Cascade autohydrolysis of Alzheimer's Aß peptides.

Protein/peptide self-assembly into amyloid structures associates with major neurodegenerative disorders such as Alzheimer's disease (AD). Soluble assemblies (oligomers) of the Aß peptide and their aggregates are perceived as neurotoxic species in AD. While screening for synthetic cleavage agents that could break down such aberrant assemblies through hydrolysis, we observed that the assemblies of Aß oligopeptides, containing the nucleation sequence Aß14-24 (H14QKLVFFAEDV24), could act as cleavage agents by themselves. Autohydrolysis showed a common fragment fingerprint among various mutated Aß14-24 oligopeptides, Aß12-25-Gly and Aß1-28, and full-length Aß1-40/42, under physiologically relevant conditions. Primary endoproteolytic autocleavage at the Gln15-Lys16, Lys16-Leu17 and Phe19-Phe20 positions was followed by subsequent exopeptidase self-processing of the fragments. Control experiments with homologous d-amino acid enantiomers Aß12-25-Gly and Aß16-25-Gly showed the same autocleavage pattern under similar reaction conditions. The autohydrolytic cascade reaction (ACR) was resilient to a broad range of conditions (20-37 °C, 10-150 µM peptide concentration at pH 7.0-7.8). Evidently, assemblies of the primary autocleavage fragments acted as structural/compositional templates (autocatalysts) for self-propagating autohydrolytic processing at the Aß16-21 nucleation site, showing the potential for cross-catalytic seeding of the ACR in larger Aß isoforms (Aß1-28 and Aß1-40/42). This result may shed new light on Aß behaviour in solution and might be useful in the development of intervention strategies to decompose or inhibit neurotoxic Aß assemblies in AD.

Protonation State of an Important Histidine from High Resolution Structures of Lytic Polysaccharide Monooxygenases.

Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the Nε2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

Copper Binding in Sweet Worts Made from Specialty Malts.

Trace levels of copper can impact the flavor stability of beer. The main source of copper is malt, and the wort copper levels are established during mashing and lautering. This study focuses on sweet worts made from experimental roasted and caramel malts. Potentiometric titrations using ion-selective electrodes combined with electron paramagnetic resonance spectroscopy have been used to investigate Cu(II) binding in worts as well as the impact of Cu(II) ions on the wort oxidative stability. High-temperature treatment during malting decreased Cu(II) binding affinities in the worts, with roasted malt worts having lower affinities than caramel malt worts of similar color and pH. Electron paramagnetic resonance spectra indicated dipeptides as the main Cu(II) chelators. A positive correlation between Cu and free amino nitrogen levels in worts is demonstrated. In dark worts with high rates of radical formation, Cu(II) had pronounced antioxidative effects. In contrast, moderate prooxidative effects were observed when adding Cu(II) to pale worts with inherently low rates of oxidation.

Spatial expression of metallothionein, matrix metalloproteinase-1 and Ki-67 in human epidermal wounds treated with zinc and determined by quantitative immunohistochemistry: A randomised double-blind trial.

Reepithelialisation is fundamental to wound healing, but our current understanding largely relies on cellular and animal studies. The aim of the present randomised double-blind three-arm controlled trial was to correlate genuine epidermal wound healing with key proteins and topical zinc treatment in humans. Sixty wounds were produced using deroofed suction blisters in 30 healthy volunteers and randomised to topical zinc sulphate (n = 20), placebo (n = 20), or control (n = 20) treatment for 4 days. All wounds with perilesional skin were processed for automatic immunostaining of paraffin tissue sections with monoclonal antibodies against Ki-67, metallothionein (MT) and matrix metalloproteinase (MMP)-1. Protein expression was quantified by automated digital image analysis. Epidermal Ki-67 and MT labelling indices were increased in keratinocytes in the neoepidermis (∼1.1 mm) and at the wound edge (0.5 mm) compared to normal skin. Increased MMP-1 immunostaining was restricted to the neoepidermis. MT was robustly upregulated in the upper dermis of the wounds. Zinc treatment enhanced MMP-1 expression beneath the neoepidermis via paracrine mechanisms and MT under the neoepidermis and in the nonepithelialised wound bed via direct actions of zinc as indicated by the induction of MT2A mRNA but not MMP-1 mRNA in cultured normal human dermal fibroblasts by zinc sulphate. The present human study demonstrates that quantitative immunohistochemistry can identify proteins involved in reepithelialisation and actions of external compounds. Increased dermal MT expression may contribute to the anti-inflammatory activities of zinc and increased MMP-1 levels to promote keratinocyte migration.

Formation and Structure of Fluorescent Silver Nanoclusters at Interfacial Binding Sites Facilitating Oligomerization of DNA Hairpins.

Fluorescent, DNA-stabilized silver nanoclusters (DNA-AgNCs) are applied in a range of applications within nanoscience and nanotechnology. However, their diverse optical properties, mechanism of formation, and aspects of their composition remain unexplored, making the rational design of nanocluster probes challenging. Herein, a synthetic procedure is described for obtaining a high yield of emissive DNA-AgNCs with a C-loop hairpin DNA sequence, with subsequent purification by size-exclusion chromatography (SEC). Through a combination of optical spectroscopy, gel electrophoresis, inductively coupled plasma mass spectrometry (ICP-MS), and small-angle X-ray scattering (SAXS) in conjunction with the systematic study of various DNA sequences, the low-resolution structure and mechanism of the formation of AgNCs were investigated. Data indicate that fluorescent DNA-AgNCs self-assemble by a head-to-head binding of two DNA hairpins, bridged by a silver nanocluster, resulting in the modelling of a dimeric structure harboring an Ag12 cluster.

Zinc Oxide Inhibits Axillary Colonization by Members of the Genus Corynebacterium and Attenuates Self-perceived Malodour: A Randomized, Double-blind, Placebo-controlled Trial.

Malodour from the axilla is commonly caused by specific microbes, and may be inhibited by zinc oxide. The aim of this study was to determine the effects of zinc oxide on the axillary microbiota, odour and pH in a randomized, double-blind, placebo-controlled trial in 30 healthy volunteers. In each participant 1 axilla was treated with zinc oxide and the other with a placebo for 13 days. The microbiota and pH were analysed before and during treatment. At the final visit, the participants judged their own axillary odour for comparison. With zinc oxide treatment total bacterial growth and, specifically, that of odour-producing Corynebacterium spp. and Staphylococcus hominis, decreased (p < 0.05), despite an increase (p < 0.0005) in skin-surface pH. Compared with the placebo, zinc oxide treatment reduced (p = 0.005) self-perceived malodour. In vitro, Corynebacterium spp. (19 isolated strains) survival was reduced (p < 0.0005) at pH 5.0 compared with pH 6.0; growth inhibition by zinc oxide occurred at ≤ 400 mg/l, and cell death occurred at ≤ 10,000 mg/l for 12 (63%) of the strains. In conclusion, application of zinc oxide reduced malodour and the counts of causative bacteria, but increased the pH of the axilla.

The role of the active site tyrosine in the mechanism of lytic polysaccharide monooxygenase.

Catalytic breakdown of polysaccharides can be achieved more efficiently by means of the enzymes lytic polysaccharide monooxygenases (LPMOs). However, the LPMO mechanism has remained controversial, preventing full exploitation of their potential. One of the controversies has centered around an active site tyrosine, present in most LPMO classes. Recent investigations have for the first time obtained direct (spectroscopic) evidence for the possibility of chemical modification of this tyrosine. However, the spectroscopic features obtained in the different investigations are remarkably different, with absorption maximum at 420 and 490 nm, respectively. In this paper we use density functional theory (DFT) in a QM/MM formulation to reconcile these (apparently) conflicting results. By modeling the spectroscopy as well as the underlying reaction mechanism we can show how formation of two isomers (both involving deprotonation of tyrosine) explains the difference in the observed spectroscopic features. Both isomers have a [TyrO-Cu-OH]+ moiety with the OH in either the cis- or trans-position to a deprotonated tyrosine. Although the cis-[TyrO-Cu-OH]+ moiety is well positioned for oxidation of the substrate, preliminary calculations with the substrate reveal that the reactivity is at best moderate, making a protective role of tyrosine more likely.

Detection and Characterization of a Novel Copper-Dependent Intermediate in a Lytic Polysaccharide Monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose which have large practical application in the process of refining biomass. The catalytic mechanism of LPMOs still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t1/2 =6-8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO-CuI reacts with sub-equimolar amounts of H2 O2 . UV/Vis absorption spectroscopy, electron paramagnetic resonance, resonance Raman and stopped-flow spectroscopy suggest that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). Additionally, activity assays in the presence of sub-equimolar amounts of H2 O2 showed an increase in the LPMO oxidation of phosphoric acid swollen cellulose. Accordingly, this suggests that the long-lived copper-dependent intermediate could be part of the catalytic mechanism for LPMOs. The observed intermediate offers a new perspective into the oxidative reaction mechanism of TaLPMO9A and hence for the biomass oxidation and the reactivity of copper in biological systems.

Copper ion / H2O2 oxidation of Cu/Zn-Superoxide dismutase: Implications for enzymatic activity and antioxidant action.

Copper ion-catalyzed oxidation of yeast SOD1 (ySOD1) was examined to determine early oxidative modifications, including oxidation of a crucial disulfide bond, and the structural and functional repercussions of these events. The study used distinct oxidative conditions: Cu2+/H2O2, Cu2+/H2O2/AscH- and Cu2+/H2O2/glucose. Capillary electrophoresis experiments and quantification of protein carbonyls indicate that ySOD1 is highly susceptible to oxidative modification and that changes can be detected within 0.1 min of the initiation of the reaction. Oxidation-induced structural perturbations, characterized by circular dichroism, revealed the formation of partially-unfolded ySOD1 species in a dose-dependent manner. Consistent with these structural changes, pyrogallol assay indicates a partial loss of enzymatic activity. ESI-MS analyses showed seven distinct oxidized ySOD1 species under mild oxidation within 0.1 min. LC/MS analysis after proteolytic digestion demonstrated that the copper-coordinating active site histidine residues, His47 and His49, were converted into 2-oxo-histidine. Furthermore, the Cu and Zn bridging residue, His64 is converted into aspartate/asparagine. Importantly, the disulfide-bond Cys58-Cys147 which is critical for the structural and functional integrity of ySOD1 was detected as being oxidized at Cys147. We propose, based on LC/MS analyses, that disulfide-bond oxidation occurs without disulfide bond cleavage. Modifications were also detected at Met85 and five surface-exposed Lys residues. Based on these data we propose that the Cys58-Cys147 bond may act as a sacrificial target for oxidants and protect ySOD1 from oxidative inactivation arising from exposure to Cu2+/H2O2 and auto-inactivation during extended enzymatic turnover.

A comparative structural analysis of the surface properties of asco-laccases.

Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1_3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function-in particular towards large substrates.

Early events in copper-ion catalyzed oxidation of α-synuclein.

Previous studies on metal-ion catalyzed oxidation of α-synuclein oxidation have mostly used conditions that result in extensive modification precluding an understanding of the early events in this process. In this study, we have examined time-dependent oxidative events related to α-synuclein modification using six different molar ratios of Cu2+/H2O2/protein and Cu2+/H2O2/ascorbate/protein resulting in mild to moderate extents of oxidation. For a Cu2+/H2O2/protein molar ratio of 2.3:7.8:1 only low levels of carbonyls were detected (0.078 carbonyls per protein), whereas a molar ratio of 4.7:15.6:1 gave 0.22 carbonyls per α-synuclein within 15 min. With the latter conditions, rapid conversion of 3 out of 4 methionines (Met) to methionine sulfoxide, and 2 out of 4 tyrosines (Tyr) were converted to products including inter- and intra-molecular dityrosine cross-links and protein oligomers, as determined by SDS-PAGE and Western blot analysis. Limited histidine (His) modification was observed. The rapid formation of dityrosine cross-links was confirmed by fluorescence and mass-spectrometry. These data indicate that Met and Tyr oxidation are early events in Cu2+/H2O2-mediated damage, with carbonyl formation being a minor process. With the Cu2+/H2O2/ascorbate system, rapid protein carbonyl formation was detected with the first 5 min, but after this time point, little additional carbonyl formation was detected. With this system, lower levels of Met and Tyr oxidation were detected (2 Met and 1 Tyr modified with a Cu2+/H2O2/ascorbate/protein ratio of 2.3:7.8:7.8:1), but greater His oxidation. Only low levels of intra- dityrosine cross-links and no inter- dityrosine oligomers were detected under these conditions, suggesting that ascorbate limits Cu2+/H2O2-induced α-synuclein modification.

The Pathogenic A2V Mutant Exhibits Distinct Aggregation Kinetics, Metal Site Structure, and Metal Exchange of the Cu2+ -Aß Complex.

A prominent current hypothesis is that impaired metal ion homeostasis may contribute to Alzheimer's disease (AD). We elucidate the interaction of Cu2+ with wild-type (WT) Aß1-40 and the genetic variants A2T and A2V which display increasing pathogenicity as A2T

Simulating Substrate Recognition and Oxidation in Laccases: From Description to Design.

To meet the very specific requirements demanded by industry, proteins must be appropriately tailored. Engineering laccases, to improve the oxidation of small molecules, with applications in multiple fields, is, however, a difficult task. Most efforts have concentrated on increasing the redox potential of the enzyme, but in recent work, we have pursued an alternate strategy to engineering these biocatalysts. In particular, we have found that redesigning substrate binding at the T1 pocket, guided by in silico methodologies, to be a more consistent option. In this work, we evaluate the robustness of our computational approach to estimate activity, emphasizing the importance of the binding event in laccase reactivity. Strengths and weaknesses of the protocol are discussed along with its potential for scoring large numbers of protein sequences and thus its significance in protein engineering.

Controlled lid-opening in Thermomyces lanuginosus lipase- An engineered switch for studying lipase function.

Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions.

Lipases That Activate at High Solvent Polarities.

Thermomyces lanuginosus lipase (TlL) and related lipases become activated in low-polarity environments that exist at the water-lipid interface where a structural change of the "lid" region occurs. In this work, we have investigated the activation of TlL (Lipase_W89) and certain lid mutants, containing either a single positive charge mutation, E87K (Lipase_K87_W89), within the lid region or a lid residue composition of both lipase and esterase character (Hybrid_W89) as a function of solvent polarity. Activation differences between the variants and TlL were studied by a combination of biophysical and theoretical methods. To investigate the structural changes taking place in the lid region upon lipase activation, we used a fluorescence-based method measuring the efficiency of Trp89 in the lid to quench the fluorescence of a bimane molecule attached in front (C255) and behind (C61) the lid. These structural changes were compared to the enzymatic activity of each variant at the water-substrate interface and to theoretical calculations of the energies associated with lid opening as a function of the dielectric constant (ε) of the environment. Our results show that the lid in Lipase_K87_W89 undergoes a pronounced structural transition toward an open conformation around ε = 50, whereas only small changes are detected for Lipase_W89 ascribed to the stabilizing effect of the positive charge mutation on the open lid conformation. Interestingly, Hybrid_W89, with the same charge as Lipase_W89, shows a stabilization of the open lid even more pronounced at high solvent polarities than that of Lipase_K87_W89, allowing activation at ε < 80. This is further indicated by measurement of the lipase activity for each variant showing that Hybrid_W89 is more quickly activated at the water-lipid interface of a true, natural substrate. Combined, we show that a correlation exists between structural changes and enzymatic activities detected on one hand and theoretical calculations on lid opening energies on the other. These results highlight the key role that the lid plays in determining the polarity-dependent activation of lipases.

The Enzymatic Activity of Lipases Correlates with Polarity-Induced Conformational Changes: A Trp-Induced Quenching Fluorescence Study.

Triacylglycerol hydrolases (EC 3.1.1.3) are thought to become activated when they encounter the water-lipid interface causing a "lid" region to move and expose the catalytic site. Here, we tested this idea by looking for lid movements in Thermomyces lanuginosus lipase (TL lipase), and in variants with a mutated lid region of esterase (Esterase) and esterase/lipase (Hybrid) character. To measure lid movements, we employed the tryptophan-induced quenching (TrIQ) fluorescence method to measure how effectively a Trp residue on the lid of these mutants (at position 87 or 89) could quench a fluorescent probe (bimane) placed at nearby site 255 on the protein. To test if lid movement is induced when the enzyme detects a lower-polarity environment (such as at the water-lipid interface), we performed these studies in solvents with different dielectric constants (ε). The results show that lid movement is highly dependent on the particular lid residue composition and solvent polarity. The data suggest that in aqueous solution (ε = 80), the Esterase lid is in an "open" conformation, whereas for the TL lipase and Hybrid, the lid remains "closed". At lower solvent polarities (ε < 46), the lid region for all of the mutants is more "open". Interestingly, these behaviors mirror the structural changes thought to take place upon activation of the enzyme at the water-lipid interface. Together, these results support the idea that lipases are more active in low-polarity solvents because the lid adopts an "open" conformation and indicate that relatively small conformational changes in the lid region play a key role in the activation mechanism of these enzymes.

Altering the activation mechanism in Thermomyces lanuginosus lipase.

It is shown by rational site-directed mutagenesis of the lid region in Thermomyces lanuginosus lipase that it is possible to generate lipase variants with attractive features, e.g., high lipase activity, fast activation at the lipid interface, ability to act on water-soluble substrates, and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region from Aspergillus terreus lipase (AtL). To investigate an altered activation mechanism for each variant compared to that of TlL, a combination of activity- and spectroscopic-based measurements were applied. The engineered variant with a lid from AtL displayed interfacial activation comparable to that of TlL, whereas variants with FAEA lid character showed interfacial activation independence with pronounced activity toward pNP-acetate and pNP-butyrate below the critical micelle concentration. For variants with lipase and esterase character, lipase activity measurements further indicated a faster activation at the lipid interface. Relative to their activity toward pNP-ester substrates in calcium-rich buffer, all lid variants retained between 15 and 100% activity in buffer containing 5 mM EDTA whereas TlL activity was reduced to less than 2%, demonstrating the lid's central role in governing calcium dependency. For FAEA-like lid variants, accessible hydrophobic surface area measurements showed an approximate 10-fold increase in the level of binding of extrinsic fluorophores to the protein surface relative to that of TlL accompanied by a blue shift in emission indicative of an open lid in aqueous solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities.

Light scattering coupled with reversed phase chromatography to study protein self-association under separating conditions.

An on-line method, coupling reversed phase chromatography with static light scattering, was developed to determine the association state of freshly eluted proteins. Under downstream process conditions, human insulin desB30 and human insulin AspB28 were tested at concentrations up to 8.5mg/mL. The refractive index increment (dn/dc) for insulin was found to depend strongly on the solvent used. A refractive index increment of 0.184±0.003mL/g was found in an aqueous buffer, pH 7.4, whereas the value was 0.155±0.003mL/g in 30%, w/w ethanol. The methodology combines on-line SLS and UV measurements with the pre-determined refractive index increment values. The developed on-line method was verified by standard off-line measurements establishing the association state at concentrations between 0.2 and 6.0mg/mL. The equipment was calibrated utilizing insulin under conditions reported to ensure either monomer or hexamer forms. The self-association of human insulin desB30 was found to be strongly suppressed in 30%, w/w ethanol at pH 7.4 in which the monomer predominates. When stabilized by zinc ions in 30%, w/w ethanol at pH 7.4, an average association number of 3.7 was found. These data demonstrate the effect of ethanol to lower strongly the energy advantage by protein self-association. Potassium chloride and/or calcium chloride in the eluents were found to be of no consequence to the association state.

Application of 204mPb perturbed angular correlation of γ-rays spectroscopy in coordination chemistry.

(204m)Pb perturbed angular correlation of γ-rays (PAC) spectroscopy has been applied successfully for the first time to detect the nuclear quadrupole interaction in a lead(II) coordination compound in a molecular crystal [tetraphenylarsonium lead(II) isomaleonitriledithiolate ([AsPh(4)](4)[Pb(2)(i-mnt)(4)])]. The recorded parameters from a powder crystalline sample are ν(Q) = 0.178(1) GHz and η = 0.970(7). The electric field gradient (EFG) was determined at the PW91/QZ4P level including relativistic effects using the two-component zeroth-order regular approximation method for both the [Pb(i-mnt)(2)](2-) monomer and the [Pb(2)(i-mnt)(4)](4-) dimer. Only the EFG for the latter compares favorably with the experimental data, indicating that the picture of this complex as a prototypical hemidirected coordination geometry with a stereochemically active lone pair on lead(II) is inadequate. Advantages and limitations of (204m)Pb PAC spectroscopy as a novel technique to elucidate the electronic and molecular structures of lead-containing complexes and biomolecules are presented.