Monitoring of Demasking of Peptide Bonds During Proteolysis by Analysis of the Apparent Spectral Shift of Intrinsic Protein Fluorescence |
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Authors: | Mikhail M Vorob��ev Vitali Vogel G��nnur G��ler Werner M?ntele |
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Institution: | (1) A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 ul. Vavilova, 119991 Moscow, Russia;(2) Institute of Biophysics, Johann Wolfgang Goethe Universit?t, Max-von-Laue-Str.1, D-60438 Frankfurt am Main, Germany |
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Abstract: | Demasking of peptide bonds during proteolysis of β-casein and β-lactoglobulin by trypsin was monitored by the measurement
of the overall spectral shift of intrinsic protein fluorescence. A clear shift of the apparent emission maxima from approximately
340–345 nm to 355–360 nm during proteolysis was observed, with a time course, which follows protein degradation and structural
opening. In contrast to procedures using extrinsic fluorescence labels, this label-free procedure does not bear the risk of
structural alterations. It is easy to perform, fast, and has a relatively high accuracy of determination. Proteolysis was
modelled as simple two-step process with consecutive demasking and hydrolysis stages. It was shown that the fluorescence shift
can be attributed to the demasking stage. Formally, kinetics of the peptide bond demasking obeys a first-order kinetic law.
Both the theoretical simulations and experiment are in accordance giving the similar dependences of the hydrolysis degree
on the degree of peptide bond demasking. |
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