An evaluation of the chemical content and microbiological contamination of Anatolian bee venom.

Bee venom is a natural substance produced by worker bees. The aim of this research paper is to determine the characteristics of Anatolian bee venom by evaluating its chemical content and microbiological properties. Physical, chemical and microbiological analyses were performed on 25 bee venom samples from different areas of Anatolia, Turkey. Data obtained by 3-replicate studies were evaluated with normality and one-way and two-way ANOVA / Tukey tests. Chemical analyses of the bee venoms revealed average melittin, apamin, and phospholipase A2 contents of 40.57%, 2.12% and 13.67%, respectively. The results suggest that Anatolian bee venom has a high phospholipase A2 content compared to the previous literature. The results for apamin content were similar to those reported in other countries. Melittin content was within the range of standard values. Bee venom samples were also observed to have a high sugar content, associated with pollen and nectar contamination. Total aerobic mesophilic bacteria counts revealed no microbial development in 11 samples of bee venom. Staphylococcus aureus was not detected in any sample. A low microbial load was associated with a high phospholipase A2 content in the bee venom composition, thus contributing to its antimicrobial character. This study presents an examination of Anatolian bee venom in terms of chemical content and microbial quality. The examination of other components in addition to phospholipase A2, melittin and apamin in future studies, together with an analysis of antimicrobial properties will further our understanding of Anatolian bee venom.

Disulfide linkage assignment based on reducing electrochemistry and mass spectrometry using a lead electrode.

The study of disulfide linkage is a crucial part of the quality assessment of biopharmaceutical products because disulfide bonds stabilize the tertiary structure of proteins and maintain protein functions. Therefore, a suitable method is highly required for disulfide linkage assignment when nested disulfide bonds formed with closely spaced cysteine residues. A novel approach for disulfide linkage assignment of disulfide-rich peptides and proteins via electrochemical reduction on a lead electrode with mass spectrometry is presented in this paper. The method features partial electrochemical reduction and alkylation of peptides followed by alkylated peptide sequencing based on tandem mass spectrometry. Lead was chosen for the first time as an electrode material for disulfide bond reduction, because it has the advantages of maintenance free (only infrequent polishing needed), easy operation in DC mode, and reusability. Without any special sample preparation and any chemical reduction agents, disulfide bond in peptides can be cleaved rapidly. The new method was successfully tested with two peptides and one protein containing nested disulfide bonds.

Quantification of melittin and apamin in bee venom lyophilized powder from Apis mellifera by liquid chromatography-diode array detector-tandem mass spectrometry.

A high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) method was developed for simultaneous determination of melittin and apamin in crude bee venom lyophilized powder (CBVLP) as the traditional Chinese medicine possessing specific biological activity. Melittin and apamin were extracted with pure water from CBVLP samples followed by HPLC-DAD-MS/MS analysis. The method was validated to demonstrate its selectivity, linearity, limit of quantification (LOQ), intraday precision, interday precision, accuracy, recovery, matrix effect, and stability. The assay was linear over the concentration ranges of 1-100 and 0.2-25 microg/ml with limit of quantifications (LOQs) of 1.0 and 0.3 microg/ml for melittin and apamin, respectively. The precision results were expressed as coefficients of variation (CVs), ranging from 2.2% to 11.4% for intraday repeatability and from 3.2% to 13.1% for interday intermediary precision. The concentrations of endogenous melittin and apamin in CBVLP samples ranged from 46% to 53% and from 2.2% to 3.7% of dry weight, respectively. This rapid, simple, precise, and sensitive method allowed the simultaneous determination of melittin and apamin to evaluate authenticity and quality of CBVLP samples.

Detection of honeybee venom in envenomed tissues by direct MALDI MSI.

A new analytical approach using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) for the study of honeybee venom is shown. In vitro and in vivo models simulating the bee sting have been developed using live honeybees and, as the envenomation sites, pig ears and rat legs; MALDI MSI has been used to map, over time, the diffusion and distribution of three venom allergens (Api m 1, Api m 4, and Api m 6) and two venom toxins (apamine and mast cell degranulating peptide). In conjunction with other classical biochemical techniques and high resolution mass spectrometry (HRMS), structural data have been obtained that contribute to current understanding of honeybee venom composition. Initial data have also been obtained demonstrating the feasibility of mapping the organism's response to the sting. The opportunity to monitor venom diffusion and the organism's response at the same time might open new pathways for in vivo preclinical studies in designing and testing new venom immunotherapy (VIT).

Toxinology of venoms from the honeybee genus Apis.

The venoms of Apis dorsata, A. cerana, A. florea, and three different populations of A. mellifera were compared for lethal activity toward mice. All venoms exhibited identical activities, a finding consistent with recent evolutionary history within the genus. Young queen honeybees use their venoms only for stinging other queens and possess a venom only half as lethal to mice as worker venom, and by the time queens are 1-2 years of age their venom has become essentially inactive. Phospholipase A2 is the most lethal of the honeybee venom peptides, whereas melittin, which is only slightly less lethal, is the most abundant. Concurrent analyses of melittin, phospholipase, and the combination of the two at their natural 3:1 mixture in bee venom revealed that the lethal activity of the mixture was about the same as native honeybee venom. This value was less than that for either melittin or phospholipase alone and indicates that synergism of the two peptides is not occurring. The results are consistent with independent lethal activities for the venom components, and show that melittin is not only the dominant, but also the main lethal component in honeybee venom.

Determination of the amino acid sequence of apamin by tandem mass spectrometry.

A new strategy has been developed for the determination of the amino acid sequence of apamin, a two disulfide-bond neurotoxic oligopeptide. Relative molecular mass up to about 2000 was determined using electrospray mass spectrometry. This technique shows that three basic amino acid residues are present in apamin. A hydrophobic tryptic digest fragment was sequenced using fast-atom bombardment combined with tandem mass spectrometry. But the full sequence has been directly observed using a tandem mass spectrometer: the daughter-ion mass spectrum of protonated molecules of dithiothreitol-reduced apamin gives the sequence.

Solution conformation of leiurotoxin I (scyllatoxin) by 1H nuclear magnetic resonance. Resonance assignment and secondary structure.

A proton NMR study at 500 MHz of leiurotoxin I in water is presented. Nearly complete sequence-specific assignments of the individual backbone and side-chain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of this toxin is inferred from a combination of short-range nuclear Overhauser enhancements, scalar couplings and proton/deuteron exchange rates. Three disulfide bridges locate the N-terminal part (that is alpha-helical from residue 6 to 16) on one side of a C-terminal two stranded antiparallel beta sheet (from Leu18 to Val29). The latter features a tight turn at Gly23-Asp24.

A micro-radioimmunoassay for apamin.

A radioimmunoassay is described which allows detection of quantities of apamin between 5 and 1200 fmoles (10-2400 pg) in 50 microliter aliquots. The assay requires a few milliliters of apamin antiserum diluted 10,000-20,000 times and 125I apamin at a specific radioactivity of 2000 Ci/mmole. This assay is specific for apamin.

Solid phase synthesis of 13-lysine-apamin, 14-lysine-apamin and the corresponding guanidinated derivatives.

In order to study the importance of arginine residues 13 and 14 in apamin, the bee venom neurotoxin, four analogues, [Lys13]-apamin, [Lys14]-apamin, [Har4, Har13]-apamin and [Har4, har14]-apamin were synthesized and tested with respect to their neurotoxicity. The two lysine-apamins were prepared by the solid phase method on benzhydrylamine resins. Before oxidation to disulphides, the (S-Acm)4-peptides were isolated and characterized. Portions of the purified lysin peptides were converted to homoarginine analogues by guanidination. The four apamin analogues were lethal, but the lethal doses differed significantly. The results demonstrate that the arginine residue at position 14 is more important for the high toxicity than is the one at position 13. The circular dichroism (CD) spectrum of [Lys13]-apamin was identical with that of apamin itself, whereas the spectrum of [Lys14]-apamin showed certain deviations.