A procedure is described for the simultaneous separation of all the peptides (other than those present in trace amounts) contained in the venom of the common European honey bee (&is mellifera). Three new peptides have been isolated: one is a fragment of the known peptide melittin. Some properties of the peptides are described.

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The sequences are given of two peptides (secapin and melittin F) whose isolation from the venom of the common European honey bee (Apis mellifera) has previously been described [Gauldie et al. (1976) Eur. J. Biochem. 61, 369--3761. Some structural studies on the known peptide 401 (MCD peptide) are described. The positions of the two disulphide bridges in peptide 401 have been determined by (an essentially) novel method.We recently described [l] a procedure for the isolation of the peptide components of the venom of the common European honey bee (Apis mell[fem also known as Apis mellifica). We found three hitherto unknown peptides: the sequence of two of these are given in this paper. We also report some structural studies on the known peptide 401 (MCD peptide). MATERIALS AND METHODS Isolation of PeptidesSecapin, melittin-F and peptide 401 were isolated in homogeneous form as previously described [l]. The amounts of these peptides present in the original venom have been estimated as 1 %, < 1 x, and 2-3 x respectively [l]. Standard ProceduresThese were amino acid analysis [2], separation of peptides by gel filtration, by chromatography and by electrophoresis using thin-layer plates of paper. They will not be described here as experimental details are readily available in the literature. Standard DeterminationA modification of the Edman method [3] was used and is described below. All reagents were carefully purified immediately prior to use.The peptides were (where appropriate) reduced and carboxymethylated. The resulting derivatives (usually in amounts in the range 10-12 mg) were dis- solved in a mixture of propan-1-01 and water (3/4, v/v) containing a buffer system consisting of dimethylallylamine (0.4 M) and enough trifluoroacetic acid to give a reading on the pH meter of 9.5. Phenyl isothiocyanate (30 pl) was then added and reaction allowed to proceed under nitrogen for 30 min at 50 "C. Excess reagent was removed by extraction with benzene and the phenylthiocarbamyl derivatives of the peptides were isolated by lyophilisation at low pressure (0.01 mm-Hg, 1.33 Pa). They were then washed with a mixture of ethylacetate and acetone (l/l, v/v) and again lyophilised. (The inclusion of this wash produces clearer chromatograms.)Cyclisation and cleavage reactions were carried out using trifluoroacetic acid as described by Edman [3]. Residual peptides were precipitated by the addition of ether and then 1,2-dichloroethane. The use of ether increases the recoveries of peptides and enables even small peptides to be recovered in good yields. The recovered peptides were washed with 1,2-dichloroethane and dried before further degradation. The thiazolinone derivatives of the cleaved N-terminal amino acids were recovered by evaporation of solvent in a stream of nitrogen. Treatment with HCl(1 M) for 10 min at 80 "C converted them into the phenylthiohydantoin derivatives. These were extracted with ethyl acetate, dried, dissolved in 1,2-dichloroethane and identified by chromatography on plates of thinlayer silica gel G (Merck AG or E...

The phospholipase A from the venom of the common European honey bee (Apis mellifica) has been completely purified. The final product (13 g from 700 g of crude venom) readily crystallizes and is homogeneous with respect to starch gel electrophoresis at pH 8.0, isoelectric focussing in polyacrylamide gel in the pH range 3–10, and sedimentation and diffusion analysis in the ultracentrifuge. Only one N‐terminal residue, isoleucine, can be detected either by the Edman or dansyl methods. Quantitative N‐terminal analysis and gel filtration on Sephadex G‐100 give values for the molecular weight of about 19000. Ultracentrifugation studies lead to a value of about 40000: in concentrated solution the molecule exists, therefore, as a dimer. The identity of the enzyme as a phospholipase of the A2 type has been confirmed since, with 1‐oleyl‐2‐isolauroyl phosphatidyl ethanolamine as substrate, isolauric acid is liberated in 100% yield whereas no oleic acid is released. A method for the assay of the enzyme based on continuous titrimetric estimation of hexanoic acid liberated from 1,2‐dihexanoyl lecithin has been used to study various aspects of the activity of the enzyme.