The Structural Biology of Catalase Evolution.

Catalases are essential enzymes for removal of hydrogen peroxide, enabling aerobic and anaerobic metabolism in an oxygenated atmosphere. Monofunctional heme catalases, catalase-peroxidases, and manganese catalases, evolved independently more than two billion years ago, constituting a classic example of convergent evolution. Herein, the diversity of catalase sequences is analyzed through sequence similarity networks, providing the context for sequence distribution of major catalase families, and showing that many divergent catalase families remain to be experimentally studied.

Injury frequency and severity in crayfish communities as indicators of physical habitat quality and water quality within agricultural headwater streams.

Crayfishes (Decapoda) are common inhabitants of agricultural headwater streams in the Midwestern USA that have been impacted by physical habitat degradation and contamination by agricultural pollutants. The frequency and severity of injuries within crayfish communities are indicators of crayfish aggression, which is influenced by physical, chemical, and biotic factors. Previous studies have not evaluated the relationships of the frequency and severity of crayfish injuries with physical habitat quality, water quality, and biotic factors within agricultural headwater streams. Understanding these relationships will assist with determining if crayfish injury variables can serve as an indicator of physical habitat quality or water quality in these small degraded streams. We sampled crayfishes, documented the frequency and type of injuries, and measured instream habitat and water chemistry in 2014 and 2015 within 12 agricultural headwater streams in Indiana, Michigan, and Ohio. We documented five native crayfish species from 1641 adult captures. The most abundant species were Faxonius rusticus, Faxonius immunis, and Faxonius propinquus. Linear mixed effect model analyses indicated that four crayfish injury response variables were positively correlated (p < 0.05) with crayfish density, physical habitat quality, and water velocity diversity and that crayfish injury response variables were more strongly correlated with crayfish density than physical habitat quality or water quality. Our results indicate that response variables describing the severity and frequency of crayfish injuries can be effective indicators of physical habitat quality in agricultural headwater streams.

Cross-class metallo-ß-lactamase inhibition by bisthiazolidines reveals multiple binding modes.

Metallo-ß-lactamases (MBLs) hydrolyze almost all ß-lactam antibiotics and are unaffected by clinically available ß-lactamase inhibitors (ßLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of ßLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both l- and d-BTZ enantiomers are micromolar competitive ßLIs of all MBL classes in vitro, with Kis of 6-15 µM or 36-84 µM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 µM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the l-BTZ enantiomers exhibit 100-fold lower Kis (0.26-0.36 µM) than d-BTZs (26-29 µM). Importantly, cell-based time-kill assays show BTZs restore ß-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate ß-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the l-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. d-BTZ complexes most closely resemble ß-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120-zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding.

Sorption of Lincomycin by Manure-Derived Biochars from Water.

The presence of antibiotics in agroecosystems raises concerns about the proliferation of antibiotic-resistant bacteria and adverse effects to human health. Soil amendment with biochars pyrolized from manures may be a win-win strategy for novel manure management and antibiotics abatement. In this study, lincomycin sorption by manure-derived biochars was examined using batch sorption experiments. Lincomycin sorption was characterized by two-stage kinetics with fast sorption reaching quasi-equilibrium in the first 2 d, followed by slow sorption over 180 d. The fast sorption was primarily attributed to surface adsorption, whereas the long-term slow sorption was controlled by slow diffusion of lincomycin into biochar pore structures. Two-day sorption experiments were performed to explore effects of biochar particle size, solid/water ratio, solution pH, and ionic strength. Lincomycin sorption to biochars was greater at solution pH 6.0 to 7.5 below the dissociation constant of lincomycin (7.6) than at pH 9.9 to 10.4 above its dissociation constant. The enhanced lincomycin sorption at lower pH likely resulted from electrostatic attraction between the positively charged lincomycin and the negatively charged biochar surfaces. This was corroborated by the observation that lincomycin sorption decreased with increasing ionic strength at lower pH (6.7) but remained constant at higher pH (10). The long-term lincomycin sequestration by biochars was largely due to pore diffusion plausibly independent of solution pH and ionic composition. Therefore, manure-derived biochars had lasting lincomycin sequestration capacity, implying that biochar soil amendment could significantly affect the distribution, transport, and bioavailability of lincomycin in agroecosystems.

Structure of the novel monomeric glyoxalase I from Zea mays.

The glyoxalase system is ubiquitous among all forms of life owing to its central role in relieving the cell from the accumulation of methylglyoxal, a toxic metabolic byproduct. In higher plants, this system is upregulated under diverse metabolic stress conditions, such as in the defence response to infection by pathogenic microorganisms. Despite their proven fundamental role in metabolic stresses, plant glyoxalases have been poorly studied. In this work, glyoxalase I from Zea mays has been characterized both biochemically and structurally, thus reporting the first atomic model of a glyoxalase I available from plants. The results indicate that this enzyme comprises a single polypeptide with two structurally similar domains, giving rise to two lateral concavities, one of which harbours a functional nickel(II)-binding active site. The putative function of the remaining cryptic active site remains to be determined.

An optimized mouse thigh infection model for enterococci and its impact on antimicrobial pharmacodynamics.

Negligible in vivo growth of enterococci and high-level dispersion of data have led to inaccurate estimations of antibiotic pharmacodynamics (PD). Here we improved an in vivo model apt for PD studies by optimizing the in vitro culture conditions for enterococci. The PD of vancomycin (VAN), ampicillin-sulbactam (SAM), and piperacillin-tazobactam (TZP) against enterococci were determined in vivo, comparing the following different conditions of inoculum preparation: aerobiosis, aerobiosis plus mucin, and anaerobiosis plus mucin. Drug exposure was expressed as the ratio of the area under the concentration-time curve for the free, unbound fraction of the drug to the MIC (fAUC/MIC) (VAN) or the time in a 24-h period that the drug concentration for the free, unbound fraction exceeded the MIC under steady-state pharmacokinetic conditions (fT(>MIC)) (SAM and TZP) and linked to the change in log10 CFU/thigh. Only anaerobiosis plus mucin enhanced the in vivo growth, yielding significant PD parameters with all antibiotics. In conclusion, robust in vivo growth of enterococci was crucial for better determining the PD of tested antibacterial agents, and this was achieved by optimizing the procedure for preparing the inoculum.

Carbonic anhydrases in industrial applications.

Carbonic anhydrases (CAs) catalyze a fundamental reaction: the reversible hydration and dehydration of carbon dioxide (CO2) and bicarbonate ([Formula: see text]), respectively. Current methods for CO2 capture and sequestration are harsh, expensive, and require prohibitively large energy inputs, effectively negating the purpose of removing CO2 from the atmosphere. Due to CA's activity on CO2 there is increasing interest in using CAs for industrial applications such as carbon sequestration and biofuel production. A lot of work in the last decade has focused on immobilizing CA onto various supports for incorporation into CO2 scrubbing applications or devices. Although the proof of principle has been validated, current CAs being tested do not withstand the harsh industrial conditions. The advent of large-scale genome sequencing projects has resulted in several emerging efforts seeking out novel CAs from a variety of microorganisms, including bacteria, micro-, and macro-algae. CAs are also being investigated for their use in medical applications, such drug delivery systems and artificial lungs. This review also looks at possible downstream uses of captured and sequestered CO2, from using it to enhance oil recovery to incorporating it into useful and financially viable products.

Ni(II) coordination to mixed sites modulates DNA binding of HpNikR via a long-range effect.

Helicobacter pylori NikR (HpNikR) is a nickel-dependent transcription factor that regulates multiple genes in the H. pylori pathogen. There are conflicting data regarding the locations of the Ni(II) sites and the role of Ni(II) coordination in DNA recognition. Herein, we report crystal structures of (i) the metal-binding domain (MBD) of HpNikR (3.08 Å) and (ii) a mutant, H74A (2.04 Å), designed to disrupt native Ni(II) coordination. In the MBD structure, four nickel ions are coordinated to two different types of nickel sites (4-coordinate, square planar, and 5/6-coordinate, square pyramidal/octahedral). In the H74A structure, all four nickel ions are coordinated to 4-coordinate square-planar sites. DNA-binding studies reveal tighter binding for target DNA sequences for holo-HpNikR compared with the affinities of Ni(II) reconstituted apo-HpNikR and H74A for these same DNA targets, supporting a role for Ni(II) coordination to 5/6 sites in DNA recognition. Small-angle X-ray scattering studies of holo-HpNikR and H74A reveal a high degree of conformational flexibility centered at the DNA-binding domains of H74A, which is consistent with disorder observed in the crystal structure of the protein. A model of DNA recognition by HpNikR is proposed in which Ni(II) coordination to specific sites in the MBD have a long-range effect on the flexibility of the DNA-binding domains and, consequently, the DNA recognition properties.

A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity.

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyrosolvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark ß8-α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved ß8-α8 loop region of these enzymes influences xylan substrate specificity but not necessarily ß-1,4-xylanase function.

Metallo-ß-lactamases withstand low Zn(II) conditions by tuning metal-ligand interactions.

A number of multiresistant bacterial pathogens inactivate antibiotics by producing Zn(II)-dependent ß-lactamases. We show that metal uptake leading to an active dinuclear enzyme in the periplasmic space of Gram-negative bacteria is ensured by a cysteine residue, an unusual metal ligand in oxidizing environments. Kinetic, structural and affinity data show that such Zn(II)-cysteine interaction is an adaptive trait that tunes the metal binding affinity, thus enabling antibiotic resistance at restrictive Zn(II) concentrations.

Anthelmintic activity of Artemisia annua L. extracts in vitro and the effect of an aqueous extract and artemisinin in sheep naturally infected with gastrointestinal nematodes.

There is no effective natural alternative control for gastrointestinal nematodes (GIN) of small ruminants, with Haemonchus contortus being the most economically important GIN. Despite frequent reports of multidrug-resistant GIN, there is no new commercial anthelmintic to substitute failing ones. Although trematocidal activity of artemisinin analogs has been reported in sheep, neither artemisinin nor its plant source (Artemisia annua) has been evaluated for anthelmintic activity in ruminants. This study evaluated the anthelmintic activity of A. annua crude extracts in vitro and compared the most effective extract with artemisinin in sheep naturally infected with H. contortus. A. annua leaves extracted with water, aqueous 0.1% sodium bicarbonate, dichloromethane, and ethanol were evaluated in vitro by the egg hatch test (EHT) and with the bicarbonate extract only for the larval development test (LDT) using H. contortus. The A. annua water, sodium bicarbonate (SBE), ethanol, and dichloromethane extracts tested in vitro contained 0.3, 0.6, 4.4, and 9.8% of artemisinin, respectively. The sodium bicarbonate extract resulted in the lowest LC99 in the EHT (1.27 µg/mL) and in a LC99 of 23.8 µg/mL in the LDT. Following in vitro results, the SBE (2 g/kg body weight (BW)) and artemisinin (100 mg/kg BW) were evaluated as a single oral dose in naturally infected Santa Inês sheep. Speciation from stool cultures established that 84-91% of GIN were H. contortus, 8.4-15.6 % were Trichostrongylus sp., and 0.3-0.7% were Oesophagostomum sp. Packed-cell volume and eggs per gram (EPG) of feces were used to test treatment efficacy. The SBE tested in vivo contained no artemisinin, but had a high antioxidant capacity of 2,295 µmol of Trolox equivalents/g. Sheep dosed with artemisinin had maximum feces concentrations 24 h after treatment (126.5 µg/g artemisinin), which sharply decreased at 36 h. By day 15, only levamisole-treated sheep had a significant decrease of 97% in EPG. Artemisinin-treated and SBE-treated sheep had nonsignificant EPG reductions of 28 and 19%, respectively, while sheep in infected/untreated group had an average EPG increase of 95%. Sheep treated with artemisinin and A. annua SBE maintained blood hematocrits throughout the experiment, while untreated/infected controls had a significant reduction in hematocrit. This is the first time oral dose of artemisinin and an aqueous extract of A. annua are evaluated as anthelmintic in sheep. Although oral dose of artemisinin and SBE, at single doses, were ineffective natural anthelmintics, artemisinin analogs with better bioavailability than artemisinin should be tested in vivo, through different routes and in multiple doses. The maintenance of hematocrit provided by artemisinin and A. annua extract and the high antioxidant capacity of the latter suggest that they could be combined with commercial anthelmintics to improve the well-being of infected animals and to evaluate potential synergism.

Neutron diffraction studies towards deciphering the protonation state of catalytic residues in the bacterial KDN9P phosphatase.

The enzyme 2-keto-3-deoxy-9-O-phosphonononic acid phosphatase (KDN9P phosphatase) functions in the pathway for the production of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, a sialic acid that is important for the survival of commensal bacteria in the human intestine. The enzyme is a member of the haloalkanoate dehalogenase superfamily and represents a good model for the active-site protonation state of family members. Crystals of approximate dimensions 1.5 × 1.0 × 1.0 mm were obtained in space group P2(1)2(1)2, with unit-cell parameters a = 83.1, b = 108.9, c = 75.7 Å. A complete neutron data set was collected from a medium-sized H/D-exchanged crystal at BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany in 18 d. Initial refinement to 2.3 Šresolution using only neutron data showed significant density for catalytically important residues.

Chicken manure biochar as liming and nutrient source for acid Appalachian soil.

Acid weathered soils often require lime and fertilizer application to overcome nutrient deficiencies and metal toxicity to increase soil productivity. Slow-pyrolysis chicken manure biochars, produced at 350 and 700°C with and without subsequent steam activation, were evaluated in an incubation study as soil amendments for a representative acid and highly weathered soil from Appalachia. Biochars were mixed at 5, 10, 20, and 40 g kg into a Gilpin soil (fine-loamy, mixed, active, mesic Typic Hapludult) and incubated in a climate-controlled chamber for 8 wk, along with a nonamended control and soil amended with agronomic dolomitic lime (AgLime). At the end of the incubation, soil pH, nutrient availability (by Mehlich-3 and ammonium bicarbonate diethylene triamine pentaacetic acid [AB-DTPA] extractions), and soil leachate composition were evaluated. Biochar effect on soil pH was process- and rate-dependent. Biochar increased soil pH from 4.8 to 6.6 at the high application rate (40 g kg), but was less effective than AgLime. Biochar produced at 350°C without activation had the least effect on soil pH. Biochar increased soil Mehlich-3 extractable micro- and macronutrients. On the basis of unit element applied, increase in pyrolysis temperature and biochar activation decreased availability of K, P, and S compared to nonactivated biochar produced at 350°C. Activated biochars reduced AB-DTPA extractable Al and Cd more than AgLime. Biochar did not increase NO in leachate, but increased dissolved organic carbon, total N and P, PO, SO, and K at high application rate (40 g kg). Risks of elevated levels of dissolved P may limit chicken manure biochar application rate. Applied at low rates, these biochars provide added nutritional value with low adverse impact on leachate composition.

The structure of Synechococcus elongatus enolase reveals key aspects of phosphoenolpyruvate binding.

A structure-function characterization of Synechococcus elongatus enolase (SeEN) is presented, representing the first structural report on a cyanobacterial enolase. X-ray crystal structures of SeEN in its apoenzyme form and in complex with phosphoenolpyruvate are reported at 2.05 and 2.30 Šresolution, respectively. SeEN displays the typical fold of enolases, with a conformationally flexible loop that closes the active site upon substrate binding, assisted by two metal ions that stabilize the negatively charged groups. The enzyme exhibits a catalytic efficiency of 1.2 × 105 M-1 s-1 for the dehydration of 2-phospho-D-glycerate, which is comparable to the kinetic parameters of related enzymes. These results expand the understanding of the biophysical features of these enzymes, broadening the toolbox for metabolic engineering applications.

Gypsum, crop rotation, and cover crop impacts on soil organic carbon and biological dynamics in rainfed transitional no-till corn-soybean systems.

Soil organic carbon (SOC), a core soil quality indicator, is influenced by management practices. The objective of our 2012-2016 study was to elucidate the impact of gypsum, crop rotation, and cover crop on SOC and several of its biological indicators under no-till in Alabama (Shorter), Indiana (Farmland), and Ohio (Hoytville and Piketon) in the USA. A randomized complete block design in factorial arrangement with gypsum (at 0, 1.1, and 2.2 Mg/ha annually), rye (Secale cereal L.) vs no cover crop, and rotation (continuous soybean [Glycine max (L) Merr., SS] vs corn [Zea mays, L.]-soybean, both the CS and SC phases) was conducted. Composite soils were collected (0-15 cm and 15-30 cm) in 2016 to analyze microbial biomass C (SMBC), SOC, total N, active C, cold and hot-water extractable C, C and N pool indices (CPI and NPI), and C management index (CMI). Results varied for main effects of gypsum, crop rotation, and cover crop on SOC pools, total N, and SOC lability within and across the sites. Gypsum at 2.2 Mg/ha increased SMBC within sites and by 41% averaged across sites. Likewise, gypsum increased SMBC:SOC, active C, and hot-water C (as indicators of labile SOC) averaged across sites. CS rotation increased SOC, active C, CPI, and CMI compared to SS, but decreased SMBC and SMBC:SOC within and across sites. CPI had a significant relationship with NPI across all sites (R2 = 0.90). Management sensitive SOC pools that responded to the combined gypsum (2.2 Mg/ha), crop rotation (CS), and cover crop (rye) were SMBC, SMBC:SOC, active C, and CMI via SMBC. These variables can provide an early indication of management-induced changes in SOC storage and its lability. Our results show that when SOC accumulates, its lability has decreased, presumably because the SMBC has processed all readily available C into a less labile form.

Visualizing the superfamily of metallo-ß-lactamases through sequence similarity network neighborhood connectivity analysis.

Protein sequence similarity networks (SSNs) constitute a convenient approach to analyze large polypeptide sequence datasets, and have been successfully applied to study a number of protein families over the past decade. SSN analysis is herein combined with traditional cladistic and phenetic phylogenetic analysis (respectively based on multiple sequence alignments and all-against-all three-dimensional protein structure comparisons) in order to assist the ancestral reconstruction and integrative revision of the superfamily of metallo-ß-lactamases (MBLs). It is shown that only 198 out of 15,292 representative nodes contain at least one experimentally obtained protein structure in the Protein Data Bank or a manually annotated SwissProt entry, that is to say, only 1.3 % of the superfamily has been functionally and/or structurally characterized. Besides, neighborhood connectivity coloring, which measures local network interconnectivity, is introduced for detection of protein families within SSN clusters. This approach provides a clear picture of how many families remain unexplored in the superfamily, while most MBL research is heavily biased towards a few families. Further research is suggested in order to determine the SSN topological properties, which will be instrumental for the improvement of automated sequence annotation methods.

Structure and functional properties of the cold-adapted catalase from Acinetobacter sp. Ver3 native to the Atacama plateau in northern Argentina.

Heme catalases remove hydrogen peroxide by catalyzing its dismutation into water and molecular oxygen, thereby protecting the cell from oxidative damage. The Atacama plateau in northern Argentina, located 4000 m above sea level, is a desert area characterized by extreme UV radiation, high salinity and a large temperature variation between day and night. Here, the heme catalase KatE1 from an Atacama Acinetobacter sp. isolate was cloned, expressed and purified, with the aim of investigating its extremophilic properties. Kinetic and stability assays indicate that KatE1 is maximally active at 50°C in alkaline media, with a nearly unchanged specific activity between 0°C and 40°C in the pH range 5.5-11.0. In addition, its three-dimensional crystallographic structure was solved, revealing minimal structural differences compared with its mesophilic and thermophilic analogues, except for a conserved methionine residue on the distal heme side, which is proposed to comprise a molecular adaptation to oxidative damage.

Evidence of adaptability in metal coordination geometry and active-site loop conformation among B1 metallo-beta-lactamases .

Subclass B1 beta-lactamases are Zn(II)-dependent hydrolases that confer bacterial resistance to most clinically useful beta-lactam antibiotics. The enzyme BcII from Bacillus cereus is a prototypical enzyme that belongs to this group, the first Zn(II)-dependent beta-lactamase to be discovered. Crucial aspects of the BcII catalytic mechanism and metal binding mode have been assessed mostly on the Co(II)-substituted surrogate. Here we report a high-resolution structure of Co(II)-BcII, revealing a metal coordination geometry identical to that of the native zinc enzyme. In addition, a high-resolution structure of the apoenzyme, together with structures with different degrees of metal occupancy and oxidation levels of a conserved Cys ligand, discloses a considerable mobility of two loops containing four metal ligands (namely, regions His116-Arg121 and Gly219-Cys221). This flexibility is expected to assist in the structural rearrangement of the metal sites during catalytic turnover, which, along with the coordination geometry adaptability of Zn(II) ions, grants the interaction with a variety of substrates, a characteristic feature of B1 metallo-beta-lactamases.

Spectroscopic signature of a ubiquitous metal binding site in the metallo-ß-lactamase superfamily.

The metallo-ß-lactamase (MßL) superfamily is a functionally diverse group of metalloproteins sharing a distinctive αß/αß fold and a characteristic metal binding motif. A large number of open reading frames identified in genomic sequencing efforts have been annotated as members of this superfamily through sequence comparisons. However, structural and functional studies performed on purified proteins are normally needed to unequivocally include a newly discovered protein in the MßL superfamily. Here we report the spectroscopic characterization of recombinant YcbL, a gene product annotated as a member of the MßL superfamily whose function in vivo remains unknown. By taking advantage of the structural features characterizing the MßL superfamily metal binding motif, we performed spectroscopic studies on Zn(II)- and Co(II)-substituted YcbL to structurally interrogate the metal binding site. The dinuclear center in Co(II)-YcbL was shown to display characteristic electronic absorption features in the visible region, which were also observed in an engineered MßL aimed at mimicking this metal site. Thus, the spectroscopic features reported herein can be employed as a signature to readily identify and characterize the presence of these ubiquitous metal binding sites.

Analysis of underivatized artemisinin and related sesquiterpene lactones by high-performance liquid chromatography with ultraviolet detection.

INTRODUCTION: Although high-performance liquid chromatography with ultraviolet detection (HPLC-PAD) is widely available, it has not been used for artemisinin (1) analysis because of the lack of UV absorption reported for this lactone. Increased Artemisia annua cultivation for production of 1 requires an affordable and reliable method to analyse 1 and its precursors dihydroartemisinic acid (2) and artemisinic acid (3) simultaneously from underivatized plant extracts. OBJECTIVE: To validate HPLC-PAD for the quantification of underivatized artemisinin from A. annua and artemisinin-based drugs. METHODOLOGY: Dried A. annua leaves were extracted with petroleum ether, dried, reconstituted in acetonitrile, and analysed by HPLC-PAD at 192 nm using an isocratic mobile phase (60:40, acetonitrile:0.1% acetic acid). HPLC-PAD was evaluated through accuracy, precision, recovery and comparison with HPLC with evaporative light scattering detection (HPLC-ELSD). RESULTS: HPLC-PAD proved accurate, precise and reproducible for the direct quantification of 1 and related compounds, and was more sensitive than ELSD for most of the compounds tested. The limit of quantification of 1-3 from plants was 0.048, 0.024 and 0.008 g/100 g dry weight, respectively. Recoveries were over 98%, with good intra- and inter-day repeatability. HPLC-PAD correlated significantly (r(2 )= 0.99, p < 0.001) with HPLC-ELSD for artemisinin analysis. HPLC-PAD was also reliable for the analysis of dihydroartemisinin, artesunate and artelinic acid. CONCLUSION: HPLC with ultraviolet detection was validated for the quantification of underivatized 1, 2, and 3 from crude plant samples, and is readily applicable for the quality control of herbals and artemisinin-related pharmaceutical compounds.