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Protein-surfactant interactions at hydrophobic interfaces studied with Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS)
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
YKI, Institute for Surface Chemistry, Stockholm.
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2008 (English)In: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 317, no 2, 449-457 p.Article in journal (Refereed) Published
Abstract [en]

The aim of this work was to study the dynamics of proteins near solid surfaces in the presence or absence of competing surfactants by means of total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Two different proteins were studied, bovine serum albumin (BSA) and Thermomyces lanuginosus lipase (TLL). A nonionic/anionic (C12E6/LAS) surfactant composition was used to mimic a detergent formulation and the surfaces used were C 18 terminated glass. It was found that with increasing surfactant concentrations the term in the autocorrelation function (ACF) representing surface binding decreased. This Suggested that the proteins were competed off the hydrophobic surface by the surfactant. When fitting the measured ACF to a model for surface kinetics, it was seen that with raised C12E6/LAS concentration, the Surface interaction rate increased for both proteins. Under these experimental conditions this meant that the time the protein was bound to the surface decreased. At 10 mu M C12E6/LAS the surface interaction was not visible for BSA, whereas it was still distinguishable in the ACF for TLL. This indicated that TLL had a higher affinity than BSA for the C 18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption.

Place, publisher, year, edition, pages
2008. Vol. 317, no 2, 449-457 p.
Keyword [en]
Hydrophobic surface; Lipase; Protein-surfactant interactions; TIR-FCS; Autocorrelation function (ACF); Hydrophobic surfaces; Protein-surfactant interactions; Total internal reflection fluorescence correlation spectroscopy (TIR-FCS); Autocorrelation; Fluorescence spectroscopy; Glass; Interfaces (materials); Lipases; Surface active agents; Proteins; bovine serum albumin; detergent; enzyme; protein; surfactant; Thermomyces lanuginosus lipase; triacylglycerol lipase; unclassified drug; adsorption; article; binding affinity; correlation function; dynamics; fluorescence analysis; hydrophobicity; kinetics; priority journal; protein interaction; spectroscopy; total internal reflection fluorescence correlation spectroscopy; Animals; Ascomycota; Binding Sites; Cattle; Fluorescent Dyes; Hydrophobicity; Lasers; Lipase; Proteins; Serum Albumin, Bovine; Spectrometry, Fluorescence; Surface Properties; Surface-Active Agents
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-6931DOI: 10.1016/j.jcis.2007.09.089ISI: 000251556100010Scopus ID: 2-s2.0-36148937163OAI: oai:DiVA.org:kth-6931DiVA: diva2:11782
Note

QC 20100818. Uppdaterad från Submitted till Published 20100818.

Available from: 2007-03-27 Created: 2007-03-27 Last updated: 2017-08-15Bibliographically approved
In thesis
1. Dynamics of Enzymes at Interfaces: Lipase adsorption and mobility on solid surfaces
Open this publication in new window or tab >>Dynamics of Enzymes at Interfaces: Lipase adsorption and mobility on solid surfaces
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis aimed to give more insight in the dynamics of enzymes at interfaces. The adsorption and mobility of adsorbed proteins can e.g. give a better understanding of structure-function properties of interfacially active enzymes. Studied enzyme was the lipase from Thermomyces lanuginosus (TLL).

Adsorption of TLL to surfaces of different hydrophobicity was studied by Dual Polarization Interferometry (DPI), Surface Plasmon Resonance (SPR) and ellipsometry. It was found that TLL had highest affinity and adsorbed to largest adsorbed amount on a hydrophobic, C18 terminated surface. Moreover, activity studies of adsorbed TLL suggested that a larger fraction of the lipases were orientated with the active site facing the surface on hydrophobic surfaces.

Mobility of adsorbed enzymes was studied by means of Fluorescence Recovery After Photobleaching (FRAP) with Confocal Laser Scanning Microscopy (CLSM). CLSM was also used as a tool to image the role of TLL in the detergency of lipids from single cotton fibers. The TLL surface mobility was measured on model surfaces of different hydrophobicity. The rate of TLL surface diffusion was strongly dependent on the surface density of lipase, which was explained by sterical hindrance and intermolecular repulsion. The diffusion was both lowest and decreased as a function of time after adsorption on the most hydrophobic surface. This was thought to be due to a larger fraction of adsorbed TLL oriented with the active site towards the hydrophobic surface and that this fraction increased as a function of time.

The presence of surfactants affected the TLL mobility on hydrophobic surfaces. The diffusion increased more than tenfold when TLL was coadsorbed with C12E6/LAS above the critical micellar concentration (cmc) of the surfactant. This was thought to be due to a surfactant induced desorption-rebinding mechanism of TLL. Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS) supported this theory and was implemented as a technique to quantify kinetic processes of protein-surfactant interactions at surfaces.

The surface mobility of TLL was higher on a trimyristin substrate surface compared to the model hydrophobic surface. Single particle tracing of lipases could be performed by conjugation of TLL to Quantum Dots (QDs). The microscopic behavior of QD-lipases on trimyristin suggested that the enzyme operated in two different modes on the surface, which gave the trajectories of single lipase molecules a “bead on a string” appearance.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. 57 p.
Series
Trita-FYS, ISSN 0280-316X ; 2007:20
Keyword
biophysics, surface chemistry, diffusion, enzymes, lipases, adsorption, mobility
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-4319 (URN)978-91-7178-604-3 (ISBN)
Public defence
2007-04-23, FB53, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100818Available from: 2007-03-27 Created: 2007-03-27 Last updated: 2010-08-18Bibliographically approved

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