociation constant obtained from this competitive relationship is 3.5 orders of magnitude greater than the constants obtained for inhibition of catalysis. By competitive displacement titrations it is shown that the primary binding site of edrophonium is distinct from that of propidium and a ternary complex with the two ligands can form on each subunit. In contrast to edrophonium, the binding of propidium is unaffected by methanesulfonylation of the active center serine and is uncompetitive with the carbamylating substrate, N-methyl-7-dimethylcarbamoxyquinolinium. Thus, it appears that propidium associates with a peripheral anionic center on the enzyme. Although propidium and edrophonium associate at separate sites on acetylcholinesterase, bis-quaternary ligands where the quaternary nitrogens are separated by 14 A displace both ligands from the enzyme with equal effectiveness.