Abstract
A novel experimental method, suitable for separate analysis of the quenching parameters characteristic of solvent-exposed and solvent-masked fluorophors of macromolecules, is described. The method is based on the modified Stern-Volmer analysis and requires simultaneous application of two kinds of quencher: one that can selectively quench the emission of exposed fluorophors (e.g., ionic quenchers) and another that is nonselective (e.g., oxygen or, in many cases, acrylamide), capable of quenching the fluorescence of both exposed and masked groups. In order to examine the accuracy of the model, a computer simulation was performed. The results showed that the errors are comparable to those arising from the conventional quenching experiments. The method is applicable to phosphorescence quenching as well and is extendable to time-resolved measurements (by replacing fluorescence intensities with lifetimes). The method was applied to resolve the quenching parameters of lysozyme fluorescence by the use of iodide as selective and acrylamide as nonselective quenchers. The determination of the acrylamide quenching constant associated with the internal fluorophor, Trp-108 (Kq = 3.5 M-1), permits specific studies on the dynamics of internal regions of the protein. The quenching constant determined for the more exposed residue Trp-62 (Kq = 1.6 M-1) provides local information about the surface independent of the electrostatic effects observed when an ionic quencher is used.
| Original language | English |
|---|---|
| Pages (from-to) | 6674-6679 |
| Number of pages | 6 |
| Journal | Biochemistry |
| Volume | 24 |
| Issue number | 23 |
| Publication status | Published - 1985 |
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- Biochemistry
Cite this
A double-quenching method for studying protein dynamics : Separation of the fluorescence quenching parameters characteristic of solvent-exposed and solvent-masked fluorophors. / Somogyi, Béla; Papp, Sándor; Rosenberg, Andreas; Seres, I.; Matkó, J.; Welch, G. Rickey; Somogyi, B.
In: Biochemistry, Vol. 24, No. 23, 1985, p. 6674-6679.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A double-quenching method for studying protein dynamics
T2 - Separation of the fluorescence quenching parameters characteristic of solvent-exposed and solvent-masked fluorophors
AU - Somogyi, Béla
AU - Papp, Sándor
AU - Rosenberg, Andreas
AU - Seres, I.
AU - Matkó, J.
AU - Welch, G. Rickey
AU - Somogyi, B.
PY - 1985
Y1 - 1985
N2 - A novel experimental method, suitable for separate analysis of the quenching parameters characteristic of solvent-exposed and solvent-masked fluorophors of macromolecules, is described. The method is based on the modified Stern-Volmer analysis and requires simultaneous application of two kinds of quencher: one that can selectively quench the emission of exposed fluorophors (e.g., ionic quenchers) and another that is nonselective (e.g., oxygen or, in many cases, acrylamide), capable of quenching the fluorescence of both exposed and masked groups. In order to examine the accuracy of the model, a computer simulation was performed. The results showed that the errors are comparable to those arising from the conventional quenching experiments. The method is applicable to phosphorescence quenching as well and is extendable to time-resolved measurements (by replacing fluorescence intensities with lifetimes). The method was applied to resolve the quenching parameters of lysozyme fluorescence by the use of iodide as selective and acrylamide as nonselective quenchers. The determination of the acrylamide quenching constant associated with the internal fluorophor, Trp-108 (Kq = 3.5 M-1), permits specific studies on the dynamics of internal regions of the protein. The quenching constant determined for the more exposed residue Trp-62 (Kq = 1.6 M-1) provides local information about the surface independent of the electrostatic effects observed when an ionic quencher is used.
AB - A novel experimental method, suitable for separate analysis of the quenching parameters characteristic of solvent-exposed and solvent-masked fluorophors of macromolecules, is described. The method is based on the modified Stern-Volmer analysis and requires simultaneous application of two kinds of quencher: one that can selectively quench the emission of exposed fluorophors (e.g., ionic quenchers) and another that is nonselective (e.g., oxygen or, in many cases, acrylamide), capable of quenching the fluorescence of both exposed and masked groups. In order to examine the accuracy of the model, a computer simulation was performed. The results showed that the errors are comparable to those arising from the conventional quenching experiments. The method is applicable to phosphorescence quenching as well and is extendable to time-resolved measurements (by replacing fluorescence intensities with lifetimes). The method was applied to resolve the quenching parameters of lysozyme fluorescence by the use of iodide as selective and acrylamide as nonselective quenchers. The determination of the acrylamide quenching constant associated with the internal fluorophor, Trp-108 (Kq = 3.5 M-1), permits specific studies on the dynamics of internal regions of the protein. The quenching constant determined for the more exposed residue Trp-62 (Kq = 1.6 M-1) provides local information about the surface independent of the electrostatic effects observed when an ionic quencher is used.
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M3 - Article
C2 - 4084551
AN - SCOPUS:0022385207
VL - 24
SP - 6674
EP - 6679
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 23
ER -