Surface fluorescence resonance energy transfer studies on interfacial adsorption of Thermomyces (humicola) lanuginosa lipase, using monomolecular films of cis-parinaric acid.
The fluorescence resonance energy transfer (FRET) technique was adapted to study the process whereby lipase is adsorbed to monomolecular lipid films spread at the air-water interface. When cis-parinaric acid (cis-PnA) was spread over an aqueous subphase before the injection of sodium taurodeoxycholate (NaTDC) and Thermomyces lanuginosa lipase (TLL), no FRET was observed. Under these conditions, no adsorption of TLL was detected using an ELISA. In contrast, FRET occurred when cis-PnA was spread over an aqueous subphase containing NaTDC and TLL. The FRET signals observed were attributed to the interactions between the adsorbed TLL and the cis-PnA monomolecular films. Comparisons between the fluorescence emission spectra corresponding to the bulk phase and the aspirated film, in the presence and absence of TLL, showed that cis-PnA was undetectable in the bulk phase. We concluded that the FRET originated from the interface and not from the bulk phase. Using surface FRET, we estimated that the surface excess of the catalytically inactive mutant, TLL(S146A), was 1.6 higher than that present in the wild-type TLL. This finding is in agreement with independent measurements of the surface excess of TLL and TLL(S146A) on monomolecular films of cis-PnA.
Yapoudjian
S
S
Laboratoire de Lipolyse Enzymatique, CNRS-IFR1, UPR 9025, 31 Chemin Joseph-Aiguier, 13402 Marseille, Cedex 20, France.
Ivanova
M
M
Douchet
I
I
Zénatti
A
A
Sentis
M
M
Marine
W
W
Svendsen
A
A
Verger
R
R
D000327:Adsorption; D000388:Air; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D004735:Energy Transfer; D005231:Fatty Acids, Unsaturated / Q000737:chemistry*; D005453:Fluorescence; D007536:Isomerism; D007834:Lasers; D008049:Lipase / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013499:Surface Properties; D013657:Taurodeoxycholic Acid / Q000378:metabolism; D014867:Water / Q000737:chemistry
D005231:Fatty Acids, Unsaturated; D014867:Water; D013657:Taurodeoxycholic Acid; D008049:Lipase; C013641:parinaric acid