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Identifier 000288936
Title Απομόνωση, χαρακτηρισμός και κρυσταλλική δομή της χαρυβδίνης, μιας νέας πρωτεΐνης, από τον οργανισμό Charybdis maritima. Aνάπτυξη βιοαισθητήρα που βασίζεται σε φωτοσύστημα για την ανίχνευση ζιζανιοκτόνων
Alternative Title Isolation, characterization and crystal structure of charybdin, a new protein from Charybdis maritima. A biosensor based on Photosystem for the detection of herbicides
Author Τουλουπάκης, Ελευθέριος
Thesis advisor Γανωτάκης, Δημήτριος
Abstract Α. In the present work, we describe the purification, characterization and structural determination of charybdin, a novel 29kDa protein from bulbs of Charybdis maritima agg. Charybdin was characterized by biochemical methods and its structure determined by X-ray crystallography. The DNA sequence, and derived amino acid sequence, revealed a significant homology with various ribosome inactivating proteins (RIPs) Although charybdin inhibited the rabbit reticulocyte translation system, the estimated IC50 value of 24nM indicates that it is not such a strong inhibitor of protein synthesis as other RIPs. Single crystals were grown in the cold room from PEG6000 solutions. Diffraction data collected to 1.6 Å resolution led to the protein structure with the Molecular Replacement method. The active site of RIPs, which contains four key amino acid residues, is highly conserved. Although the three of the four key residues are present at the active site of charybdin, the fourth residue, which is an invariant Tyr80 (ricin numbering) among more than 360 RIP sequences known to date, is substituted by Val. Β. A screen-printed electrodes based sensor, with immobilised photosynthetic material was successfully developed and tested for detection of herbicides inhibiting photosynthesis. Upon illumination of the photosynthetic material a signal from a reduced mediator was recorded amperometrically. The added herbicide inhibits the photosynthetic process and decreases the reduction of the mediator. The decrease in the measured current is proportional to the herbicide concentration. The spinach thylakoid membranes successfully immobilised onto the sensor surface using cross-linking with glutaraldehyde and bovine serum albumin, gave reproducible and sensitive measurements. The developed system allowed reliable detection of herbicides with a detection limit ranging from 10 to 300nM, depending on the type of herbicide and with a measured lifetime up to 23 hours. In parallel and compared to the thylakoid membranes, Chlamydomonas reinhardtii mutant cells were immobilised and tested to assure a higher biological stability of the biosensor. Preliminary analysis of river water samples using this sensor was also performed indicating good correlation between the data obtain with the biosensor and the ones obtained after GC-MS analysis.
Language Greek
Issue date 2006-12-06
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Chemistry--Doctoral theses
  Type of Work--Doctoral theses
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