Plasma-Derived Hemopexin as a Candidate Therapeutic Agent for Acute Vaso-Occlusion in Sickle Cell Disease: Preclinical Evidence.

People living with sickle cell disease (SCD) face intermittent acute pain episodes due to vaso-occlusion primarily treated palliatively with opioids. Hemolysis of sickle erythrocytes promotes release of heme, which activates inflammatory cell adhesion proteins on endothelial cells and circulating cells, promoting vaso-occlusion. In this study, plasma-derived hemopexin inhibited heme-mediated cellular externalization of P-selectin and von Willebrand factor, and expression of IL-8, VCAM-1, and heme oxygenase-1 in cultured endothelial cells in a dose-responsive manner. In the Townes SCD mouse model, intravenous injection of free hemoglobin induced vascular stasis (vaso-occlusion) in nearly 40% of subcutaneous blood vessels visualized in a dorsal skin-fold chamber. Hemopexin administered intravenously prevented or relieved stasis in a dose-dependent manner. Hemopexin showed parallel activity in relieving vascular stasis induced by hypoxia-reoxygenation. Repeated IV administration of hemopexin was well tolerated in rats and non-human primates with no adverse findings that could be attributed to human hemopexin. Hemopexin had a half-life in wild-type mice, rats, and non-human primates of 80-102 h, whereas a reduced half-life of hemopexin in Townes SCD mice was observed due to ongoing hemolysis. These data have led to a Phase 1 clinical trial of hemopexin in adults with SCD, which is currently ongoing.

Pharmacological Inhibition of Factor XIIa Attenuates Abdominal Aortic Aneurysm, Reduces Atherosclerosis, and Stabilizes Atherosclerotic Plaques.

BACKGROUND: 3F7 is a monoclonal antibody targeting the enzymatic pocket of activated factor XII (FXIIa), thereby inhibiting its catalytic activity. Given the emerging role of FXIIa in promoting thromboinflammation, along with its apparent redundancy for hemostasis, the selective inhibition of FXIIa represents a novel and highly attractive approach targeting pathogenic processes that cause thromboinflammation-driven cardiovascular diseases. METHODS: The effects of FXIIa inhibition were investigated using three distinct mouse models of cardiovascular disease-angiotensin II-induced abdominal aortic aneurysm (AAA), an ApoE-/- model of atherosclerosis, and a tandem stenosis model of atherosclerotic plaque instability. 3F7 or its isotype control, BM4, was administered to mice (10 mg/kg) on alternate days for 4 to 8 weeks, depending on the experimental model. Mice were examined for the development and size of AAAs, or the burden and instability of atherosclerosis and associated markers of inflammation. RESULTS: Inhibition of FXIIa resulted in a reduced incidence of larger AAAs, with less acute aortic ruptures and an associated fibro-protective phenotype. FXIIa inhibition also decreased stable atherosclerotic plaque burden and achieved plaque stabilization associated with increased deposition of fibrous structures, a >2-fold thicker fibrous cap, increased cap-to-core ratio, and reduction in localized and systemic inflammatory markers. CONCLUSION: Inhibition of FXIIa attenuates disease severity across three mouse models of thromboinflammation-driven cardiovascular diseases. Specifically, the FXIIa-inhibiting monoclonal antibody 3F7 reduces AAA severity, inhibits the development of atherosclerosis, and stabilizes vulnerable plaques. Ultimately, clinical trials in patients with cardiovascular diseases such as AAA and atherosclerosis are warranted to demonstrate the therapeutic potential of FXIIa inhibition.

Small heat-shock proteins: paramedics of the cell.

The small heat-shock proteins (sHSPs) comprise a family of molecular chaperones which are widespread but poorly understood. Despite considerable effort, comparatively few high-resolution structures have been determined for the sHSPs, a likely consequence of their tendency to populate ensembles of inter-converting conformational and oligomeric states at equilibrium. This dynamic structure appears to underpin the sHSPs' ability to bind and sequester target proteins rapidly, and renders them the first line of defence against protein aggregation during disease and cellular stress. Here we describe recent studies on the sHSPs, with a particular focus on those which have provided insight into the structure and dynamics of these proteins. The combined literature reveals a picture of a remarkable family of molecular chaperones whose thermodynamic and kinetic properties are exquisitely balanced to allow functional regulation by subtle changes in cellular conditions.

Dissecting heterogeneous molecular chaperone complexes using a mass spectrum deconvolution approach.

Small heat-shock proteins (sHSPs) are molecular chaperones that prevent irreversible aggregation through binding nonnative target proteins. Due to their heterogeneity, these sHSP:target complexes remain poorly understood. We present a nanoelectrospray mass spectrometry analysis algorithm for estimating the distribution of stoichiometries comprising a polydisperse ensemble of oligomers. We thus elucidate the organization of complexes formed between sHSPs and different target proteins. We find that binding is mass dependent, with the resultant complexes reflecting the native quaternary architecture of the target, indicating that protection happens early in the denaturation. Our data therefore explain the apparent paradox of how variable complex morphologies result from the generic mechanism of protection afforded by sHSPs. Our approach is applicable to a range of polydisperse proteins and provides a means for the automated and accurate interpretation of mass spectra derived from heterogeneous protein assemblies.

Gold-mediated C-I bond activation of iodobenzene.

Controversy resolved! A combination of gas-phase ion-molecule reactions and theoretical studies confirm bisligated mononuclear Au(I) complexes are unable to undergo oxidative addition of iodobenzene for Sonogashira coupling, but that the ligated gold clusters [Au(3)L(n)](+) (L=Ph(2)P(CH(2))(n)PPh(2); n=3-6) activate the C-I bond. DFT calculations on the transition states show that the linker size n tunes the cluster reactivity.

The polydispersity of αB-crystallin is rationalized by an interconverting polyhedral architecture.

We report structural models for the most abundant oligomers populated by the polydisperse molecular chaperone αB-crystallin. Subunit connectivity is determined by using restraints obtained from nuclear magnetic resonance spectroscopy and mass spectrometry measurements, enabling the construction of various oligomeric models. These candidate structures are filtered according to their correspondence with ion-mobility spectrometry data and cross-validated by using electron microscopy. The ensuing best-fit structures reveal the polyhedral architecture of αB-crystallin oligomers, and provide a rationale for their polydispersity and facile interconversion.

Quaternary dynamics of αB-crystallin as a direct consequence of localised tertiary fluctuations in the C-terminus.

The majority of proteins exist in vivo within macromolecular assemblies whose functions are dependent on dynamical processes spanning a wide range of time scales. One such assembly is formed by the molecular chaperone αB-crystallin that exists in a variety of exchanging oligomeric states, centred on a mass of approximately 560 kDa. For many macromolecular assemblies, including αB-crystallin, the inherent dynamics, heterogeneity and high mass contribute to difficulties in quantitative studies. Here, we demonstrate a strategy based on correlating solution-state nuclear magnetic resonance spectroscopy and mass spectrometry data to characterize simultaneously the organization and dynamics of the polydisperse αB-crystallin ensemble. We show that protomeric dimers assemble into oligomers via the binding of extended C-termini, with each monomer donating and receiving one terminus. Moreover, we establish that the C-termini undergo millisecond fluctuations that regulate the interconversion of oligomeric forms. The combined biophysical approach allows construction of an energy profile for a single monomer that completely describes the equilibrium dynamics of the ensemble. It also facilitates an analysis of dynamics spanning the millisecond to hour time scales and secondary to quaternary structural levels, and provides an approach for, obtaining simultaneously detailed structural, thermodynamic and kinetic information on a heterogeneous protein assembly.

αB-crystallin polydispersity is a consequence of unbiased quaternary dynamics.

The inherent heterogeneity of many protein assemblies complicates characterization of their structure and dynamics, as most biophysical techniques require homogeneous preparations of isolated components. For this reason, quantitative studies of the molecular chaperone αB-crystallin, which populates a range of interconverting oligomeric states, have been difficult, and the physicochemical basis for its polydispersity has remained unknown. Here, we perform mass spectrometry experiments to study αB-crystallin and extract detailed information as to its oligomeric distribution and exchange of subunits under a range of conditions. This allows a determination of the thermodynamic and kinetic parameters that govern the polydisperse ensemble and enables the construction of a simple energy profile for oligomerization. We find that the quaternary structure and dynamics of the protein can be explained using a simple model with just two oligomer-independent interactions (i.e., interactions that are energetically identical in all oligomers from 10mers to 40mers) between constituent monomers. As such, the distribution of oligomers is governed purely by the dynamics of individual monomers. This provides a new means for understanding the polydispersity of αB-crystallin and a framework for interrogating other heterogeneous protein assemblies.

Preference for bridging versus terminal ligands in magnesium dimers.

Magnesium dimers play important roles in inorganic and organometallic chemistry. This study evaluates the inherent bridging ability of a range of different ligands in magnesium dimers. In the first part, the Cambridge Structural Database is interrogated to establish the frequency of different types of ligands found in bridging versus terminal positions in two key structural motifs: one in which there are two bridging ligands (the D(2h) "Mg(2)(µ-X(2))" structure); the other in which there are three bridging ligands (the C(3v) "Mg(2)(µ-X(3))" structure). The most striking finding from the database search is the overwhelming preference for magnesium dimers possessing two bridging ligands. The most common bridging ligands are C-, N-, and O-based. In the second part, DFT calculations (at the B3LYP/6-311+G(d) level of theory) are carried out to examine a wider range of structural types for dimers consisting of the stoichiometries Mg(2)Cl(3)R and Mg(2)Cl(2)R(2), where R = CH(3), SiH(3), NH(2), PH(2), OH, SH, CH(2)CH(3), CH=CH(2), C≡CH, Ph, OAc, F and Br. Consistent with the database search, the most stable magnesium dimers are those that contain two bridging ligands. Furthermore, it was demonstrated that the electronic effect of the bridging ligands is important in influencing the stability of the magnesium dimers. The preference for a bridging ligand, which reflects its ability to stabilize a magnesium dimer, follows the order: OH > NH(2) > C≡CH > SH > Ph > Br > PH(2) = CH=CH(2) > CH(2)CH(3) > CH(3) > SiH(3). Finally, the role that the ether solvent Me(2)O has on the stability of isomeric Mg(2)Cl(2)Me(2) dimers was studied. It was found that the first solvent molecule stabilizes the dimers, while the second solvent molecule can either have a stabilizing or destabilizing effect, depending on the isomer structure.

How does acetylcholine lose trimethylamine? A density functional theory study of four competing mechanisms.

Low-energy collision-induced dissociation (CID) of acetylcholine (ACh) yields only two fragment ions: the dominant C(4)H(7)O(2)(+) ion at m/z 87, arising from trimethylamine loss; and protonated trimethylamine at m/z 60. Since the literature is replete with conflicting mechanisms for the loss of trimethylamine from ACh, in this article density functional theory (DFT) calculations are used to assess four competing mechanisms: (1) Path A involves a neighboring group attack to form a five-membered ring product, 2-methyl-1,3-dioxolan-2-ylium cation; (2) Path B is a neighboring group attack to form a three-membered ring product, 1-methyl-oxiranium ion; (3) Path C involves an intramolecular elimination reaction to form CO protonated vinylacetate; and (4) Path D is a 1,2-hydride migration reaction forming CH(2)-protonated vinylacetate. At the MP2/6-311++G(2d,p)//B3-LYP/6-31+G(d,p) level of theory path A is the kinetically favored pathway, with a transition-state energy barrier of 37.7 kcal mol(-1) relative to the most stable conformer of ACh. The lowest energy pathway for the formation of protonated trimethylamine was also calculated to proceed via path A, involving proton transfer within the ion-molecule complex intermediate, with the exocylic methyl group being the proton donor. To confirm the site of proton transfer, low-energy CID of acetyl-d(3)-choline (d(3)-ACh) was carried out, which revealed loss of trimethylamine and the formation of Me(3)ND(+).

Energetics and dynamics of the fragmentation reactions of protonated peptides containing methionine sulfoxide or aspartic acid via energy- and time-resolved surface induced dissociation.

The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64 Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility in these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (nonmobile proton conditions) to lysine (partially mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFECs) reveal that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1-2 orders of magnitude lower than nonselective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to nonselective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these processes are much slower compared to amide bond cleavage, explaining why these selective bond cleavages are not observed if fragmentation is performed under mobile proton conditions. This study further affirms that fragmentation of peptide ions in the gas phase are predominantly governed by entropic effects.

Comparison of collision-induced dissociation and electron-induced dissociation of singly protonated aromatic amino acids, cystine and related simple peptides using a hybrid linear ion trap-FT-ICR mass spectrometer.

The gas-phase fragmentation reactions of singly protonated aromatic amino acids, their simple peptides as well as simple models for intermolecular disulfide bonds have been examined using a commercially available hybrid linear ion trap-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Low-energy collision-induced dissociation (CID) reactions within the linear ion trap are compared with electron-induced dissociation (EID) reactions within the FT-ICR cell. Dramatic differences are observed between low-energy CID (which occurs via vibrational excitation) and EID. For example, the aromatic amino acids mainly fragment via competitive losses of NH(3) and (H(2)O+CO) under CID conditions, while side-chain benzyl cations are major fragment ions under EID conditions. EID also appears to be superior in cleaving the S-S and S-C bonds of models of peptides containing an intermolecular disulfide bond. Systematic studies involving fragmentation as a function of electron energy reveal that the fragmentation efficiency for EID occurs at high electron energy (more than 10 eV) compared with the low-electron energy (less than 0.2 eV) typically observed for electron capture dissociation fragmentation. Finally, owing to similarities between the types of fragment ions observed under EID conditions and those previously reported in ultraviolet photodissociation experiments and the electron-ionization mass spectra, we propose that EID results in fragmentation via electronic excitation and vibrational excitation. EID may find applications in analyzing singly charged molecular ions formed by matrix-assisted laser desorption ionization.

Can metal ions be used as gas-phase disulfide bond cleavage reagents? A survey of coinage metal complexes of model peptides containing an intermolecular disulfide bond.

The role that a metal ion can have in promoting disulfide bond cleavage has been assessed by surveying the tandem mass spectra of the following metal complexes of model peptides containing an intermolecular disulfide bond: [M--H+Cu(II)](+); [M--H+Cu(II)(bipy)](+); [M+Ag(I)](+); and [M+Au(I)(PMe(3))](+). In comparison to previously studied protonated peptides, these binary and ternary metal complexes generally yield more abundant S--S and/or C--S bond cleavage. In general, [M--H+Cu(II)](+) ions cleave the adjacent C--S bond more readily, while the [M+Au(I)(PMe(3))](+) ion cleaves the S--S bond more readily. The ternary metal complex [M--H+Cu(II)(bipy)](+), on the other hand, fragments by exclusive loss of the bipyridyl ligand for the larger model peptides studied. Of all coinage metal systems studied, Me(3)PAu(+) is superior in promoting disulfide bond cleavage.

A novel salt bridge mechanism highlights the need for nonmobile proton conditions to promote disulfide bond cleavage in protonated peptides under low-energy collisional activation.

The gas-phase fragmentation mechanisms of small models for peptides containing intermolecular disulfide links have been studied using a combination of tandem mass spectrometry experiments, isotopic labeling, structural labeling, accurate mass measurements of product ions, and theoretical calculations (at the MP2/6-311 + G(2d,p)//B3LYP/3-21G(d) level of theory). Cystine and its C-terminal derivatives were observed to fragment via a range of pathways, including loss of neutral molecules, amide bond cleavage, and S-S and C-S bond cleavages. Various mechanisms were considered to rationalize S-S and C-S bond cleavage processes, including charge directed neighboring group processes and nonmobile proton salt bridge mechanism. Three low-energy fragmentation pathways were identified from theoretical calculations on cystine N-methyl amide: (1) S-S bond cleavage dominated by a neighboring group process involving the C-terminal amide N to form either a protonated cysteine derivative or protonated sulfenyl amide product ion (44.3 kcal mol(-1)); (2) C-S bond cleavage via a salt bridge mechanism, involving abstraction of the alpha-hydrogen by the N-terminal amino group to form a protonated thiocysteine derivative (35.0 kcal mol(-1)); and (3) C-S bond cleavage via a Grob-like fragmentation process in which the nucleophilic N-terminal amino group forms a protonated dithiazolidine (57.9 kcal mol(-1)). Interestingly, C-S bond cleavage by neighboring group processes have high activation barriers (63.1 kcal mol(-1)) and are thus not expected to be accessible during low-energy CID experiments. In comparison to the energetics of simple amide bond cleavage, these S-S and C-S bond cleavage reactions are higher in energy, which helps rationalize why bond cleavage processes involving the disulfide bond are rarely observed for low-energy CID of peptides with mobile proton(s) containing intermolecular disulfide bonds. On the other hand, the absence of a mobile proton appears to "switch on" disulfide bond cleavage reactions, which can be rationalized by the salt bridge mechanism. This potentially has important ramifications in explaining the prevalence of disulfide bond cleavage in singly protonated peptides under MALDI conditions.

Neighbouring group processes in the deamination of protonated phenylalanine derivatives.

The gas-phase fragmentation of protonated phenylalanine and a series of its derivatives (tyrosine, 4-methylphenylalanine, 4-aminophenylalanine, 4-methoxyphenylalanine, 4-tert-butylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 4-bromophenylalanine, 4-iodophenylalanine, 4-cyanophenylalanine, 4-nitrophenylalanine, 3-fluorophenylalanine, and 3,4-dichlorophenylalanine) were examined using a combination of low energy CID in a quadrupole ion trap mass spectrometer as well as DFT calculations and RRKM modelling. In particular, the relationship between the electron-donating ability of the substituent and the competitive losses of H2O + CO and NH3 were explored through the application of the Hammett equation. It was found that electron-donating substituents promote the loss of NH3, while electron-withdrawing substituents suppress the loss of NH3 and favour the H2O + CO loss fragmentation channel instead. These observations are consistent with a neighbouring group pathway operating for the loss of NH3. Molecular orbital calculation (at the B3LYP/6-31+G(d,p) level of theory) were also performed for a range of derivatives to compare the relative transition state energy barriers for three competing mechanisms: (i) the combined loss of H2O + CO, which is triggered by an initial intramolecular proton transfer from the ammonium group to hydroxyl OH, followed by the combined loss of H2O and CO to form an immonium ion; (ii) loss of NH3 via an aryl assisted neighbouring group pathway to yield a phenonium ion; (iii) loss of NH3 via a 1,2-hydride migration process, which results in the formation of a benzyl cation. The relative energy barriers for H2O + CO loss remain nearly constant, while that for both NH3 pathways increase as the substituent moves from electron-donating to electron-withdrawing. The relative transition state energy for loss of NH3 via the aryl assisted neighbouring group pathway is always lower than that of the 1,2-hydride migration process. RRKM modelling of the DFT predicted barrier heights suggest that the rate constants for H2O + CO loss are insensitive to the substituent on the ring, while the NH3 loss channels are greatly affected by the substituent. These theoretical results are consistent with the experimental observation of the relative yields of the competing fragmentation channels. Finally, comparisons with published gas phase and condensed phase studies on related systems are made.

Atomic force microscopy study of the interaction between adsorbed poly(ethylene oxide) layers: effects of surface modification and approach velocity.

The interaction forces between layers of the triblock copolymer Pluronic F108 adsorbed onto hydrophobic radio frequency glow discharge (RFGD) thin film surfaces and hydrophilic silica, in polymer-free 0.15 M NaCl solution, have been measured using the atomic force microscope (AFM) colloid probe technique. Compression of Pluronic F108 layers adsorbed on the hydrophobic RFGD surfaces results in a purely repulsive force due to the steric overlap of the layers, the form of which suggests that the PEO chains adopt a brush conformation. Subsequent fitting of these data to the polymer brush models of Alexander-de Gennes and Milner, Witten, and Cates confirms that the adsorbed Pluronic F108 adsorbs onto hydrophobic surfaces as a polymer brush with a parabolic segment density profile. In comparison, the interaction between Pluronic F108 layers adsorbed on silica exhibits a long ranged shallow attractive force and a weaker steric repulsion. The attractive component is reasonably well described by van der Waals forces, but polymer bridging cannot be ruled out. The weaker steric component of the force suggests that the polymer is less densely packed on the surface and is less extended into solution, existing as polymeric isolated mushrooms. When the surfaces are driven together at high piezo ramp velocities, an additional repulsive force is measured, attributable to hydrodynamic drainage forces between the surfaces. In comparing theoretical predictions of the hydrodynamic force to the experimentally obtained data, agreement could only be obtained if the flow profile of the aqueous solution penetrated significantly into the polymer brush. This finding is in line with the theoretical predictions of Milner and provides further evidence that the segment density profile of the adsorbed polymer brush is parabolic. A velocity dependent additional stepped repulsive force, reminiscent of a solvation oscillatory force, is also observed when the adsorbed layers are compressed under high loads. This additional force is presumably a result of hindered drainage of water due to the presence of a high volume fraction of polymer chains between the surfaces.

A mass spectrometric and molecular orbital study of H2O loss from protonated tryptophan and oxidized tryptophan derivatives.

Protonated N-acetyltryptophan, oxindolylalanine (a mono-oxidized derivative of tryptophan), and N-acetyloxindolylalanine, as well as several di- and tripeptide derivatives containing oxindolylalanine, undergo a range of fragmentation reactions in the gas phase, including the loss of water. In order to elucidate the sites of water loss within these ions, and to determine the mechanisms associated with these processes, we have conducted a series of experiments employing multistage tandem mass spectrometry (MS/MS and MS(3)) in a quadrupole ion trap mass spectrometer, regiospecific structural labeling, and independent solution-phase syntheses of proposed product ion structures, coupled with the use of molecular orbital calculations at the B3LYP/6-31G* level of theory. We demonstrate that the loss of H(2)O from the amide carbonyl group of protonated N-acetyltryptophan O-methyl ester occurs via a "side-chain-backbone" neighboring group reaction to yield a protonated carboline derivative. In contrast, the loss of water from the O-methyl ester of protonated oxindolylalanine results in the formation of a tricyclic structure by "backbone-side-chain" nucleophilic attack from the amino nitrogen to the C2 position of the indole ring. The O-methyl ester of protonated N-acetyloxindolylalanine was found to dissociate via the loss of water from both possible sites, i.e. from the side-chain indolyl oxygen and the backbone amide carbonyl group. An estimate of the relative preference for water loss from each site was obtained from the abundances of product ions formed from MS(3) analysis of regiospecifically labeled derivatives of N-acetyloxindolylalanine, and from the results of molecular orbital calculations. These studies indicate the absence of a characteristic 'signature' ion or neutral loss for peptides containing oxindolylalanine residues under low-energy ion trap CID conditions.

Gas-phase reactions of protonated tryptophan.

The gas phase reactions of protonated tryptophan have been examined in a quadrupole ion trap using a combination of collision induced dissociation, hydrogen-deuterium exchange, regiospecific deuterium labeling and molecular orbital calculations (at the B3LYP/6-31G* level of theory). The loss of ammonia from protonated tryptophan is observed as the primary fragmentation pathway, with concomitant formation of a [M + H - NH(3)](+) ion by nucleophilic attack from the C3 position of the indole side chain. Hydrogen-deuterium exchange and regiospecific deuterium labeling reveals that scrambling of protons in the C2 and C4 positions of the indole ring, via intramolecular proton transfer from the thermodynamically preferred site of protonation at the amino nitrogen, precedes ammonia loss. Molecular orbital calculations have been employed to demonstrate that the activation barriers to intramolecular proton transfer are lower than that for NH(3) loss.