Poison, plants and Palaeolithic hunters. An analytical method to investigate the presence of plant poison on archaeological artefacts

In this paper we present the development of a method for the detection of toxic substances on ancient arrow points. The aim is to go back in time until the Palaeolithic period in order to determine if poisonous substances were used to enhance the hunting weapons. The ethnographic documentation demonstrates that hunters of every latitude poisoned their weapons with toxic substances derived from plants and occasionally from animals. This highlights that often the weapons would be rather ineffective if the tips were not poisoned. The fact that toxic substances were available and the benefits arising from their application on throwing weapons, suggests that this practice could be widespread also among prehistoric hunters. The project reviewed the research of the toxic molecules starting from current information on modern plants and working backwards through the ages with the study of ethnographic and historical weapons. This knowledge was then applied to the archaeological material collected from International museum collections. Results have shown that using this method it is possible to detect traces of toxic molecules with mass spectrometry (MS) and hyphenated chromatographic techniques even on samples older than one hundred years, which we consider a positive incentive to continue studying plant poisons on ancient hunting tools

Simultaneous Determination of Binding Constants for Multiple Carbohydrate Hosts in Complex Mixtures

We describe a simple method for the simultaneous determination of association constants for a guest binding to seven different hosts in a mixture of more than 20 different oligosaccharides. If the binding parameters are known for one component in the mixture, a single NMR titration suffices to determine binding constants for all other detectable and resolvable hosts. With the use of high-resolution ¹H–¹³C HSQC experiments, complexes of amphiphiles with more than 10 different maltooligosaccharides can be resolved. Hereby, the binding capabilities of a set of structurally related hosts can be quantitatively studied to systematically explore noncovalent interactions without the need to isolate each host

Fast and Accurate Quantitation of Glucans in Complex Mixtures by Optimized Heteronuclear NMR Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is a widely used technique for mixture analysis, but it has shortcomings in resolving carbohydrate mixtures due to the narrow chemical shift range of glycans in general and fragments of homopolymers in particular. Here, we suggest a protocol toward fast spectroscopic glycan mixture analysis. We show that a plethora of oligosaccharides comprising only α-glucopyranosyl residues can be resolved into distinct quantifiable signals with NMR experiments that are substantially faster than chromatographic runs. Conceptually, the approach fully exploits the narrow line widths of glycans (ν_1/2 < 3 Hz) in the ¹³C spectral dimension while disregarding superfluous spectral information in compound identification and quantitation. The acetal (H₁C₁) groups suffice to spectroscopically resolve ∼20 different starch fragments in optimized ¹H–¹³C NMR with a narrow ¹³C spectral width (3 ppm) that allows sampling the indirect ¹³C dimension at high resolution within 15 min. Rapid quantitations by high-resolution NMR data are achieved for glycans at concentrations as low as 10 μg/mL. For validation, comparisons were made with quantitations obtained by more time-consuming chromatographic methods and yielded coefficients of determination (<i>R</i>²) above 0.99

Probing Interactions between β‑Glucan and Bile Salts at Atomic Detail by ¹H–¹³C NMR Assays

Polysaccharides are prospective hosts for the delivery and sequestration of bioactive guest molecules. Polysaccharides of dietary fiber, specifically cereal (1 → 3)(1 → 4)-β-glucans, play a role in lowering the blood plasma cholesterol level in humans. Direct host–guest interactions between β-glucans and conjugated bile salts are among the possible molecular mechanisms explaining the hypocholesterolemic effects of β-glucans. The present study shows that ¹H–¹³C NMR assays on a time scale of minutes detect minute signal changes in both bile salts and β-glucans, thus indicating dynamic interactions between bile salts and β-glucans. Experiments are consistent with stronger interactions at pH 5.3 than at pH 6.5 in this in vitro assay. The changes in bile salt and β-glucan signals suggest a stabilization of bile salt micelles and concomitant conformational changes in β-glucans

Functional and structural characterization of plastidic starch phosphorylase during barley endosperm development

<div><p>The production of starch is essential for human nutrition and represents a major metabolic flux in the biosphere. The biosynthesis of starch in storage organs like barley endosperm operates via two main pathways using different substrates: starch synthases use ADP-glucose to produce amylose and amylopectin, the two major components of starch, whereas starch phosphorylase (Pho1) uses glucose-1-phosphate (G1P), a precursor for ADP-glucose production, to produce α-1,4 glucans. The significance of the Pho1 pathway in starch biosynthesis has remained unclear. To elucidate the importance of barley Pho1 (<i>Hv</i>Pho1) for starch biosynthesis in barley endosperm, we analyzed <i>Hv</i>Pho1 protein production and enzyme activity levels throughout barley endosperm development and characterized structure-function relationships of <i>Hv</i>Pho1. The molecular mechanisms underlying the initiation of starch granule biosynthesis, that is, the enzymes and substrates involved in the initial transition from simple sugars to polysaccharides, remain unclear. We found that <i>Hv</i>Pho1 is present as an active protein at the onset of barley endosperm development. Notably, purified recombinant protein can catalyze the <i>de novo</i> production of α-1,4-glucans using <i>Hv</i>Pho1 from G1P as the sole substrate. The structural properties of <i>Hv</i>Pho1 provide insights into the low affinity of <i>Hv</i>Pho1 for large polysaccharides like starch or amylopectin. Our results suggest that <i>Hv</i>Pho1 may play a role during the initiation of starch biosynthesis in barley.</p></div

Abundance levels and enzymatic activity of <i>Hv</i>Pho1 during barley endosperm development.

<p>(A) <i>Hv</i>Pho1 protein abundance from barley endosperm extracts analyzed via immunoblot at 2 d intervals. Only one band is visible just above 100 kDa in accordance with an expected mass of 105 kDa. (B) Relative quantification of the data from panel A (blue line) and activity from panel C (H₂O control; red line). (C) Starch phosphorylase activity probed in 2 day intervals as for panel A but with native gels and Lugol coloring of activity products. Strong synthetic activity appears as a dark stained band and is marked with a black arrow. White bands and smears represent amylolytic activities. All gels include a recombinant <i>Hv</i>Pho1 control as the right-most band. The different redox treatments are indicated next to each gel. (D) Immunoblot (top) and native gel (bottom) analysis of <i>Hv</i>Pho1 protein abundance on buffer soluble (S) protein and buffer insoluble (P) protein fractions of barley endosperm between 0 and 8 DAF. Numbers indicate the DAF. Arrows mark the position of the two relevant bands in the immunoblot and the position of the (single) activity band in the zymogram.</p

Structural details of the active site of <i>Hv</i>Pho1 and acceptor recognition.

<p>(A) Mode of binding of maltotetraose in the active site of <i>Hv</i>Pho1 (which is depicted as a semi-transparent ribbon). Maltotetraose is shown as ball and stick with yellow carbons and can also be seen in the same color in panel C. A modeled maltose is in ball and stick with orange carbons. Contacting amino acids are shown as stick models with green carbons, while those involved in stacking interactions with the glucose units are depicted with gray carbons. The pyridoxal phosphate is also depicted, with pink carbons, in the lower left corner. (B) Movement of a loop of <i>Hv</i>Pho1 in response to maltotetraose binding. Residues 422–427 of the <i>Hv</i>Pho1 complex with maltotetraose are highlighted with all atom sticks (orange carbons) and the maltotetraose is shown as sticks with cyan carbons. The thicker ribbons represent the α-carbon trace of <i>Hv</i>Pho1 bound to maltotetraose (orange), <i>Hv</i>Pho1 in the native structure (green), <i>Hv</i>Pho1 in complex with acarbose (gray), <i>Ec</i>MalP in complex with maltopentaose (yellow) and rabbit muscle glycogen phosphorylase (white). (C) Superposition of maltopentaose in the binding site of MalP (white, from PDB_code 1e4o), maltotetraose in <i>Hv</i>Pho1 and acarbose in <i>Hv</i>Pho1; plus PLP groups and selected details from the native <i>Hv</i>Pho1 structure. For clarity, only the mentioned groups, the pyridoxal-5’-phosphates (PLP) and, in the case of all three structures of <i>Hv</i>Pho1 reported here, Tyr900 and Tyr905 are depicted. Details from the maltotetraose complex are shown with yellow carbons, details from the acarbose complex with cyan carbons and details from the native structure with pink carbons. The four glucose units in the maltotetraose complex overlap well with the MalP structure for sub-sites +1 to +4.</p

Model describing the effect of the L78 insertion on polysaccharide binding to <i>Hv</i>Pho1.

<p>(A) <i>Hv</i>Pho1 forms a homodimer in solution with an enlarged molecular size. The formation of the dimer is brought about by the crystallographic dimer interface. The flexible nature of the L78 insertion could block access of larger glucans to the protein´s surface. (B) The specific degradation products of <i>Hv</i>Pho1 are the F50s which probably lack L78. Our crystal structure does not contain the L78 insertion and might therefore represent the F50s rather than the full-length enzyme. The F50s provide better access to larger polysaccharides like starch or amylopectin. (C) <i>Hv</i>Pho1ΔL78 lacks the L78 insertion but it also lacks a break in the protein chain. Affinity of larger polysaccharides is similar to full-length <i>Hv</i>Pho1 as the main protein backbone is closed and restricts access to this area.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Crystal structure and modes of polysaccharide binding.

<p>(A) Thin Layer Chromatography (TLC) of a crystallization drop of <i>Hv</i>Pho1 containing 10 mM maltoheptaose. A ladder with 1 mM sugar markers is included and two dilutions from the crystallization drop were applied next to it. 1 μl of solution was loaded in each lane. (B) Overall structure of the <i>Hv</i>Pho1 dimer in the crystals. One monomer is shown as a semitransparent gray surface with the <i>α-</i>carbon trace as a stick model. The second monomer is shown as a cartoon model. Colors are in rainbow from dark blue in the N-terminus through light blue, green, yellow and orange to red at the C-terminus. The protein co-factor pyridoxal phosphate group is show as spheres (pink carbons) and lies at the interface between both, the N-terminal and C-terminal subdomains. The missing loops are indicated with dashed green lines. (C) Structural overlay of the native <i>Hv</i>Pho1 structure (green) and rabbit glycogen phosphorylase B (PDB-code 1P2B). 1P2B has maltoheptaose (shown as ball and sticks) bound in the glycogen storage site of glycogen phosphorylase B, although only maltopentaose has been explicitly modelled. 2 different angles are presented. Parts of <i>Hv</i>Pho1 corresponding to the L78 insert are colored pink with the last modeled residues indicated by sequence numbers. (D) Recombinant <i>Hv</i>Pho1, when stored at 15°C, degrades over time into specific degradation products called F50 and F50s. Left: initial degradation products after 1 week. Middle: a stable F50 band, indicated with a square, was apparent after 4 weeks incubation. It was excised from SDS-PAGE, gel eluted, trypsin digested and the resulting proteolytic fragments analyzed by MALDI-TOF. The sequence to the right shows full-length <i>Hv</i>Pho1. Labelled in red are MALDI-TOF recorded trypsin fragments (they do not cover the L78 insertion). (E) Superposition of <i>Hv</i>Pho1 (green carbon backbone) on the maltotriose binding site of <i>At</i>PHS2 (gray carbon backbone). The maltotriose from the <i>At</i>PHS2 crystal, not present in the <i>Hv</i>Pho1 structure, is shown with cyan carbons. For clarity, only side chains are shown except for W405/W363. Hydrogen bonds to the maltotriose in <i>At</i>PHS2 are highlighted with yellow dashed lines and the corresponding residues are labelled in the figure, first with the Pho1 residue numbering, then with the <i>At</i>PHS2 numbering.</p