Tyrosine absorptivity

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. 

Of course, the reason for the improvement in the calibration model when the second term is included is that A21 serves to compensate for the absorbance due to the tyrosine since X21 is in the spectral region of a tyrosine absorption band with little interference from tryptophan. Figure 6. In general, the selection of variables for multivariate regression analysis may not be so obvious. 

Thus, knowledge of the transition moment direction of a phenol band could help in interpreting the fluorescence spectrum of a tyrosine chromophore in a protein in terms of orientation and dynamics. The absorption spectrnm of the first excited state of phenol was observed around 275 nm with a fluorescence peak aronnd 298 nm in water. The tyrosine absorption was reported at 277 nm and the finorescence near 303 nm. Fluorescent efficiency is about 0.21 for both molecules. The fluorescent shift of phenol between protic and aprotic solvents is small, compared to indole, a model for tryptophan-based protein, due to the larger gap between its first and second excited states, which resnlts in negligible coupling . ... 

The bands at 384 and 331 nm in the metchloro derivative must also be attributable to some kind of electronic transition involving one or both of the Fe(III) ions. Their positions and intensities can be rationalized if they are assigned to SPE excitations in an oxobridged dimer. Alternatively, ligand Fe(III) charge transfer transitions are also possible explanations. At wavelengths lower than 331 nm, the tyrosine absorption takes over and additional peaks owing to Fe(III) are presumably buried. 

One additional spectral feature to be pointed out is the accessible wavelength range possible with the diamond OTEs. The spectrum shows that the region between 200 and 400 nm contains two absorption bands, the N and L bands, which, like the Soret and a-bands, are attributed to -it IT transitions of the heme Fe [183]. Many characteristic amino acid absorption transitions occur below 400 nm, such as tyrosine absorption at 280 nm. This range has been exploited in spectroelectrochemical measurements of ferrocene at diamond OTEs [127]. 

Important structural transitions occur in extreme pH values, i.e., below pH 4.0 and above pH 10.5, The reversibility of the transition which occurs between pH 3 and 4 was demonstrated by following different parameters (e.g., fluorescence of the unique tryptophan, fluorescence of tyrosine, tyrosine absorption, molar ellipticity, and reduced viscosity). The same transition curve is observed with all parameters, with a midpoint corresponding to pH 3.9 (Fig. 5.5). 

Fig. 5,5. Reversible acid induced transition of staphylococcal nuclease observed by different methods (a) emission fluorescence of Trp 140 (according to Epstein et aL, 1971a) (b) changes in reduced viscosity ( , ) and molar ellipticity (A A) at 220 nm ( , A) measurements made on decreasing pH ( , A), measurements made upon increasing pH (from Anfinsen et aL, 1972) (c) emission fluorescence of Trp 140 ( ), tryrosine ( ), and tyrosine absorption at 287 nm ( ) (from Anfinsen et aL, 1972) (d) areas of imidazole C2 proton resonances of each of the four histidine H-1, H-2, H-3, H-4 (from Anfinsen et aL, 1972).

Procedure. The method can be tested using the matrix of concentrations, in micromoles per liter (pmol L ), of tryptophan and tyrosine at 280 nrrr suitably rrrodified to take into account constant absorption at 280 nrrr of some absorber that is neither tryptophan nor tyrosine... 

Absorption of proteins in the 230-300 nm range is determined by the aromatic side chains of tyrosine (Xmax = 274 am), tryptophan (Xmax = 280 nm), and phenylalanine (Xmax = 257 nm). Because the difference in the absorption spectra of native and unfolded protein molecules is generally small, difference spectra can... 

Tyrosine contains a phenolic side chain with a pKa of about 9.7-10.1. Due to its aromatic character, tyrosine is second only to tryptophan in contributing to a protein s overall absorptivity at 275-280nm. Although the amino acid is only sparingly soluble in water, the ionizable nature of the phenolic group makes it often appear in hydrophilic regions of a protein—usually... 

Protein peroxidation Modified tyrosines GC/MS, HPLC, immunoassays Protein carbonyls Atomic absorption spectroscopy, fluorescence spectroscopy, HPLC... 

L-Amino acid oxidase has been used to measure L-phenylalanine and involves the addition of a sodium arsenate-borate buffer, which promotes the conversion of the oxidation product, phenylpyruvic acid, to its enol form, which then forms a borate complex having an absorption maximum at 308 nm. Tyrosine and tryptophan react similarly but their enol-borate complexes have different absorption maxima at 330 and 350 nm respectively. Thus by taking absorbance readings at these wavelengths the specificity of the assay is improved. The assay for L-alanine may also be made almost completely specific by converting the L-pyruvate formed in the oxidation reaction to L-lactate by the addition of lactate dehydrogenase (EC 1.1.1.27) and monitoring the oxidation of NADH at 340 nm. 

The aromatic amino acids each have two major absorption bands in the wavelength region between 200 and 300 nm (see reviews by Beaven and Holiday(13) and Wetlaufer(14). The lower energy band occurs near 280 nm for tryptophan, 277 nm for tyrosine, and 258 nm for phenylalanine, and the extinction coefficients at these wavelengths are in the ratio 27 7 l.(14) As a result of the spectral distributions and relative extinction coefficients of the aromatic amino acids, tryptophan generally dominates the absorption, fluorescence, and phosphorescence spectra of proteins that also contain either of the other two aromatic amino acids. 

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