Alternative Reaction Schemes Research Articles

Rapid, Quantitative Nuclear Magnetic Resonance Test for Oxygen-17 Enrichment in Water.

Nuclear magnetic resonance (NMR) studies involving 17O are increasingly important in molecular biology, material science, and other disciplines. A large number of these studies employ H217O as a source of 17O, and this reliance can be limiting because the high cost of H217O. To overcome this constraint, a recent study proposed a distillation scheme capable of producing significant quantities of H217O at a low cost. Although this method is reported to be effective, the reactions proposed to quantify percent of 17O enrichment are either time intensive or have a risk of errors due to the isotope effect. Here, an alternative reaction scheme is described to measure 17O water that ultimately creates methyl benzoate as the sole 17O-containing product. The proposed reaction is completed in a matter of minutes at room temperature, produces only one 17O product, and requires no clean-up step. The large isotope shift observed in solution NMR between the 13C═16O and 13C═17O resonances allows for integration of the individual peaks. This 13C NMR analysis is found to be highly accurate over a wide enrichment range and is accessible to most NMR spectroscopists.

Strongly Coupled Redox-Linked Conformational Switching at the Active Site of the Non-Heme Iron-Dependent Dioxygenase, TauD.

2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. We characterize an in situ structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD) by using a combination of spectroelectrochemical and semiempirical computational methods, demonstrating that the Fe(III/II) transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement alters the apparent redox potential of the active site between -127 mV for reduction of the ferric state and +171 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex. Structural perturbations exhibit limited sensitivity to mediator concentrations and potential pulse duration. Similar changes were observed in the Fe-TauD and taurine-2OG-Fe-TauD complexes, thus attributing the reorganization to the protein moiety rather than the cosubstrates. Redox-difference infrared spectra indicate a reorganization of the protein backbone in addition to the involvement of carboxylate and histidine ligands. Quantitative modeling of the transient redox response using two alternative reaction schemes across a variety of experimental conditions strongly supports the proposal for intrinsic protein reorganization as the origin of the experimental observations.

Ionization and Conformational Equilibria of Citric Acid: Delocalized Proton Binding in Solution.

The microspeciation of citric acid is studied by analyzing NMR titration data. When the site binding (SB) model, which assumes fully localized proton binding to the carboxylic groups, is used to obtain microscopic energy parameters (dissociation constants, pair and triplet interaction energies between charged carboxylate groups), contradictory results are obtained. The resulting macroscopic constants are in very good agreement with the values reported in the literature using potentiometry. However, the found pair interaction energy between the terminal carboxylates and the triplet interaction energy are physically meaningless. To solve this apparent contradiction, we consider the possibility of delocalized proton binding, so that the proton can be exchanged at high velocity in the NMR time scale through short, strong, low-barrier (SSLB) hydrogen bonds. With this aim, ab initio MP2 calculations using the SMD polarizable continuum model for the solvent were performed and the fully roto-microspeciation elucidated. First, fully localized proton binding was assumed, and the resulting microstate probabilities are in reasonable agreement with those reported in previous works that use selective blocking of the carboxylic groups. They are, however, in clear disagreement with the microstate probabilities derived from the NMR titration data, which predict, within a very narrow confidence interval, a unique microspecies for the symmetric di-ionized form. Moreover, counterintuitively, the interaction between terminal charged groups is much larger than that between central and terminal groups. As a consequence, we have explored the possibility of delocalized proton binding by calculating the energy of intermediate proton positions between two carbolxylic groups. The results reveal that the exchange of the proton through the hydrogen bonds is in some cases produced without energetic barrier. This effect is specially relevant in the di-ionized form, with all the most stable conformations forming a SSLB, which together would constitute the only microstate detected by NMR. An alternative reaction scheme for the ionization process, based on proton delocalization, is proposed.

Kinetics of ATP and TNP-ATP Binding to the Active Site of CheA from Thermotoga maritima

The mechanism of nucleotide binding to the active site of Thermotoga maritima CheA was investigated using stopped-flow fluorescence experiments that monitored binding of ATP and TNP-ATP to the catalytic domain (P4) of CheA that had been engineered to include a tryptophan residue as a fluorescent reporter group at the active site (P4(F487W)). Rapid decreases in protein intrinsic fluorescence and increases in TNP-ATP fluorescence were observed during binding reactions, and time courses were analyzed to define the kinetic mechanisms for ATP and TNP-ATP binding. This analysis indicated that binding of ATP(Mg(2+)) to P4(F487W) involves a single reversible step with a k(on) of 0.92 +/- 0.09 microM(-1) s(-1), a k(off) of 1.9 +/- 0.4 s(-1), and a K(d) of 1.5-2.1 microM (all values determined at 4 degrees C). Binding of TNP-ATP(Mg(2+)) to P4(F487W) involves a more complicated mechanism, requiring at least three sequential steps. Computer simulations and nonlinear regression analysis were used to estimate the rate constants of the forward and reverse reactions for each of the three steps in the reaction scheme [Formula: see text] Similar analysis indicated that an alternative reaction scheme, involving a rate-limiting conformational change in P4 prior to TNP-ATP binding, did an equally good job of accounting for all of the kinetics results:[Formula: see text] In both models, steps 2 and 3 have slow reversal rates that contribute to the high affinity of the active site for TNP-ATP (K(d) = 0.015 microM). These results highlight the dramatic effect of the TNP moieties on CheA-nucleotide interactions, and they provide the first detailed information about the kinetic mechanism underlying interaction of a protein histidine kinase with this tight-binding inhibitor.

Glycerol Etherification over Highly Active CaO‐Based Materials: New Mechanistic Aspects and Related Colloidal Particle Formation

Glycerol is an attractive renewable building block for the synthesis of di- and triglycerols, which have numerous applications in the cosmetic and pharmaceutical industries. In this work, the selective etherification of glycerol to di- and triglycerol was studied in the presence of alkaline earth metal oxides and the data are compared with those obtained with Na2CO3 as a homogeneous catalyst. It was found that glycerol conversion increased with increasing catalyst basicity; that is, the conversion increases in the order: MgO<CaO<SrO<BaO. The best selectivity values for (di- + tri-) glycerol (>90 % at 60 % conversion) are obtained over CaO, SrO, and BaO. For these catalysts no substantial acrolein formation was observed. Furthermore, at the start of the reaction mainly linear diglycerol was produced, whereas at higher conversion degrees branched diglycerol started to form. In another series of experiments different types of CaO materials were prepared. It was found that these CaO-based materials not only differed in their surface area and number of basic sites, but also in their Lewis acid strength. Within this series the CaO material possessing the strongest Lewis acid sites had the highest catalytic activity, comparable to that of BaO, pointing towards the important role of Lewis acidity for this etherification reaction. Based on these observations a plausible alternative reaction scheme for glycerol etherification is presented, which considers the facilitation of the hydroxyl leaving process. Finally, the stability of the catalytic solids under study was investigated and it was found that colloidal CaO particles of about 50-100 nm can be spontaneously generated during reaction. Catalytic testing of these CaO colloids, after isolation from the reaction medium, revealed a very high etherification activity. Understanding the nature of these Ca-based colloids opens new opportunities for investigating supported colloidal particle catalysts to take advantage of both their hetero- and homogeneous nature.

Characterisation of metal oxide films deposited by non‐hydrolytic ALD

AbstractAtomic layer deposition (ALD) has become an established technique for producing thin films of a wide range of materials. Of particular note is the extensive research on the use of ALD metal oxide films as the replacement for the high‐k SiO2 dielectric layer in future semiconductor devices. In much of this work, metal halides or metallorganics combined with water, peroxide or ozone have served as precursors for the sequential deposition process that forms the basis of ALD. Alternative reaction schemes, such as those in which metal halides are reacted with metal alkoxides, have also been investigated with some success though higher temperatures are often required for film deposition. However, the effect of alternative oxidising species in ALD has not been extensively studied in terms of both their efficacy and their influence on the film growth process. Here we describe recent work on the use of t‐butyl alcohol (tBA) and di‐isopropyl ether (DIPE) for the ALD of titania films from titanium tetrachloride. A quartz crystal microbalance (QCM) was used to monitor film growth in real time. Films were also grown on silicon wafers for post‐deposition analysis. Spectroscopic ellipsometry, atomic force microscopy (AFM), X‐ray diffraction (XRD) and secondary ion mass spectrometry (SIMS) were used for this purpose. The results obtained from this combination of surface analytical techniques will be discussed in detail in order to illustrate the strength of this approach for the elucidation of ALD film growth by means of non‐hydrolytic reactions. Copyright © 2006 John Wiley &amp; Sons, Ltd.

Substrate Specificity and Structure-Function Analysis of the 3′-Phosphoesterase Component of the Bacterial NHEJ Protein, DNA Ligase D

DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.

Hydroxyl radical formation by O-O bond homolysis in peroxynitrous acid.

Peroxynitrite decay in weakly alkaline media occurs by two concurrent sets of pathways which are distinguished by their reaction products. One set leads to net isomerization to NO(3)(-) and the other set to net decomposition to O(2) plus NO(2)(-). At sufficiently high peroxynitrite concentrations, the decay half-time becomes concentration-independent and approaches a limiting value predicted by a mechanism in which reaction is initiated by unimolecular homolysis of the peroxo O-O bond, i.e., the following reaction: ONOOH --> (*)OH + (*)NO(2). This dynamical behavior excludes alternative postulated mechanisms that ascribe decomposition to bond rearrangement within bimolecular adducts. Nitrate and nitrite product distributions measured at very low peroxynitrite concentrations also correspond to predictions of the homolysis model, contrary to a recent report from another laboratory. Additionally, (1) the rate constant for the reaction ONOO(-) --> (*)NO + (*)O(2)(-), which is critical to the kinetic model, has been confirmed, (2) the apparent volume of activation for ONOOH decay (DeltaV() = 9.7 +/- 1.4 cm(3)/mol) has been shown to be independent of the concentration of added nitrite and identical to most other reported values, and (3) complex patterns of inhibition of O(2) formation by radical scavengers, which are impossible to rationalize by alternative proposed reaction schemes, are shown to be quantitatively in accord with the homolysis model. These observations resolve major disputes over experimental data existing in the literature; despite extensive investigation of these reactions, no verifiable experimental evidence has been advanced that contradicts the homolysis model.

Synthesis and structure-activity relationships of novel antiseptics

For the purpose of developing new antiseptics, we searched for compounds having high biocidal activity covering both gram-positive and gram-negative bacteria. Accordingly, we designed 1,5-disubstituted biguanides and synthesized them using two alternative efficient reaction schemes. The bactericidal activity of these biguanides was assayed by the micro titration plate method. Among the biguanides synthesized, 3,4-dichlorobenzyl derivatives were found to exhibit particularly high bactericidal activity. Ultimately, compound 11 was chosen as a candidate novel antiseptic, which is currently under continued evaluation.

Proteinase Specificity and Functional Diversity in Point Mutants of Plasminogen Activator Inhibitor 1

Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily. The alternative behavior of PAI-1 as an inhibitor, a non-inhibitory substrate, or a non-reactive latent form has been shown to be dependent on the initial conformation. In this study, we have evaluated the effect of a substitution outside the reactive site loop (P18) or in the reactive site loop (P6 and P10) on proteinase specificity and conformational transitions in PAI-1. Wild-type PAI-1 (wtPAI-1) revealed the same conformational distribution pattern toward tissue-type plasminogen activator (t-PA) as toward urokinase-type plasminogen activator (u-PA) (i.e. 53 +/- 6. 9% active, 36 +/- 6.8% latent, and 12 +/- 1.9% substrate). Inactivation of wtPAI-1 resulted in the conversion of the labile active form into the latent form while the stable substrate form remained unchanged. PAI-1-P6 (Val --> Pro at P6) revealed a target specificity for t-PA (39 +/- 7% versus 3 +/- 2% of the theoretical maximal value toward t-PA and u-PA, respectively), PAI-1-P10 (Ser --> Pro at P10) was 4-fold more active toward u-PA than toward t-PA, and PAI-1-P18 (Asn --> Pro at P18) exhibited inhibitory properties exclusively toward u-PA (41 +/- 10%). Surprisingly, inactivation of these mutants revealed functional and conformational transitions distinct from those observed for wtPAI-1. Inactivation of PAI-1-P6(Val --> Pro) resulted in a total conversion of the active form into the latent form and in a partial conversion of the substrate form into the latent form. The active forms of both PAI-1-P10(Ser --> Pro) and PAI-1-P18(Asn --> Pro) are also labile but, in contrast to the active form of wtPAI-1, convert into substrate forms. Based on the existence of various conformations of PAI-1, we propose an alternative reaction scheme describing the putative interactions between serpins and their target proteinases. The unusual conformational and functional flexibility of PAI-1 that, according to the current study, appears not to be restricted to the reactive site loop further underlines the importance of potential structural rearrangements (e.g. upon binding to cofactors) in PAI-1 (or serpins in general) for its functional behavior at particular biological sites.

Oxidation of Glyoxylic Acid by Cerium(IV): Oxygen-Induced Enhancement of the Primary Radical Concentration

In order to help understand the role of oxygen in Ce(IV)-induced oxidation of small carbonic acids, we investigated the reaction of glyoxylic acid (HCOCOOH) and Ce(IV) in 1 M sulfuric acid. Spectrophotometric data showed that in excess of glyoxylic acid the consumption of Ce(IV) obeys pseudo-first-order kinetics, with a rate constant of 8.8 L mol-1 s-1 at 25 °C and an activation energy of 80 kJ mol-1. Rapid-flow EPR measurements revealed an approximately 1:2:1 triplet with a g value of 2.0071 ± 0.0005 and a hyperfine splitting of 7.1 ± 0.2 G, assignable to the primary radical formed by abstraction of a hydrogen atom from hydrated glyoxylic acid. The rate constant for the anaerobic self-decay of the radical was measured as approximately 3.7 × 109 L mol-1 s-1. Surprisingly, oxygen had no effect on the Ce(IV) kinetics, while the radical decay was significantly inhibited under aerobic conditions (ratio of experimental rate constants ≈6.3). Amperometric measurements revealed accompanying oxygen consumption. Analyses based on numerical simulations show that the observed oxygen-induced increase in radical concentration cannot be explained in the framework of standard autooxidation mechanisms. An alternative reaction scheme is suggested which reproduces the observed aerobic radical kinetics and which thus could be relevant to similar oxidation reactions.

Comment on “the kinetics of sterane biological marker release and degradation processes during the hydrous pyrolysis of vitrinite kerogen” by G. D. Abbott, G. Y. Wang, T. I. Eglinton, A. K. Home and G. S. Petch

In order to further develop understanding of the observable changes in sterane isomer ratios, Abbott et al. (1990) conducted a series of hydrous pyrolysis experiments on mineral-free vitrinite kerogen. Using a deuterated model compound, they found that direct chiral isomerization appears to be relatively unimportant. They derived a kinetic model comprising consecutive release and degradation processes for both isomers and, using a bi-exponential concentration-time function, they were able to determine Arrhenius parameters for four individual reactions. Abbott et al. (1990) claimed that these four reactions account for the change in sterane diastereomer ratios in hydrous pyrolysis experiments. Since it has been observed by a number of people that the so-called steraine isomerization is not a unidirectional reaction, an alternative reaction scheme would be most welcome. Although Abbott and coworkers do not claim that the published kinetic data hold true in the natural environments, the question arises as to whether the kinetic data obtained from these hydrous pyrolysis experiments are applicable to sedimentary processes which occur over geological time.

Formal total synthesis of (+)-12-deoxyscalarolide

An effective formal total synthesis of (+)-12-deoxyscalarolide is described, starting from natural manool. Two alternative reaction schemes have been investigated for the synthesis of the tetra-carbocyclic key intermediate (–)-methyl 17-oxo-24-norscalaran-25-oate. The unsaturated lactonic ring is then created from this ester in three steps to afford, in high yield, the (–)-scalar-16-en-25,24-olide which is easily isomerised under basic conditions to (+)-12-deoxyscalarolide.

Energetics of order/order transitions in nucleic acids

Besides the temperature induced canonical helix to random coil transition, which serves as a prototype of an order/disorder transition, there is a second group of conformational changes in helical polynucleotides, which will be termed order/order transitions. Depending on the environmental conditions and the sequence of the polynucleotides involved, there are four alternative reaction schemes: (i) The inversion of the helical handedness from a right-handed to a left-handed conformation, (ii) the disproportionation of a double helix into a single strand and a triple helix, (iii) the addition of a single strand to a double helix, and (iv) the single/double strand displacement reactions, that is, the displacement of one of the two strands of the helix by a matching single-stranded polynucleotide or the mutual exchange of one strand each between two helical structures. The first three reactions are considered reversible while the last reaction is irreversible; that is, one cannot regain the initial state by inversing the course of the change, here the change in temperature. Both ribo and deoxyribo polynucleotides can undergo these reactions. In the following paper at least one example is given for each of these reactions.

The brusselator: it does oscillate all the same

The occurrence of temporal oscillations in the Brusselator model is perfectly compatible with such basic physicochemical laws as detailed balance at equilibrium and the law of conservation of mass for closed chemical systems. The recent erroneous claims asserting the contrary are refuted. We also discuss the mathematical conditions, ensuring that the model's dynamics faithfully approximate those of an alternative reaction scheme involving only mono- and bi-molecular chemical steps.

Reversible Photoisomerisierungen an Sulfophthalein‐Indikatorfarbstoffen. IV. Stopped‐Flow‐Untersuchungen zum Mechanismus der baseninduzierten Sultonringöffnung

Reversible Photoisomerizations of Sulfophthalein Indicator Dyes. IV. Stopped Flow Investigations on the Mechanism of the Base Induced Sulton Ring OpeningThe colour formation of rapidly mixed solutions of bromo thymol blue and organic bases in toluene obeys a biexponential rate law. The most probable reaction sequence is: Sulton (colourless) + base ⇌ ring opened H‐bridged complex (λmax ≈ 420 nm) ⇌ H‐bridged ion pair (λmax ≈ 400 nm). Alternative reaction schemes of the same complexity could be ruled out though the underlying kinetic problem is underdetermined. As a generalizing conclusion it is stated that valuable mechanistic and kinetic information may be derived from underdetermined kinetic problems by calculating the ranges of physically meaningful solutions for the unknowns which are compatible with the particular kinetic schemes instead of the unavailable singular solutions.

Molecular weight distributions of poly(methyl methacrylate) produced at high viscosities

AbstractThe molecular weight distribution of poly(methyl methacrylate), which is produced by the free‐radical polymerisation of methyl methacrylate, is influenced by the presence of poly(methyl methacrylate) which is dissolved in the monomer prior to the polymerisation. An experimental technique has been devised which permits a distinction to be made between polymer which is present at the start of the reaction and the polymer which is formed by the reaction. Mathematical analyses of alternative reaction schemes have been made. These predicted changes that would occur in the molecular weight distribution of the new polymer when the solution viscosity increased. Comparison with experimental measurements of molecular weights makes possible the distinction between the alternative reaction schemes. The results are compatible with a reaction mechanism in which the mobilities of the growing radicals are not influenced equally by the solution viscosity. The results are not those which would be expected by the simple inclusion of primary radical termination in the reaction scheme. In some cases, analysis by gel permeation chromatography showed that the molecular weight distribution of the poly(methyl methacrylate) formed by the reaction is bimodal. This is consistent with predictions from the mathematical analysis.

ChemInform Abstract: THE HOMOGENEOUS HYDROGEN‐EVOLVING SYSTEMS (MO2FE6S8(SPH)9)4‐,5‐/C6H5SH: REACTION CHARACTERISTICS, KINETICS, AND POSSIBLE MECHANISMS

AbstractDie Titelcluster (I) sowie das Derivat (II) entwickeln aus Lösungen von Benzolthiol in Dimethylacetamid bei Raumtemp. H2.

The homogeneous hydrogen-evolving systems [Mo2Fe6S8(SPh)9]4-,5-/C6H5SH: reaction characteristics, kinetics, and possible mechanisms

~([4-1, [5-1) and [Fe4S4(SPh),13- evolve H2 from benzenethiol in solutions of purified N,N'-dimethylacetamide at ambient temperature. With 500 equiv of thiol clusters [4-] and [5-] afford 86% and -100% H2 yields, respectively, after 24 h. Cluster [5-] also produces H2 from a variety of other protic sources including an 83% yield from 2 equiv of Et3NH+ after 24 h. Both [5-]/PhSH and [4-]/PhSH systems generate a common intermediate (Im), which is the last kinetically resolvable species prior to H2 evolution, experimentally expressible as d[H2]/df = K[Im][PhSH]. Analysis of H2 evolution rates in the [4-]/PhSH system leads to the experimental rate law d[H,]/df = [4-]2[PhSH]2/(2300( [3-] + 0.00043) [PhS-] + 0.014[PhSH]). The kinetics of a large number of reaction schemes were theoretically analyzed. Acceptable schemes are those that conform to the preceding rate laws and other experimental constraints. From these the simplest chemically reasonable schemes 'are proposed for Im formation in the [5-]/PhSH system and H2 evolution in the [4-]/PhSH system. The latter is considered to be RSH I4-1 Rs13-1 [4-] V [4-].H+ e [5-].H+ + Im - H2 involving cluster protonation, electron transfer, Im formation, and H2 evolution. The reaction sequence is presumably driven by the demonstrated irreversibility of the overall reaction 2[4-] + 2PhSH - 2[3-] + 2PhS- + H2. Details of the kinetic analysis and alternative schemes are presented. The potential advantage of a two-electron carrier for reduction of H+ (to hydride) and other two-electron substrates is noted. In this work this advantage is expressed in the proposed and alternative reaction schemes and is experimentally demonstrated by a 42% H2 yield from the one-electron reductant [ Fe4S4(SPh),13and -500 equiv of PhSH after 24 h.

Equilibrium binding of acetylcholine to the membrane-bound acetylcholine receptor.

We have studied the binding of acetylcholine to membrane-bound acetylcholine receptor from Torpedo marmorata employing a highly accurate airfuge assay procedure. At equilibrium the receptor displays two classes of acetylcholine binding sites; these interact with only weak positive cooperativity. As a further difference to binding data deduced from electrophysiological dose/response curves, the equilibrium constants for the two classes of sites (Kd1 = 25 nM, Kd2 = 8 nM) are orders of magnitude lower than the concentration required for half-maximal response. Both the weaker-than-expected cooperativity of sites and the high binding affinities are likely to be due to desensitisation of the receptor during the period of incubation. The positively cooperative interaction of acetylcholine binding sites is only observed with membrane preparations obtained in the presence of appropriate chelating, sulfhydryl-blocking and active-serine-blocking agents. Aged membrane preparations loose the ability of site interactions while only small changes in the total number of binding sites are observed. In the absence of divalent ions, the affinity of binding of acetylcholine to the receptor is reduced. To assess the significance of the binding data obtained, several alternative reaction schemes for non-random binding to two sites at the receptor are considered. In addition, the effects of possible sources of experimental error on the shape of Scatchard plots are analysed.