Analysis as a sum of Gaussian bands

In this analysis, we have proposed for each spectrum, one, two or three maxima and we asked the program to draw the best Gaussian curves we can obtain from our spectra. 

The best fit was obtained with the sum of two bands. At low concentration of calcofluor, one band is obtained at a maximum of 326 nm and a bandwidth of 23 nm. The second band has a maximum at 347 nm and a bandwidth of 40 nm (Fig. 8.29). Although the positions of the maxima are significant and correspond to those found for the hydrophobic and surface Trp residues, respectively (Fig. 8.26), the values of the bandwidths do not correspond to those known for Trp residues (48 and 59 nm for the hydrophobic and surface residues, respectively). This means that it is difficult to separate the real spectra one from each other, i.e., in presence of low calcofluor concentration, a i-acid glycoprotein has kept its folded structure. 

Spectrum (d) shows the sum of spectra (c) and (b). This spectrum overlaps the original spectrum (a) indicating how well the two components fit the original spectrum. 

Performing the fits for the spectrum obtained at high concentrations of calcofluor, yields also two bands (Fig. 8.30). One has a maximum at 324 nm and a bandwidth of 28 nm. The second shows a maximum at 349 nm and a bandwidth of 53 nm. We notice that only the bandwidth of the second band is close to that of fluorescence spectrum of Trp residue near the protein surface. Thus, binding of calcofluor at a concentration much higher than that of the protein, induces a modification in the fluorescence spectrum of the surface Trp residue. This modification seems not to be observed for the hydrophobic Trp residues. 

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