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Fluorescein, acrylodan and pyrene conjugates of horse plasma gelsolin : responses to calcium, actin and tropomyosin

University of British Columbia

Gelsolin, a protein involved in the regulation of actin filament length, was isolated from horse plasma and modified with the amine-selective reagent fluorescein-5-isothiocyanate (FITC). The extent of FITC incorporation per gelsolin molecule was 4.8 ± 0.6, based upon 4 labelling trials. FITC-gelsolin analysed by SDS-PAGE revealed a single fluorescent band under UV light prior to staining, and then a single Coomassie Blue stained band. Viscosity studies showed that gelsolin’s F-actin severing activity was not impaired upon reaction with FITC, but incorporation of the fluorophores did increase the the temperature at which the onset of irreversible precipitation occurred. Under physiological ionic conditions, fluorescence studies failed to detect a C2a effect on FITC-gelsolin’s spectroscopic characteristics, but in low ionic strength solutions, C2a increased the fluorescence intensity by approximately 25%. A calcium effect was also evident in bimolecular fluorescence quenching studies with KI, which showed that C2a rendered the fluorescence more susceptible to quenching. Subsequent protein denaturation with guanidine-HC1 resulted in further exposure of the fluorescein chromophores to iodide in solvent. An investigation of reactivity with the thiol selective reagents acrylodan (ACR) and pyrene iodoacetamide (HA) revealed that native gelsolin exposes 1 to 2 cysteine groups for reaction. Under denaturing conditions, the extent of reaction increased to approximately 3 with ACR and 4 with PTA. In comparison, the average number of thiol groups available for reaction with 5,5’-dithiobis(2-nitrobenzoic acid) was approximately 1, and 3 to 4 in native and denaturing conditions, respectively. Actin-gelsolin complexation was revealed by fluorescence titrations and fluorescence polarization measurements. Addition of actin to FITC-labelled gelsolin increased the fluorescence intensity of the sample in a Ca2 specific manner, while binding of actin to ACR-gelsolin resulted in a fluorescence decrease. Fluorescence polarization values increased on addition of actin to both FITC- and ACR-labelled gelsolin solutions only in the presence of Ca2, establishing the calcium requirement for actin binding. Tropomyosin binding to gelsolin was evident from an increase in the fluorescence intensity of FITC-labelled gelsolin, and from retention of labelled gelsolins on a tropomyosin-agarose affinity column. Quantitative assessment of a direct titration of FI’l’C-labelled gelsolin with tropomyosin yielded two independent tropomyosin binding sites on gelsolin, with a dissociation constant of approximately 0.6 IIM. Both of these effects required the presence of micromolar Ca2. Photoactivation of solutions of benzophenone-4-isothiocyanate labelled gelsolin and tropomyosin resulted in the appearance of high molecular weight bands on SDS-PAGE, indicative of intermolecular crosslinking.

Thesis/Dissertation

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2008-12-17

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http://hdl.handle.net/2429/3066

Chemistry

University of British Columbia

UBCV

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