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Local environment perturbations in alpha1-antitrypsin monitored by a ratiometric fluorescent label

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Abstract

The complex multistep inhibition of proteinases by alpha1-antitrypsin (α1-AT) was investigated by covalently labeling its unique Cys residue with a ratiometric environment-sensitive fluorescent dye, 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone (BMFC). The binding of BMFC-labeled α1-AT with pancreatic elastase led to significant changes in the dual emission of BMFC. The 8 nm blue shift of one of the bands and ca. 65% change in the intensity ratio of the two emission bands suggested an increased exposure of the labeled Cys-232 residue to the bulk water on complex formation. In contrast, the bacterial V8 proteinase-induced cleavage of the reactive center loop of BMFC-labeled α1-AT did not generate any significant change in the Cys-232 region. Similar experiments with elastase and α1-AT conjugated to the classical environment-sensitive dye, IANBD, confirmed these results but led to much smaller modifications in the emission spectrum. Stopped-flow investigation of the reaction between BMFC-labeled α1-AT and elastase showed both a well-described fast and a new slow step of the inhibition process. The latter step is probably associated with the structural reorganization aimed at stabilizing the final complex. These results present a convenient fluorescence ratiometric approach based on the BMFC label for studies of protein conformational changes.

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Abbreviations

α1-AT:

α1-Antitrypsin (α1-proteinase inhibitor)

PPE:

Porcine pancreatic elastase

ESIPT:

Excited state intramolecular proton transfer

RCL:

Reactive center loop

BMFC:

6-Bromomethyl-2-(2-furanyl)-3-hydroxychromone

IANBD:

N,N′-Dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine

Hepes:

4-(2-Hydroxyethyl) piperazine-1-ethane sulfonic acid

DMF:

N,N-Dimethylformamide

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Authors and Affiliations

  1. Laboratoire de Biophotonique et Pharmacologie, UMR-CNRS 7213, Faculté de Pharmacie, 74, route du Rhin, 67401, Illkirch-Cedex, France

    Christian Boudier, Andrey S. Klymchenko, Yves Mely & Anny Follenius-Wund

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  1. Christian Boudier
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Correspondence to Anny Follenius-Wund.

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Boudier, C., Klymchenko, A.S., Mely, Y. et al. Local environment perturbations in alpha1-antitrypsin monitored by a ratiometric fluorescent label. Photochem Photobiol Sci 8, 814–821 (2009). https://doi.org/10.1039/b902309g

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