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Sensitive Quantification of Small Molecular Weight Thiolswith Dieosin Glutathione Disulfide (Di-E-GSSG)
WINTER 2021
, Abstract:
This experiment is based on the principle of Fluorescence Spectroscopy. It will be used to
determine the concentration of small molecular weight thiols using the initial rates method. A
fluorogenic reagent dieosin glutathione disulfide (Di-E-GSSG) will be used to determine the role
of thiol pKa in the reactivity of the biological thiols H2S, HCys, Cys, and glutathione.
Phenomenon involved is fluorescence self-quenching that occurs when two identical fluorescent
molecules are brought close to one another resulting in intermolecular interactions between the
fluorophores, causing their fluorescence emission to be quenched.
Theory
Fluorescence is a type of luminescence caused by photons exciting a molecule, raising it to an
electronic excited state. It’s brought about by absorption of photons in the singlet ground state
promoted to a singlet-excited state. As the excited molecule returns to ground state, emits a
photon of lower energy, which corresponds to a longer wavelength, than the absorbed photon.
Fluorescence spectroscopy analyzes fluorescence from a molecule based on its fluorescent
properties.
The jablonski diagram is a representation of the transition of electronic state of a molecule
during the fluorescence phenomenon. The left axis shows increasing energy, where a typical
fluorescent molecule has an absorbance spectrum. This spectrum shows the energy or
wavelengths, where the molecule will absorb light.[1]
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