Fluorescence study of steroid hormone binding activity of Helix pomatia agglutinin.
Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin, found in the reproductive gland of a Roman snail. The present study has shown that HPA, in addition to its carbohydrate binding capacity possesses a hydrophobic binding activity. This protein binds with high affinity (k(D)=1.9-2.4 microM) steroid hormones: testosterone and progesterone, identified as putative ligands for the animal lectin HPA. Additionally, we have found that this lectin also interacts with adenine (k(D)=5.4+/-0.5 microM) and arylaminonaphthalene sulfonate TNS (k(D)=12+/-0.3 microM). Binding of HPA to hormones and adenine was accompanied by a significant increase of the intrinsic Trp fluorescence (up to 50%), characterizing the conformational changes in the lectin molecule. The hyperbolic shape of the binding curves indicated one high affinity site for the two steroid hormones and adenine, and more than one hydrophobic site for TNS, showed by the sigmoidal curve fit and Hill coefficient of (n(H)=1.5+/-0.2). Hormones and adenine compete for an identical binding site, suggested to occupy the central hydrophobic cavity of the HPA hexamer. Fluorescence resonance energy transfer (FRET) was applied to calculate the intramolecular distance between TNS and Trp chromophores.
Bogoeva
Vanya P
VP
Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, Bl. 21, Sofia 1113, Bulgaria. vanya.bogoeva@gmail.com
Russev
George C
GC
D000225:Adenine / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D031541:Fluorescence Resonance Energy Transfer; D006372:Helix, Snails; D006388:Hemagglutinins / Q000378:metabolism*; D006728:Hormones / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D009282:Naphthalenesulfonates / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013739:Testosterone / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
C038095:Helix lectin; D006388:Hemagglutinins; D006728:Hormones; D037102:Lectins; D008024:Ligands; D009282:Naphthalenesulfonates; D013739:Testosterone; D011374:Progesterone; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D014364:Tryptophan; D000225:Adenine