Proteins derivative spectra

A cofactor can be extracted from the iron-molybdenum protein, using Af-methylformamide. This cofactor (called FeMoCo) has many spectroscopic properties in common with the native protein, especially the EXAFS spectrum, and activates the inactive large protein derived from Azobacter vinelandii UW45 mutant which cannot incorporate molybdenum. The cofactor contains no protein or peptide, but does contain molybdenum, iron, and sulfur in atomic ratios of 1 6-8 4-9. It is believed to contain the dinitrogen-binding site (presumably molybdenum) but there is no definitive proof of this. 

Fig. 13 indicates spectra of various samples of serum albumin treated with 2-mercaptoethanol or ferrous salt, or both. The sample treated with 2-mercaptoethanol alone contains no iron and no labile sulfur, whereas the sample treated with ferrous salt alone contains iron but not labile sulfur. Further, the sample treated with both 2-mercaptoethanol and ferrous salt exhibits a visible absorption and it contains 81.3 mp.-atoms of iron per mg of protein and 82.4 mjxmoles of labile sulfur per mg of protein. The spectrum of methylene blue derived from labile sulfur in the artificial iron protein by Lath s reaction is identical with that derived from a standard solution of Na2S or from native adrenodoxin. 

For example, ESI/MS allowed the analysis of various proteins derived from the HIV virus and obtained by genetic engineering, such as the pl8 protein [116]. Beyond the main peak series corresponding to the protein with a measured molecular mass of 14 590 Da (as opposed to a calculated molecular mass of 14 589 Da), the spectrum shown in Figure 8.20 contains two other series. The first minor series (8 %) indicated by the letter T and giving a measured mass of 12651 Da corresponds to the C-terminal side of pl8 cleaved at position 111-112, whereas the second series (3 %) indicated by the letter D with a measured mass of 29 175 Da corresponds to the dimer formed by the linkage of two cysteines of two different proteins. 

Such derivative techniques can reveal small changes in spectra and hence in protein secondary structure which will be picked up in the second derivative spectrum of the protein as gain or loss of characteristic band intensities (Susi and Byler, 1983 Alvarez et al., 1987). 

The electronic spectrum of the B2 subunit ( ax = 455, 485, and 615 nm) closely resembles those of Mn-catalase and synthetic tribridged Mn 0 complexes (8). The metal site was thus proposed (229) to be analogous to the diiron center of the enzyme from E. coli. This analogy may be reasonable as iron restores 50-70% of the activity in protein derived from Mn-deprived cells (230). Similar to the enzyme from E. coli, the Mn-containing ribonucleotide reductase is inhibited by hydroxyurea and au-... 

The second derivative spectra of tyrosine and tryptophan are flat bet veen 245 and 270 nm, while the spectrum of phenylalanine shows its characteristic spectral bands (Xniax 249, 256, 260, 262, 266 and 270 nm and A trough 247, 252, 258, 261, 264 and 268 nm bet veen 245 and 270 nm). Ichika va and Terada (1977) used the spectral properties of the second derivative spectrum bet veen 245 and 270 nm to study phenylalanine in proteins. 

In fact, the authors found that in different proteins, the second derivative spectra below 270 nm were essentially the same as the second derivative spectrum of phenylalanine (Ichikawa and Terada, 1979). The absorption at certain peaks and troughs of the second derivative spectrum of phenylalanine are found dependent on the microenvironment of the phenylalanine. Denaturation of the proteins by urea or guanidine modifies the intensities of the spectral bands of phenylalanine without inducing a significant shift in their positions. Table 1.3 shows the difference between peaks and troughs in the second derivative spectrum of phenylalanine under various conditions and Table 1.4 shows the difference observed for four proteins in the native and denatured states. 

Other studies performed on the second-derivative spectrum (Ragone et al. 1984) have shown that the ratio r between two peak to trough distances (287-283 and 295-290) was related to the tyrosine / tryptophan ratio and was found dependent on the surrounding medium polarity of tyrosine. Table 1.5 shows the values of r for different proteins measured in the native (r ) and denatured (r ) states. We can notice that r increases when the proteins are denatured, i.e., tyrosines are much more exposed to the solvent in the denatured state. The increase of r with the protein deiiaturation is in principle dependent on the tyrosines locations in the proteins in the native state. 

Figure 1.36 displays more detailed parts of the second derivative spectrum from 245 and 270 nm (a) and from 270 to 300 nm (b). One can notice that the general feature of the derivative spectra corresponds very well with those of the three amino acids (Tip, Tyr and Phe), responsible for the protein absorption. 

Fig.3 Room temperature absorption spectrum of the isolated pigment-binding protein. The inset shows the second derivative spectrum.

To obtain this detailed structural information, it is necessary to enhance the resolution of the protein amide I band, which usually appears as a single broad absorbance contour (Figure 1). The widths of the overlapping component bands are often greater than the separation between the absorbance maxima of neighboring bands. Because the band overlapping is beyond instrumental resolution, several mathematical band-narrowing methods (i.e., resolution enhancement methods) have been developed to overcome this problem [11,50-52,54]. For studies of lyophilization-induced structural transitions, calculation of the second derivative spectrum is recommended [11]. This method is completely... 

An illustration of the procedure to compute a second derivative of a spectrum is shown in Figure 10.7. A single Lorentzian band is transformed to the Fourier domain, where the signal is multiplied by a parabolic function. Upon transformation back to the spectral domain, the second derivative spectrum is shown. The FWHH of the second derivative band is approximately 2.5 times smaller than that of the original spectrum. Examples of second derivatives of protein spectra are shown in Figure 10.8. 

Neural networks have been applied to IR spectrum interpreting systems in many variations and applications. Anand [108] introduced a neural network approach to analyze the presence of amino acids in protein molecules with a reliability of nearly 90%. Robb and Munk [109] used a linear neural network model for interpreting IR spectra for routine analysis purposes, with a similar performance. Ehrentreich et al. [110] used a counterpropagation network based on a strategy of Novic and Zupan [111] to model the correlation of structures and IR spectra. Penchev and co-workers [112] compared three types of spectral features derived from IR peak tables for their ability to be used in automatic classification of IR spectra. 

Figure 5.19 MALDI-ToF mass spectrum, providing a molecular-weight profile of the tryptic peptides derived from spot 22 (see Figure 5.18) of the silver-stained two-dimensional gel of the proteins extracted from the yeast S. cerevisiae. From Poutanen, M., Salusjarvi, L., Ruohonen, L., Penttila, M. and KaUddnen, N., Rapid Commun. Mass Spectrom., 15, 1685-1692, copyright 2001. John Wiley Sons Limited. Reproduced with permission.

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