Open AccessPhospholipase A from Bee Venom
Author(s) -
Shipolini Rudolf A.,
Callewaert George L.,
Cottrell Richard C.,
Doonan Shawn,
Ver Charles A.,
Banks Barbara E. C.
Publication year - 1971
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1971.tb01414.x
Subject(s) - chromatography , chemistry , isoelectric point , sephadex , lecithin , isoelectric focusing , venom , phospholipase a , biochemistry , sedimentation equilibrium , ultracentrifuge , edman degradation , phospholipase a2 , enzyme , peptide sequence , gene
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 A 2 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.