Quenching of tryptophan fluorescence in a highly scattering solution: Insights on protein localization in a lung surfactant formulation.

Luca Ronda,Stefano Bettati,Fabrizio Salomone,Silvia Catinella,Barbara Pioselli,Marialaura Marchetti,Patrick Van Der Wel

doi:10.1371/journal.pone.0201926

Abstract

CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic surfactant developed for respiratory distress syndrome replacement therapy in pre-term newborn infants. CHF5633 contains two phospholipids (dipalmitoylphosphatidylcholine and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoglycerol sodium salt), and peptide analogues of surfactant protein C (SP-C analogue) and surfactant protein B (SP-B analogue). Both proteins are fundamental for an optimal surfactant activity in vivo and SP-B genetic deficiency causes lethal respiratory failure after birth. Fluorescence emission of the only tryptophan residue present in SP-B analogue (SP-C analogue has none) could in principle be exploited to probe SP-B analogue conformation, localization and interaction with other components of the pharmaceutical formulation. However, the high light scattering activity of the multi-lamellar vesicles suspension characterizing the pharmaceutical surfactant formulation represents a challenge for such studies. We show here that quenching of tryptophan fluorescence and Singular Value Decomposition analysis can be used to accurately calculate and subtract background scattering. The results indicate, with respect to Trp microenvironment, a conformationally homogeneous population of SP-B. Trp is highly accessible to the water phase, suggesting a surficial localization on the membrane of phospholipid vesicles, similarly to what observed for full length SP-B in natural lung surfactant, and supporting an analogous role in protein anchoring to the lipid phase.

Highlights

Mammalian lung surfactants are mixtures of 90% lipids and about 10% of four surfactant-specific proteins named SP-A, SP-B, SP-C, and SP-D [1] with the role of reducing surface tension in the alveolar space, allowing breathing, uniform lung inflation and preventing alveolar collapse during expiration [2, 3]
Only the single the fluorophore (Trp) residue of SP-B analogue is expected to contribute significantly to fluorescence emission by the synthetic surfactant, since no Trp is present in the sequence of SP-C analogue
The slow decay in signal intensity, which is reversible upon mixing of the sample, is perfectly consistent with the hypothesis that within the synthetic surfactant the predominant form of SP-B analogue is not that of freely diffusing monomers or oligomers, but rather it is stably adherent to the multilamellar lipid vesicles of the formulation

Summary

Introduction

Mammalian lung surfactants are mixtures of 90% lipids and about 10% of four surfactant-specific proteins named SP-A, SP-B, SP-C, and SP-D [1] with the role of reducing surface tension in the alveolar space, allowing breathing, uniform lung inflation and preventing alveolar collapse during expiration [2, 3]. Pre-term newborns, lacking functional pulmonary surfactant, experience respiratory distress syndrome (RDS). This condition is clinically treated with surfactant. Quenching of tryptophan fluorescence in a highly scattering lung surfactant formulation. Chiesi employee authors are articulated in the “authors contributions” section

Methods

Results

Conclusion