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Biophysical and Biochemical Characterization of Peptide, Protein, and Bioconjugate Products
Published in Sandeep Nema, John D. Ludwig, Parenteral Medications, 2019
Tapan K. Das, James A. Carroll
For practical applications of Trp fluorescence in characterization as well as for comparability purposes, steady-state fluorescence studies are quite sufficient to probe conformational changes or unfolding of a therapeutic biologic candidate due to high sensitivity of fluorescence signal to local environment of Trp and high signal-to-noise ratio of fluorescence signal. The major goal in the application of Trp fluorescence spectroscopy in a comparability study is to interpret the fluorescence properties such as emission maxima and fluorescence intensity in terms of changes in protein structure. In other words, it is expected that comparison of fluorescence spectra will detect any significant changes in folding and structure of a biologics candidate arising from manufacturing and process. Fluorescence quenching studies using acrylamide and sodium iodide provides valuable information on surface exposure of Trp. A conformational transition may change the exposure of Trp to solute quenchers (acrylamide, iodide, or CsCl), and hence can be monitored by measuring Trp quenching [89]. Steady-state fluorescence anisotropy is another fluorescence protocol that can be used to study rigidity (or lack of) of a protein segment and relative size of protein. Anisotropy value can change upon unfolding of a protein or complexation of a protein (e.g., aggregation, antigen binding).
Förster Resonance Energy Transfer Microscopy for Monitoring Molecular Dynamics in Living Cells
Published in Guy Cox, Fundamentals of Fluorescence Imaging, 2019
Vinod Jyothikumar, Yuansheng Sun, Ammasi Periasamy
Optimal expression levels of vectors are important. Experiments based on time courses of expression need to select the time when the expression is sufficient to measure FRET, when the vectors are still localized in the same areas as in their endogenous wild-type counterparts, where their functional activity can be established. The over expression of exogenous proteins in experiments can also lead to homo-FRET, i.e., FRET between like chromophores. This process can only be monitored by fluorescence anisotropy [39] and is used to study protein structures, oligomerization and the organization of membrane proteins in sub-microdomains at the cell surface.
Review of Nanoscale Spectroscopy in Medicine
Published in Sarhan M. Musa, Nanoscale Spectroscopy with Applications, 2018
Chintha C. Handapangoda, Saeid Nahavandi, Malin Premaratne
is a quantity that characterizes the polarization of fluorescence (Prasad 2003). Fluorescence anisotropy measurements provide information on the size and shape of proteins and the rigidity of various molecular environments. These measurements have been used to measure protein-protein associations, fluidity of membranes and for immunoassays of various substances (Lakowicz 2010).
Solvatochromic behavior of a pyrene-pyrimidine-based Schiff base and detection of heavy metal ions in aqueous media
Published in Journal of Coordination Chemistry, 2021
Swadesh Ghosh, Dipti Singharoy, Saugata Konar, Jnan Prakash Naskar, Subhash Chandra Bhattacharya
In addition steady state anisotropy of PYPH has been investigated in fully saturated condition of different types of solvent (polar protic, H2O; polar aprotic, ACN and nonpolar, n-hexane (n-hex)). Steady state fluorescence anisotropy helps to understand the motional restriction of the molecule due to interaction with different solvents. The anisotropy of PYPH is 0.12 in aqueous media whereas the anisotropy of PYPH is 0.02 and 0.05, respectively, in acetonitrile and n-hex (Figure S5, supplementary material). Hence, anisotropy of PYPH in water is greater than that of polar aprotic ACN and nonpolar n-hex solvents, supporting that strong intermolecular hydrogen bonding interaction takes place. Hydrophobic interaction of PYPH in non-polar solvent indicates higher anisotropy of PYPH than polar aprotic solvents. Time-resolved fluorescence measurements were performed for PYPH in three different solvents to characterize the spectral properties and dynamics, considering the polarity in the excited state (Table S1, supplementary material). The excited state decay profiles in different homogeneous environments are shown in Figure S6 (supplementary material) which has been fitted with a single exponential decay with χ2 value near one (Table S1, supplementary material). Fluorescence lifetime values of PYPH increases with polarity of solvents and lies between 3.8 and 4.7 ns. Lifetime value of PYPH in water is higher than that in other solvents, which supports the higher fluorescence intensity of PYPH in water.