Interfacial protein fluorescence

Interfacial protein fluorescence is an in situ method that can provide real time data with a resolution of 0.1 s. This technique is a major advantage in that the protein adsorption-desorption dynamics may be determined without resorting to sample manipulation prior to analysis. Figure 9 illustrates adsorption-desorption dynamics for both BSA and 7-globulin at bulk equimolar concentrations of 6.671xM/L. The 7-globulin required 40 min to reach... 

The fluorescence lifetime is sensitive to the environment of the fluorophore, and in membranes this usually means the surrounding fatty acyl chains or the membrane protein interfacial region (see summary in Table 5.3). Generally, the lifetime of membrane-bound fluorophores is rather less sensitive to the types of subtle alterations which are encountered in membranes as compared to the fluorescence anisotropy parameters. The gel-to-liquid crystalline phase transition is a notable exception where most fluorophores show an alteration in lifetime properties. Although, again, the anisotropy (see below) is the most sensitive parameter in this regard, the fluorescence lifetime has been used with considerable success in the study of phase transitions and lateral phase separations. Fluorophores used to yield information on the... 

The fast reaction rate between Zn(II) ion with Hocqn at the 1-butanol-water interface was measured by two-phase sheath flow method [33]. The formation of a fluorescence complex at the interface was measured in the period less than 5 ms after the two-phase contact as shown in Fig. 18. By the help of digital simulation, the initial process of the interfacial reaction between Zn(II) and Hqn was analyzed (Fig. 18). This approach is promising for the measurement of rapid interfacial reactions such as protein folding and luminescence lifetime as well [36]. 

To determine the t) e of interfacial structure formed it is necessary to label and locate different proteins. Although AFM can visualize individual proteins it is actually quite difficult to distinguish one protein from another. A better approach is to tag proteins and fluorescent tags are particularly useful. It has to be established that tagging does not alter the functional properties of the proteins (Gunning et al., 2001). Once this has been shown then fluorescent tagging can be used to visualize network structure. 

Figure 1 Relationships of S with interfacial tension and emulsifying activity of proteins. I, bovine serum albumin 2, /3-lactoglobulin 3. trypsin 4, ovalbumin 5, conalbuntin 6, lysozyme 7, K-casein 8, 9, I0, II, and 12, denatured ovalbumin by heating at 85°C for l, 2, 3, 4, and 5 min respectively 13, 14, 15, 16. 17, and 18. denatured lysozyme by heating at 85"C for l, 2, 3, 4, 5, and 6 min respectively 19, 20, 21, 22, and 23, ovalbumin bound with 0.2, 0.3, 1.7, 5.7, and 7.9 mol of sodium dodecyl sulfate/mol of protein respectively 24, 25, 26, 27, and 28, ovalbumin bound with 0.3, 0.9, 3.1,4.8, and 8.2 mol of linoleate/mol of protein respectively. Interfacial tension measured at corn oil/0.20c protein interface with a Fisher Surface Tensiontat Model 21. Emulsifying activity index calculated from the absorbance at 500 nm of the supernatant after centrifuging blended mixtures of 2 ml of corn oil and 6 ml of 0.5% protein in 0.01 M phosphate buffer, pH 7.4 S initial slope of fluorescence intensity (FI) vs. percent protein plot. 10 /al of 3.6 mM m-parinaric acid solution was added to 2 ml of 0.002 to 0.1% protein in 0.01 M phosphate buffer, pH 7.4, containing 0.002% SDS. FI was measured at 420 nm by exciting at 325 nm. (From Ref. 2. Reprinted by permission.)...

Interfacial fluorescence provides an excellent means of studying protein adsorption. The maximum energy available for excitation is localized within a few hundred angstroms of the surface where most of the protein is concen-... 

Interfacial fluorescence signals did not yield direct information on the amount of adsorbed protein. This lack of quantitation appears to be the major weakness of the method. Calibration studies have been attempted (6), but the ideal solution would be a second, independent quantitative technique such as FTIR or use of unaltered radiolabeled protein. An independent calibration method is now being developed. 

Figure 11.15 Fluorescent protein as a mechanophore at the fibre-epoxy resin interface in self-reporting fibre-reinforced composites, (a) The formation of microdamages promotes interfacial debonding between resin and fibre, therefore causing the protein to unfold and to lose its fluorescence. (b) Confocal fluorescence microscopy image of a damaged glass fibre-eYFP/epo>y composite, (c) Z-stack projection of confocal fluorescence microscopy images of a damaged carbon fibre-eYFP/ epoxy composite. (F yellow fluorescence channel, O overlay of fluorescence and transmission images).

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