Roberto Arreguín

PURIFICATION AND CHARACTERIZATION OF MULTIPLE FORMS OF ASCLEPAIN G FROM ASCLEPIAS GLAUCESCENS H.B.K.

Abstract Ten asclepain forms were found to be present in the latex of milkweed (Asclepias glaucescens H.B.K.) plants. Four of them were purified by high performance liquid chromatography on a cation exchange column, and characterized. Asclepains Ag3, Ag6, Ag7 and Ag8 were isolated as homogeneous proteins of very similar molecular weights (approx. 23 000) with isoelectric points greater than 9.0. These forms possess nearly identical secondary structures, as judged from their circular dichroism spectra. Interestingly, one of these forms (Ag3) markedly differed from the other three forms with respect to its specificity toward ester and amide substrates.

A Conus regularis Conotoxin with a Novel Eight-Cysteine Framework Inhibits CaV2.2 Channels and Displays an Anti-Nociceptive Activity

A novel peptide, RsXXIVA, was isolated from the venom duct of Conus regularis, a worm-hunting species collected in the Sea of Cortez, México. Its primary structure was determined by mass spectrometry and confirmed by automated Edman degradation. This conotoxin contains 40 amino acids and exhibits a novel arrangement of eight cysteine residues (C-C-C-C-CC-CC). Surprisingly, two loops of the novel peptide are highly identical to the amino acids sequence of ω-MVIIA. The total length and disulfide pairing of both peptides are quite different, although the two most important residues for the described function of ω-MVIIA (Lys2 and Tyr13) are also present in the peptide reported here. Electrophysiological analysis using superior cervical ganglion (SCG) neurons indicates that RsXXIVA inhibits CaV2.2 channel current in a dose-dependent manner with an EC50 of 2.8 μM, whose effect is partially reversed after washing. Furthermore, RsXXIVA was tested in hot-plate assays to measure the potential anti-nociceptive effect to an acute thermal stimulus, showing an analgesic effect in acute thermal pain at 30 and 45 min post-injection. Also, the toxin shows an anti-nociceptive effect in a formalin chronic pain test. However, the low affinity for CaV2.2 suggests that the primary target of the peptide could be different from that of ω-MVIIA.

Preliminary assessment of protein associated with airborne particles in Mexico City

The protein associated with airborne particles was measured during 1991 as an indicator of airborne biological material in different outdoor urban environments. Fifty air samples were collected simultaneously at three sampling sites, located in the north, south and downtown Mexico City, using a PM10 high-volume sampler (particles<10 μm). The air filters were weighed and protein extracted using a phosphate buffer. Protein concentrations were determined by Lowry assay. The extracts were also analysed by SDS electrophoresis and IEF using a Phastsystem. High concentrations of airborne particles were recorded at the sampling sites with a geometric mean of 70.2 μg/m3 in the south (residential area), 95.5 μg/m3 in the center (urban-commercial area), and with the highest value of 108.9 μg/m3 in the north (urban-industrial area). No statistically significant difference (P>0.05) was observed among the protein concentrations from the sampling sites and the concentrations ranged from non-detectable to 2.54 μg/m3. However, the protein concentrations presented significant difference (P<0.05) with respect to rainy and dry seasons. The Spearman correlation coefficient between protein concentration and airborne particles concentration was statistically significant (r=0.50). The molecular weights (MW) and isoelectric points (pI) for the proteins present in some of the extracts were determined. The values ranged from approximately 8000 to 106 000 Da and the pI values from nearly 4.0 to 5.85. This is important because the major allergens from inhalants are mostly acidic proteins with molecular weights in the range of 20 000–40 000 Da.

Isolation and characterization of a protease from the marine sponge Spheciospongia vesparia.

A protein that showed activity against proteic (casein and hide powder azure) and synthetic (BAEE and HLPA) substrates was isolated from the marine sponge Spheciospongia vesparia. The protease was purified from an aqueous extract by ammonium sulfate precipitation, gel filtration, hydrophobic and HPLC‐anion exchange chromatographies. The purified protease showed a single band in SDS‐PAGE minigels and had a molecular weight of 29,600, but when submitted to isoelectric focusing it showed 2 bands with isoelectric points of 4.56 and 4.43. Its catalytic action was inhibited by EDTA and 1,10‐phenanthroline, so it seemed to be a metalloprotease.

PhcrTx2, a New Crab-Paralyzing Peptide Toxin from the Sea Anemone Phymanthus crucifer

Sea anemones produce proteinaceous toxins for predation and defense, including peptide toxins that act on a large variety of ion channels of pharmacological and biomedical interest. Phymanthus crucifer is commonly found in the Caribbean Sea; however, the chemical structure and biological activity of its toxins remain unknown, with the exception of PhcrTx1, an acid-sensing ion channel (ASIC) inhibitor. Therefore, in the present work, we focused on the isolation and characterization of new P. crucifer toxins by chromatographic fractionation, followed by a toxicity screening on crabs, an evaluation of ion channels, and sequence analysis. Five groups of toxic chromatographic fractions were found, and a new paralyzing toxin was purified and named PhcrTx2. The toxin inhibited glutamate-gated currents in snail neurons (maximum inhibition of 35%, IC50 4.7 µM), and displayed little or no influence on voltage-sensitive sodium/potassium channels in snail and rat dorsal root ganglion (DRG) neurons, nor on a variety of cloned voltage-gated ion channels. The toxin sequence was fully elucidated by Edman degradation. PhcrTx2 is a new β-defensin-fold peptide that shares a sequence similarity to type 3 potassium channels toxins. However, its low activity on the evaluated ion channels suggests that its molecular target remains unknown. PhcrTx2 is the first known paralyzing toxin in the family Phymanthidae.