Lysozyme difference spectra

Fig. 5. pH-Dependence of the (lysozyme + hexa-NAG)-Iysozyrae difference spectrum in 40% methanol at -20 C. 

Fig. 32. IR spectrum of dry lysozyme and difference spectrum for 0.07 k at 27°C. (Top) Dry sample (bottom) difference spectrum at 0.07 h. From Careri et al. (1979b).

Figure 3.X The methyl region of a 270 MHz HNMR spectrum of human leukaemia lysozyme (5 mM, 55°C). The upper trace is the normal spectrum, the lower trace (a) is a convolution difference spectrum. Note the additional fine structure present in (a) which aids...

The photo-CIDNP difference spectrum of 2 mM lysozyme in D2O in the presence of 50 mM NAG is presented in Figure 9a. The spectrum of Figure 9b was taken under exactly the same conditions but in the absence of NAG. Remarkably, an einhanaement of the CIDNP intensities occurs in the presence of NAG. Furthermore, the polarized lines are shifted. This can be clearly seen in Figure 9c, which is the difference of spectrum (a) (at half the vertical scale) and spectrum (b). The most pronounced shifts occur for the lines of Trp 62 an upfield shift for the C-2 H at 7.1 ppm, a splitting of the B-CH2 protons at 1.9 ppm and upfield shift of the a-CH at 4.5 ppm. The emission of this latter proton is also present in Figure 8c, but at the higher temperature it is obscured by the HDO line. 

Fig. 2. Low-field region of the proton decoupled natural abundance spectrum of HEW Lysozyme (a) 30 °C, 53000 transients (b) 20 °C, 76000 scans (c) difference spectrum obtained by subtraction of normalized free induction decays of b and a followed by Fourier transformation. See details under Materials and Methods .

Further information about the abnormal carboxyl groups in lysozyme has been obtained from studies of its ultraviolet difference spectrum. Unlike insulin (Laskowski et al., 1960a) and ribonuclease (Scheraga, 1957), where the difference spectrum is due to perturbations of the tyrosyl chromo-phore, there is no difference spectrum arising from perturbations of the tyrosyl groups of lysozyme by acid treatment. Instead, the acid treatment produces a difference spectrum in lysozyme which is due to perturbation of the tryptophan chromophore. 

A comparison of the difference spectrum of lysozyme (Fig. 152) with that of tryptophan or glycyltryptophan (Figs. 119 and 120) shows that the difference spectrum of the protein appears to be due entirely to the indole chromophore below pH 7. Neither the peaks at 280 and 287 m i, characteristic of the phenolic chromophore, nor the peaks near 260 m/i, characteristic of the benzene chromophore, are observed. These may possibly be masked by the indole spectrum. Therefore, it is not possible to obtain information about the abnormal tyrosyl groups from the difference spectrum. 

Fig. 152. The ultraviolet difference spectrum of 0.23% solutions of lysozyme at 25°C. and ionic strength 0.15. Sample pH 5.04 reference pH 1.15 (Donovan et al., 1961).

Fig. 153. The pH dependence of the difference spectrum of 0.24% (1.6 X 10 M) solutions of lysozyme at 0.15 ionic strength. Reference pH s are approximately 1.2. Open circles,25 C. filled squares, 1 C. Open squares, the pH dependence of the tyrosine ionization in the methylated lysozyme. Dashed curve is a calculated difference spectrum for tyrosine, with p i t 10.80 for the three groups, and 0.080 for w. The solid curves are calculated difference spectra for the following groups 1 group with pKi t 4.20, = 0, AD = 0.070 1 group with pjTint 6.85, Aff = 3 kcal., AD = 0.060 3 groups with pKint 10.80, AD = 6 kcal., AD s= 1.10...

Figure 2 shows the DCDR spectra of lysozyme fibrils (a), native lysozyme (b), and their difference spectrum (c). As can be seen in the fibril spectrum, there is a considerable relative intensity increase and shift of the amide I ( 1660 cm ) and amide III ( 1260 cm ) bands. These blue and red shifts of the amide I and amide 111 bands, respectively, are consistent with an increase in p-sheet... 

Another example of protein-ligand binding is shown in Figure 8, which compares the DCDR spectra of the human serum albumin-suramin complex (a) and the human serum albumin control (b). The resulting difference spectrum (c) clearly reveals an intensity increase broad peak at around 1333 cm as well as other peaks that clearly resemble those of the suramin control sample (d). In this case, as with the lysozyme-nitrate complex, there is again no evidence of significant suramin induced protein secondary structure change. 

A wide range of reversible adsorption kinetic rates was also found by TIR/FRAP for another protein, lysozyme, on a substrate with a different surface charge, alkylated silicon oxide.(61) It is possible that the wide range of rates results from a spectrum of surface binding site types and/or formation of multilayers of adsorbed protein. 

Ruggiero et al. (1986) measured the ESR spectra of samples of lysozyme, myoglobin, and hemoglobin with covalently bound spin labels and noncovalently bound spin probes, in solution and in the partially hydrated powder, over the temperature range 120—260 K. The several proteins behaved similarly. The solution samples differed from the powders in showing a change in spectrum shape at 210 K, understood to represent freezing of water in the hydration shell. 

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