Sage Journals: Discover world-class research

Abstract

A novel application for pre-fluorescent probes in the detection of peptide- and protein-based radicals is proposed. Pre-fluorescent probes combine a fluorescent moiety labeled with a paramagnetic nitroxide that acts as a fluorescence quencher. Trapping of a radical by the nitroxide group restores the fluorescence properties. The increase in fluorescence intensity with time reflects the formation and quenching of free radicals and can be employed for the quantitative evaluation of yields and kinetics. In this test system, the pre-fluorescent probe 4-(9-acridinecarbonate)-2,2,6,6-tetramethylpiperidinyl-1-oxyl radical (Ac-Tempo), in which an acridine moiety was labeled with 2,2,6,6-tetramethylpiperidinyl-1-oxy (Tempo), was employed to probe peptide- and protein-based radicals. Peptide-based radicals were generated through the reaction between horseradish peroxidase (HRP)/H₂O₂ and a derivative of tyrosine. Protein-based radicals were generated through the reaction between myoglobin (Mb) and H₂O₂. In both cases the Ac-Tempo was found, using a combination of high-performance liquid chromatography (HPLC) and mass spectrometry, to be converted into fluorescent acridine (Ac)-piperidine (4-(9-acridinecarbonate)-2,2,6,6-tetramethylpiperidine).

Keywords

Pre-fluorescent probe Protein radicals Peptide radicals Nitroxide

Get full access to this article

View all access options for this article.

References

Moad

Shipp

D. A.

Smith

T. A.

and Solomon

D. H

, J. Phys. Chem. A 103, 6580 (1999).

Augusto

De Menezes

S. L.

Linares

Romero

Radi

and Denicola

, Biochemistry 41, 14323 (2002).

Nakao

L. S.

Iwai

L. K.

Kalil

and Augusto

, FEBS Lett. 547, 87 (2003).

Gunther

M. R.

, Free Radical Biol. Med. 36, 1345 (2004).

Augusto

Bonini

M. G.

and Trindade

D. F

, Free Radical Biol. Med. 36, 1224 (2004).

Davies

M. J.

and Hawkins

C. L

, Free Radical Biol. Med. 36, 1072 (2004).

Detweiler

C. D.

Deterding

L. J.

Tomer

K. B.

Chignell

C. F.

Germolec

and Mason

R. P

, Free Radical Biol. Med. 33, 364 (2002).

Ramirez

D. C.

Chen

Y. R.

and Mason

R. P

, Free Radical Biol. Med. 34, 830 (2003).

Y. Y.

Ramirez

D. C.

Detweiler

C. D.

Mason

R. P.

and Chignell

C. F

, Photochem. Photobiol. 77, 585 (2003).

10.

Mason

R. P.

, Free Radical Biol. Med. 36, 1214 (2004).

11.

Wright

P. J.

and English

A. M

, J. Am. Chem. Soc. 125, 8655 (2003).

12.

Sobek

Martschke

and Fischer

, J. Am. Chem. Soc. 123, 2849 (2001).

13.

Perkins

M. J.

, Advances in Physical Organic Chemistry (Elsevier, Toronto, 1980), p. 1.

14.

Blough

N. V.

and Simpson

D. J

, J. Am. Chem. Soc. 110, 1915 (1988).

15.

Garcia

B. O.

Maretti

Saatre

and Scaiano

J. C

, Macromolecules 34, 6184 (2001).

16.

Karatekin

Landis

Lem

O'Shaughnessy

and Turro

N. J

, Macromolecules 34, 8187 (2001).

17.

Coenjarts

Garcia

Llauger

Palfreyman

Vinette

A. L.

and Scaiano

J. C

, J. Am. Chem. Soc. 125, 620 (2003).

18.

Ivan

M. G.

and Scaiano

J. C

, Photochem. Photobiol. 78, 416 (2003).

19.

Stubbe

and Van der Donk

W. A

, Chem. Rev. 98, 705 (1998).

20.

Yin

W. J.

Mitra

Stearns

R. A.

Baillie

T. A.

and Kumar

, Biochemistry 43, 5455 (2004).

21.

Fenwick

C. W.

and English

A. M

, J. Am. Chem. Soc. 118, 12236 (1996).

22.

Davies

M. J.

Gilbert

B. C.

and Haywood

R. M

, Free Radical Res. Commun. 15, 111 (1991).

23.

Maillard

Ingold

K. U.

and Scaiano

J. C

, J. Am. Chem. Soc. 105, 5095 (1983).

New Approach for the Detection of Peptide- and Protein-Based Radicals Using a Pre-Fluorescent Probe

Abstract

Keywords

Get full access to this article

References