Tyrosine difference spectra

Most studies have concentrated on those conditions where reversible transitions can be demonstrated. However, at neutral pH the thermal transition temperature is high enough to introduce difficulties. Ribo-nuclease kept at 95° at pH 7 for 20 min is irreversibly denatured both in its spectral properties and enzymic activity (337). Tramer and Shugar showed that RNase inactivated at pH 7.8 by heating for 30 min has normalized all of its tyrosine residues as far as alkaline spectrophoto-metric titration is concerned. However, the magnitude of the acid difference spectrum is unaffected although the midpoint has shifted from pH 2 to 3. 

Cathou et al. (459) found that the Cotton effect near 270 nm in the ORD spectrum of RNase disappeared on interaction with either 2 -CMP or 3 -CMP. The X-ray studies (120) (see Fig. 23) clearly show that no tyrosine residues are in close contact with the substrate. Thus the change in rotatory behavior must reflect either (1) a shift in protein structure on association of the nucleotide or (2) the induction of a Cotton effect of the opposite sign in the bound nucleotide. In the independent spectral and chemical studies of Irie and Sawada (480), the reduced nucleotide 5,6-dihydrouridine-2 (3 )-phosphate, known to interact with the enzyme, showed no difference spectrum. With nucleotides containing... 

The additional effects in the aromatic region of the difference spectrum (250-300 nm) are probably caused by aromatic transitions which are influenced by the redox state of the copper. The shoulder at 270 nm, which occurs in all three proteins, could result from an increase in tyrosine absorption. In this context, it is interesting to recall that Tyr 108 (azurin numbering), which is relatively close to the proposed copper ligands Cys 112 and Met 121, is completely invariant both in azurin and plastocyanin and may therefore be an obligatory constituent of the copper site. 

The disruption of the hydrophobic interior and exposure of the tyrosine residues in the enzyme and alcohol-modified protein is revealed by the appearance of the positive peaks in the region of 276-278 nm and 284 nm in the UV difference spectrum (Figure 3). The subsequent refolding to a helical or other ordered structure is shown by the strong positive peak at 233 nm. 

Model compound and difference spectral studies have been carried out on tyrosine and related compounds by Wetlaufer (1956), Laskowski (1957), Edelhoch (1958), Chervenka (1959), Bigelow and Geschwind (1960), Yanari and Bovey (1960), and Foss (1961) (see also Section VI,Z)). Studies of the fluorescence of tyrosine—activation spectrum, fluorescence emission spectrum, and quantum yield—have been reported by Teale and Weber (1957). The pH-dependence of the fluorescence of tyrosine and related compounds was studied by White (1959) fluorescence-polarization studies from the same laboratory were reported by Weber (1960a) for simple compounds, and for proteins (Weber, 1960b). Teale (1960) has carried out extensive studies on the fluorescence characteristics of a score of proteins. 

Because the 2570 A band of phenylalanine is weak, it is often obscured in proteins by the much stronger tyrosine and tryptophan absorptions. It is occasionally visualized in protein spectra as ripples (fine structure) in the spectral region 2500-2700 A. These ripples can be amplified by the difference spectral technique, as is shown in Fig. 13. A typical phenylalanine difference spectrum, obtained in a comparison of the isoelectric amino acid with a solution of the same concentration at pH 1 is shown in Fig. 12. Difference spectra for phenylalanine in various solvents have been measured by Bigelow and Geschwind (1960), Yanari and Bovey (1960), and Donovan et al. (1961). Fluorescence activation and emission spectra for phenylalanine were measured by Teale and Weber (1957). 

Fig. 9. Spectra of tyrosine in various states of ionization. Above, spectra of isoelectric tyrosine (pH 6), and the divalent anion (pH 13) which is dominated by the phenolate ion. Center, the spectral change from pH 6 to pH 13 represented as a difference spectrum. Bottom, for comparison with center, the difference spectrum generated by measuring tyrosine at pH 6 versus tyrosine at pH 0.5. Note the disparity of ordinate scales for the three levels of the figure. The scale for the bottom figure above 2430 A is on the right. (S. Malik, 1961.)...

Ca + binding affects the environments of the tryptophan and the tyrosine residues. Figure 4 shows the Ca +-induced difference spectrum of 67-kDa protein. 

Figure 5.21. Upper Aromatic region of 500 MHz H-NMR spectrum of the complex between 2,4-diaminopyrimidine and selectively deuterated dihydrofolate reductase, in which the only aromatic protons remaining were the 2 6 -protons of the five tyrosine residues. Lower Difference spectrum showing the NOE effects observed on irradiating resonance in this sample.

Figure 6. (a) Aromatic region of the 360 MHz nmr spectrum of 1 5 X 10 N rlbonuclease A In P2O, pH = 7.0, 38 0, 2000 pulses. The resolution Is Increased by multiplication of the FID by a sine bell, (b) photo-CIWP difference spectrum of BNase A (25 pulses), (c) photo-CIDNP difference spectrum of BNase S (same conditions). Pairs of doublets Indicated by Yl, Y2 and Y3 belong to tyrosines. 

In Fig. 6A and 6B the aromatic region of the normal and the photo-CIDBP difference spectrum of gene-5 protein containing deuterated phenylalanyl residues Is shown. The photo-CIDBP difference spectrum obtained from the gene-5 protein tetranucleotlde complex Is given in Fig. 6C. The tyrosine emission signals are completely quenched. To ensure that the total amount of protein was ccinplexed to the... 

CIDNP difference spectrum of protein tetranucleotlde cosqplex (D) CIDNP difference spectrum recorded after adding N-acetyl tyrosine to sample of spectrum C. 

DNA fragment an excess of tetranucleotlde was present In solution (0.30 mM protein versus 0.46 mM d(pC-G-C-G). To exclude the possibility that the flavin dye Is rendered Inactive by the presence of the tetranucleotlde extra N-ace-tyl tyrosine (0.28 mM) was added to the solution. Under these conditions the emission signal of a free tyrosine appears In the CIDNP difference spectrum (Fig. 6D). This observation makes an Inactivation of the flavin dye by the presence of the tetranucleotlde very unlikely. In a separate experiment it was shown that the tetranucleotlde does not show any CIDNP effects. Thus the experiments presented in Fig. 6 demonstrate that upon binding of the tetranucleotlde the hydroxyl groups of the tyrosines 26, 41 and 56 are no longer available for reaction with the flavin dye. This we interprets to mean that these tyrosines are Involved in the protein-DNA interaction. The present re-... 

There is also spectroscopic evidence about the properties of the interior of CA. Thus the spectrum of 4-tert-butylphenol complexed with cyclohexaamylose (C6A) in aqueous solution was superimposable on that of the phenol in dioxane (29), thus indicating the dioxane-like interior of the C6A cavity. Further, the difference spectrum between an aqueous mixture of C7A and N-acetyl tyrosine ethyl ester (ATEe) and ATEe in water alone was essentially identical to that between ATEe in ethyleneglycol and ATEe in water (35), which also underlines the similarity between the CA cavity and the structure of Sephadex. 

Unfortunately, it will be difficult to detect histidyl difference spectra in proteins because of the high optical densities in the low wavelength region of the histidyl difference spectrum caused by tyrosine, tryptophan, and phenylalanine. 

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...

The difference spectrum of unfolded versus folded proteins in general indicates several minima which reflect the variations of the environment of the different chromophores. Figure 6.1 shows an example of perturbation of amino acids by a change of their environment. Tryptophan has a contribution with a first minimum at 292 nm and a second at 284 nm for tyrosine the main peak is at 286 nm, the other at 278 nm for phenylalanine, there are different peaks, the main at 253 nm, the others at 260, 265, and 270 nm. Table 6.1 gives the variation in molar extinction coefficient at the wavelength of the main peak for various chromophores. There are small variations in these values for particular proteins. 

In the solid, dynamics occurring within the kHz frequency scale can be examined by line-shape analysis of 2H or 13C (or 15N) NMR spectra by respective quadrupolar and CSA interactions, isotropic peaks16,59-62 or dipolar couplings based on dipolar chemical shift correlation experiments.63-65 In the former, tyrosine or phenylalanine dynamics of Leu-enkephalin are examined at frequencies of 103-104 Hz by 2H NMR of deuterated samples and at 1.3 x 102 Hz by 13C CPMAS, respectively.60-62 In the latter, dipolar interactions between the 1H-1H and 1H-13C (or 3H-15N) pairs are determined by a 2D-MAS SLF technique such as wide-line separation (WISE)63 and dipolar chemical shift separation (DIP-SHIFT)64,65 or Lee-Goldburg CP (LGCP) NMR,66 respectively. In the WISE experiment, the XH wide-line spectrum of the blend polymers consists of a rather featureless superposition of components with different dipolar widths which can be separated in the second frequency dimension and related to structural units according to their 13C chemical shifts.63... 

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