Žmogaus augimo hormono kokybinio ir kiekybinio nustatymo fluorescenciniais metodais galimybės

Show simple item record Jarienė, Giedrė 2017-08-21T08:17:49Z 2017-08-21T08:17:49Z 2006
dc.description.abstract Abbrevations t – fluorescence lifetime j – rotational correlational time ([Ru(bpy)2(phen-ITC)]2+) – Bis-(bipyridine)-5-(isotiocyanatophenantrolin)-Ru(PF6-)2 Ab – antibody AF – Alexa Fluor 660 AF-hGH – with Alexa Fluor 660 labelled human growth hormone FITC – fluorescein isothiocyanate FITC-Ab – FITC labelled antibody FP – fluorescence polarization FRET – fluorescence resonance energy transfer hGH – human growth hormone PMI – N-(3-pyrene)maleimide PMI-hGH – human growth hormone labelled N-(3-pyrene)maleimide PVPPB – poly(N-vinyl-2-pyrolidone) with phenylboronic acid copolymer r – anisotropy RITC – rodamine B isothiocyanate RITC-hGH – with RITC labeled human growth hormone SDS-PAGE – sodium dodecylsulfate-polyacrylamide gel electrophoresis INTRODUCTION Relevance of the study. Human growth hormone (hGH) is a heterogeneous protein with several molecular forms (isoforms). Human growth hormone measurements are complicated because of the heterogeneous nature of hGH. Specific assays for each isoform are currently unavailable. Because the different antibodies used in immunoassays bind to a different spectrum of hGH isoforms, hGH concentrations measured by immunoassay are likely to depend on the particular antibody used. Moreover, because the distribution of the different hGH isoforms varies among individuals, the results from different immunoassays cannot be interrelated easily by using a single conversion factor. Fluorescence is by far the most important optical spectroscopic method for measuring protein–protein interactions because fluorophores are highly sensitive probes of environmental changes. Fluorescence labels using fluorescence methods have some advantages in comparison with the methods using labels of other kind. New fluorescent labels and new fluorescence detection methods – such as time-resolved fluorescence polarization – open new possibilities of the estimation of the molecular size of proteins and their complexes with antibodies. These possibilities can be very useful in quantitative and qualitative determination of hGH and isoforms of hGH. Aim and tasks. The aim of our study was to establish possibilities of the quantitative and qualitative determination of the human growth hormone using fluorescence methods, to estimate the possibilities to use these methods in case of presence of this hormone in buffer solutions, in immunological systems and synthetic medium. To achieve this goal, we set up the following tasks: 1. To investigate the possibilities of the use of the fluorescence phenomenon for the determination of the human growth hormone, advantages of the different fluorescence methods, if possible, compare it with the methods using labels of other kind; to choose usable for the determination of the hormone fluorescence methods and immunoassay varieties. 2. To carry out experiments approving the possibility of the correct attaching of the fluorescence label to the hormone or antibody with regard to chosen fluorescence methods and immunoassay varieties; to approve possibility of the donor-acceptor interaction of two fluorescence labels in the immunorecognition of the hormone. 3. To determine the human growth hormone in the buffer solutions, in immunological systems and synthetic medium. Significance and novelty of the study. For the first time, fluorescence features of the originally obtained hGH monomeric and polymeric molecular forms were investigated using tryptophan residue fluorescence. For the first time it was shown that different correlation time of different molecular forms of hGH can be estimated using a suitable long-lifetime fluorescence label and fluorescence polarization. As a result, a new method of estimation of hGH isoforms with a different molecular size using different correlation time of these molecules was proposed. The new method has no analogues between methods of determination of molecular forms of hormones-proteins. New possibilities of the quantitative determination of the fluorescent labelled hGH in various situations and medium were investigated using long-visible-wavelength excited fluorescent label. For the first time the possibilities to use so labelled hGH in a in vitro investigation of immobilization and controlled release of human growth hormone were investigated. Structure of the dissertation. The dissertation consists of Introduction (containing Preface and Review of literature), Material and Methods, Results and Discussion (containing 5 sub-chapters), Defended conclusions, References (88 positions) and the list of the author’s publications containing the materials of the dissertation. The text of the dissertation takes 81 pages, contains 6 tables and 22 figures. The language of the work is Lithuanian. MATERIALS AND METHODS The main materials. Human growth hormone (hGH) as a monomeric form or a mixture of different oligomeric forms as obtained in the previous works by Professor L.Lašas was used. Mass-spectroscopy, HPLC and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of monomeric hGH show sample to be greater than 98% pure and molecular weight of 22 kDa. Oligomeric hGH forms were separated by the means of size exclusion chromatography. SDS-PAGE of two sample-fractions revealed that samples’ molecular weights were respectively 66 kDa and 110 kDa. Mass-spectroscopy and SDS-PAGE performed under reducing conditions revealed that oligomeric hGH forms consist of 22 kDa macromonomer units and are more than 98% pure. Fluorescent labels. Fluorescein isothiocyanate (FITC), N-(3-pyren)maleimide (PMI) and rhodamine B isothiocyanate (RITC) were obtained from Aldrich, Alexa Fluor 660 (AF) was obtained from Molecular Probes, bis-(bipyridine)-5-(isothiocyanatophenanthrolin)-Ru(PF6-)2 ([Ru(bpy)2(phen-ITC)]2+) was obtained from Fluka. Antibodies. Immobilized antibodies against hGH were obtained from bioMerieux Sa, monoclone antibodies against hGH - from Biotechna. Fluorescence experiments. Fluorescence experiments were performed on a Hitachi 650-40 fluorescence spectrometer, special experiments with a front surface accessory, polarization accessory and fiber-optic accessory were performed on Perkin Elmer luminescence spectrometer LS-5B. Fluorescence intensities were measured with 2 nm excitation and emission slits (in case of measurements with fiber-optic accessory these slits were 5 nm). Fluorescence anisotropy was measured with 10 nm excitation and emission slits. Unpolarized fluorescence spectrums were recorded with 5 nm excitation and emission slits, polarized spectrums – with 15 nm (for monochromator with fixed wavelength) and 5 nm (for monochromator with changeable wavelength) slits. Fluorescence anisotropy method. One of the possible ways to estimate the monomeric and oligomeric molecular forms of this hormone is fluorescence polarization immunoassay with possibility to determinate a molecular size of the hGH form. The molecular size and the rotational volume of fluorescent labeled molecules or bioconjugates are directly proportional to rotational correlation time and can be determined by the measurement of fluorescence anisotropy (r), (1) where III and I┴ are the components of the fluorescence intensity that are parallel and perpendicular to the electric vector of the excitation light, and IT is the total fluorescence intensity. The dependence of the anisotropy with the rotational diffusion process: (2) where ro is the anisotropy in the absence of diffusion, τ is the fluorescence lifetime (in this time the fluorescence anisotropy decreases up to ro·e- 1), and φ is the rotational correlation time of the fluorophore. In the absence of diffusion, the anisotropy is given by the relative orientation of the excitation and emission moments of the fluorophore, taking values between -0.2 and 0.4. In solution, the excited molecules can rotate, thereby changing their orientation. The angle θ between the average orientation of the molecules at a given time with respect to the original orientation depends on the time, the temperature, the solvent properties, and the size and the shape of the molecule. This process, known as rotational diffusion, can be characterized by a parameter called rotational correlation time (φ), defined as the time after which cos θ = e-1 . In a fluorescent conjugate that behaves in solution as a rigid rotator, the parameters ro and τ depend on the photophysical properties of the fluorophore, whereas φ depends mainly on the size and shape of the macromolecule. The rotational correlation time φ can be expressed by the equation, (3) where η is the solvent viscosity coefficient, k is the Boltzman constant, T is temperature, and V is the volume of the rotating particle. This approximation appears to be valid for proteins. To obtain constant viscosity for all samples special mixtures of aqueous glycerol solution (25%) and sample substances with viscosity of 2 cP (twofold higher than viscosity of H2O) were prepared. Because the polypeptides and buffer concentrations are very low, sample viscosities can be calculated from the glycerol concentration. Every sample was prepared in duplicate. Fluorescence anisotropy was measured in the mixture of sample in phosphate buffer (pH 7,2) and glycerol solution at 20 °C, setting the excitation at 467 nm and the emission at 612 nm with bandwidths of 10 nm. Every measurement was repeated three times. Anisotropy was calculated using equation 4, including the instrument G factor defined as the ratio of sensitivities of the detection system for the vertically and horizontally polarized light. (4) where H and V refer to the horizontal and vertical positions, respectively, of the excitation (first subscript) and emission (second subscript) polarization filters. Samples of the hormone were labelled with [Ru(bpy)2(phen-ITC)]2+ . The labeling degrees of the samples were: 1,056 ± 0,09 (hGH monomer), 1,051 ± 0,11(hGH trimer), 1,046 ± 0,09 (hGH pentamer). Only one la
dc.language.iso lit
dc.subject Molecular forms of human growth hormone
dc.subject Žmogaus augimo hormono molekulinės formos
dc.subject Fluorescencija
dc.subject Anisotropy
dc.subject Anizotropija
dc.subject Žmogaus augimo hormonas
dc.subject Human growth hormone
dc.subject Fluorescence
dc.title Žmogaus augimo hormono kokybinio ir kiekybinio nustatymo fluorescenciniais metodais galimybės
dc.title.alternative Possibilities of qualitative and quantitative determination of the human growth hormone using fluorescence methods
dc.type Daktaro disertacija

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