Introduction: Antimicrobial lectins (L) and (glyco)oxidases (O) in biological sources are represented as systems (LS and OS, respectively). The aim was to identify OS and then LS in plants selected for biologically active additives. Reagents and methods: Concanavalin A (ConA, Sigma), pseudopolysaccharides on the base of biotinylated polyacrylamide (Sugar-PAA-b; http://www.lectinity.com/). Concentrates of high molecular mass (> 27 kD) components were plant crude fractions [1]: PG-II from mixture of 7 plants for “PhytoGoR”; IT-I,II,III from leaves of “Ivan-Tea”; PC2-I,II from plant roots and birch fungus for “Phytocomposition No 2”; CV-II from birch fungus for “ChagoVit”. Fractions were separated by isoelectric focusing (IEF) withinрН2-6 in the block of PAA gel in the presence of urea and saccharose [2]. Components were blotted on Immobillon-P membranes. OS on Immobillon was initiated with chemiluminescent substrate BioWest (UVP). LS was treated with Sugar-PAA-b: Man-PАА-b followed by additional staining with Man-6-P-PAA-b and then Gal-PАА-b. Glycoconjugate bound was treated with Streptavidin-Peroxidase and BioWest. Chemiluminescence was registered in the BioChemi System (UVP) in regime of live imagination. Results: 1.ОS within pI 3.2-3.7 (fractions P2-II,CV-II) were identified. 2. Synergistic action of Man-PАА-b and Man-6-P-PAA-b in detection of extended LS was observed. Pseudo(phospho)mannan-binding LS (ConA and P2-II in the region of pI 5-6 were distributed in similar manner (ConA revealed maximal number of forms). Additional staining with pseudogalactan did not extend LS regions. 3. IEF-PААgel and electroblotting resulted in significant visual purification and separation of LS and OS. Conclusions: Approaches developed indicate usefulness of procedures described for detection of L, O, LS and OS in plant fractions.
References. 1.Lakhtin, V.M.; Korsun V.F.; Lakhtin, M.V.; Korsun E.V. Practical Phytotherapy (Moscow). 2004, N 3, 9-16. 2.Lasne, F.; Martin, L.; Crepin, N.; de Ceauriz, J. Anal.Biochem. 2002, 311, 119-126.
