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Quantitation of biotin groups by a new competitive binding assay with avidin and biotin-4-fluorescein / submitted by Eva Krenn
VerfasserKrenn, Eva
GutachterGruber, Hermann
ErschienenLinz, 2017
Umfang121 Blätter : Illustrationen
HochschulschriftUniversität Linz, Univ., Masterarbeit, 2017
Schlagwörter (DE)Biotin / Avidin / Fluoreszenz Methode / Calmodulin
Schlagwörter (GND)Anorthit / Avidin / Fluoreszenzanalyse / Calmodulin
URNurn:nbn:at:at-ubl:1-13507 Persistent Identifier (URN)
 Das Werk ist gemäß den "Hinweisen für BenützerInnen" verfügbar
Quantitation of biotin groups by a new competitive binding assay with avidin and biotin-4-fluorescein [2.79 mb]
Zusammenfassung (Englisch)

A major aim of this master thesis was to find a method that enables the reliable determination of the concentration of d-biotin and d-biotin derivatives in an unknown sample. The idea of the assay came up when CMBD peptides (i.e., peptides containing a calmodulin-binding domain) were coupled to biotin-PEG-maleimide linkers with different PEG chain lengths. During the purification of the coupling products by HPLC reversed phase chromatography it turned out that detection and identification of the biotinylated peptides in the HPLC chromatogram was very difficult. For that reason a fluorecence assay was developed that enables the determination of the unknown d-biotin concentrations. Therefore the samples with unknown biotin concentrations were first mixed with an always equal amount of avidin and then mixed with an always equal amount of B4F. The assay was applied to free d-biotin, as well as to d-biotin derivatives like biotin DNA with 30 bases or biotin peptides with 17 amino acids or digested BSA fragments. After the application of the assay to different d-biotin derivatives following conculsions could be made: 1. Increasing amounts of d-biotin occupy an increasing amount of the biotin-binding sites of the tetrameric avidin molecules. Thus, less B4F can bind to the binding sites and less B4F molecules get quenched. 2. The increase in fluorescence with increasing amounts of biotin is nonlinear. The quenching of B4F is extenuated in the end, when most of the biotin binding sites are already pre-blocked by free d-biotin molecules. 3. When the binding sites are saturated by biotin, there is an intersection point and then a plateau with approximately constant fluorescence can be observed. 4. The method can also be applied to larger biotin derivatives such as biotin DNA with 30 bases or biotin peptides with 17 amino acid residues. 5. Digestion of BSA (1 h, 6 M HCl, 100 C) gives peptide fragments with <500 Da. 6. From no. 4 and no. 5 it follows that this digestion condition is sufficient to obtain so small fragments for biotin-BSA that four biotin peptides can easily bind per avidin under the assay conditions. 7. Application of the biotin assay to biotin-BSA resulted in 4.4 biotin residues per BSA molecule. 8. For comparison: For the biotinylation, 2.666 mol of biotin-cap-NHS per mole of BSA were offered. This means that 106 %( 15 %) of the offered biotin-cap-NHS molecules were actually coupled. We assume that most likely the BSA contains associated material. Therefore the result is more than 100 %.