Lens culinaris agglutinin

Figure 10.6 shows fluorescence emission spectra of lens culinaris agglutinin (LCA) (a) (Lmax = 330 nm), of inaccessible Trp residues (b) (A.max = 324 nm) obtained by extrapolating to [I-] = oo, and of quenched Trp residues (c) obtained by subtracting spectrum (b) from spectrum (a). The emission maximum of accessible Trp residues is located at 345 nm, a characteristic of emission from Trp residues near the protein surface. Thus, both classes of Trp residues contribute to the fluorescence spectrum of LCA (Albani 1996). The presence of five Trp residues makes the analysis by the modified Stern-Volmer equation very approximate nevertheless, a selective quenching method allows the percentage of accessible fluorophores to the quencher to be determined. 

Albani, J.R. (1996). Dynamics of Lens culinaris agglutinin studied by red-edge excitation spectra and anisotropy measurements of 2-p-loluidinylnaphlhalcnc-6-sulfonale (TNS) and of tryptophan residues. Journal of Fluorescence, 6, 199-208. 

Lens culinaris Agglutinin Dynamics and Binding Studies... 

Albani, J.R., Debray, H., Vincent, M. and Gallay, J. (1997). Role of the carbohydrate moiety and of the alpha 1-fucose in the stabilization and the dynamics of the Lens culinaris agglutinin-glycoprotein complex. A fluorescence study. Journal of Fluorescence, 7, 293-298. 

The fluorescence excitation spectrum characterizes the electron distribution of the molecule in the ground state. Excitation is equivalent to absorption since upon absorption, the molecule reaches the excited state Sn- The fluorescence excitation spectrimi is obtained by fixing the emission wavelength and by running the excitation monochromator. Figure 2.9 displays the fluorescence excitation spectrum of Lens culinaris agglutinin. Fluorescence occurs from the Trp residues of the protein. 

Composed of two a and p chains (MW = 5710 and 20572, respectively), Lens culinaris agglutinin (LCA) is a tetramer p2 with a molecular weight equal to 52570 (Loris et al. 1993). LCA contains five Trp residues, three embedded in the protein matrix (Trp 152p, Trp 19a and Trp 40a) and two near the protein surface ( Trp 53p and Trp 128p). 

Figure 4.15. Determination of the binding constant of a - methyl glucose on Lens culinaris agglutinin. The dissociation constant obtained from the slope of the plot is equal to 4.25 + 1.5 niM, an associaion constant equal to 2.35 x 10 M . The plot shown is from 9 experiments. Xex = 280 nm and Xem = 330 nm. The same data were obtained when the emission wavelength was 310 or 350 nm. This value of the association constant for the glucose-LCA complex is close to that found for the LCA-mannose complexe (Ka 5.6 x 10" M ).

The same studies performed on the 5 Trp residues of the protein Lens culinaris agglutinin yield a thermal structoal fluctuation value equal to -1.45% per °C (Fig. 

When the fluorescence spectrum is recorded as a function of temperature, one can notice that emission bandwidth increase with temperatme is dependent upon the fluorophore environment and the fluorophore itself Figure 4.38 displays the fluorescence emission spectrum of Trp residues of the protein Lens culinaris agglutinin. One can notice that in the range of studied temperatures, a shift to the red was not observed and thus we are far from denaturing temperatures. Also, the emission bandwidth (54 nm) does not change with the temperature. 

Figure 4.39 displays the fluorescence emission spectrum of TNS bound to the protein Lens culinaris agglutinin recorded at different temperatiues. The bandwidth of the spectra increases from 78 to 98 nm upon temperature increase. 

Figure 4.39. Fluorescence spectra of TNS bound to Lens culinaris agglutinin as function of temperature. Xqx 320 nm. Spectrum 1 6.5°C. Spectrum 2 9.5°C. Spectrum 3 2°C. Spectrum 4 15°C. Spectrum 5 17.8°C. Spectrum 6 20.4°C. Spectrum 7 23°C.

Figure 5.2. Fluorescence anisotropy spectra of Lens culinaris agglutinin LCA (squares) and Vida fava agglutinin VFA (triangles) obtained at 20°C. Xem 330 nm. The instrument used is a Perkin-Eliner LS 5B.

The anisotropies of both Trp-residues of apacid glycoprotein measured at 20°C are very close (Table 8.7), (a result identical to that obtained with the Weber method, see Table 8.6) indicating that the two Trp residues are highly mobile. This result is confirmed by the values of the rotational correlation times (3 and 5 ns) lower than the global rotational correlation time of a i-acid glycoprotein. When the Trp residue is embedded in the protein core and does not show any residual motions such as in the Lens culinaris agglutinin, its rotational con elation time will be close to that of the protein and its anisotropy will be higher than that of the surface Trp residue. 

Table 8.7. Comparison of the anisotropies of the two classes of Trp residues of ai-acid glycoprotein and Lens culinaris agglutinin. Measurements were performed at 20°C with the Quenching Resolved Emission Anisotropy method.

We measured recently the anisotropies of the Trp residues buried in the protein core of the lectin Lens culinaris agglutinin (LCA) and of those at its surface. We found that the protein core has restricted motions compared to the protein surface (the values of the anisotropies of the surface and of the buried Trp residues are 0.112 and 0.257, respectively) (Albani, 1996). 

Albani, J. R, 1998, Lens culinaris agglutinin - lactotransferrin and serotransferrin complexes, followed by fluorescence intensity quenching of fluorescein (FITC) with iodide and temperature. Biochimica Biophysica Acta 1425, 405-410. 

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