Tryptophan ultraviolet absorption

A number of investigators have studied the effect of ozone on the ultraviolet absorption spectra of proteins and amino acids. A decrease in the absorption of 280-nm light in a number of proteins was originally reported ly Giese et aV to be a consequence of ozone exposure they suggested that this was due to an interaction of ozone with the ring structures of tyrosine and tryptophan. Exposure of a solution of tryptophan to ozone resulted in a decrease in 280-nm absorption, whereas the extinction coefficient of tyrosine increased. Similar results with tyrosine were reported by Scheel et who also noted alterations in the ultraviolet spectra of egg albumen, perhaps representing denaturation by ozone. 

Amino acids do not give any very useful ultraviolet absorption spectra unless they possess aromatic groups as in phenylalanine, tryptophan, and tyrosine. The absorption characteristics of these groups are more useful in monitoring chemical and conformational changes in proteins than they are in the simple amino acids. 

Ultraviolet absorption spectra of tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) at pH 6. The molar absorptivity is reflected in the extinction coefficient, with the concentration of the absorbing species expressed in moles per liter. (Source From D. B. Wetlaufer, Adv. Protein Chem. 17 303-390, 1962.)... 

Ultraviolet Absorption The ultraviolet absorption characteristics of the aromatic side chains of the primary amino acid sequence are often used to estimate an extinction coefficient for a protein. The extinction coefficient is calculated based upon the number of tyrosine, tryptophan, and phenylalanine (aromatic) residues in the protein. Phenylalanine has a relatively weaker absorptivity and may not be included in the calculations. One advantage of this approach is the fact that it is relatively nondisruptive, leaving protein conformation undisturbed. In addition, the same solution used for the concentration assessment can be removed from the cuvette and used for additional experiments. The measurement is based upon Beer s law ... 

The application of low-temperature techniques to the investigation of protein spectra in the ultraviolet region was initiated by Lavin and Northrop (1935) who investigated the ultraviolet absorption spectra of pepsin, serum albumin, and ovalbumin in glycerol, and showed that the fine structure of the protein spectrum was enhanced at — 100°C. Preliminary reports of similar work have been published by Randall and Brown (1949) on thin films of sublimed tryptophan and phenylalanine at 90°C., and by Sinsheimer et al. (1949) for tryptophan at 77.6°K. Loof-bourow and his coworkers (Sinsheimer et al., 1950) have begun publication of a series of papers reporting much more comprehensive work on the influence of low temperature on the spectra of amino acids and proteins in thin films and in solid solution. Beaven et al. (1950) have reported a few results on thin Aims of the aromatic amino acids. 

For human serum albumin Tanford (1950) found by spectrophotometry that the ionization of the tyrosine hydroxyl groups was completely reversible up to pH 12. Measurements at the wavelength of the tyrosine anion maximum (2930 A.), uncorrected for the small tryptophan contribution, gave a pK of 11.7 for this process. Both the ultraviolet absorption and titration data for this protein could be quantitatively interpreted on the basis of complete freedom of all the 18 tyrosine hydroxyl groups in the molecule to ionize. In this respect human serum albumin thus resembles insulin and not ovalbumin. 

Fig. 2. Ultraviolet absorption spectra of tryptophan, tyrosine, and phenylalanine at pH 6 (from Wetlaufer >).

Fig. 6. Ultraviolet absorption spectra of phenylalanine and tryptophan (a) and tyrosine (pH 7 and pH10)(b)...

Assays for melanocyte-stimulating hormone were carried out by Mr. S. Kulovich using the in vitro frog skin assay (1). Subcutaneous assay for adrenocorticotropic activity (2), based on ascorbic acid depletion in hypophysectomized rats, was performed by Dr, J.D. Fisher of the Armour Laboratories. Acid hydrolysates (constant boiling HCl, deaerated, 22 hours, 110°) of these fractions were characterized by automatic amino acid analysis with a Spinco model 120B analyzer. The number of tryptophan residues in the intact peptide was estimated from the ultraviolet absorption curves made with a Cary model 15 spectrophotometer. Electrophoresis was carried out on Whatman paper No. 1 with pyridine-acetate buffer, pH 6.5, and 4 molar urea for 3 hours at 26 volts per cm. Peptides were detected with bromphenol blue(3). 

Auer, H. E. Far-Ultraviolet Absorption and Circular Dichroism Spectra of L-Tryptophan and Some Derivatives. J. Amer. Chem. Soc. 95, 3003-3011 (1973). 

Side chains of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan absorb ultraviolet light in the 240- to 300-nm region, while histidine and cystine absorb to a lesser extent. Figure 3-13 shows the absorption spectrum of a "reference compound" for tyrosine. There are three major absorption bands, the first one at 275 nm being a contributor to the well-... 

The aromatic amino acids phenylalanine, tyrosine, and tryptophan all possess absorption maxima in the near-ultraviolet (fig. 3.7). These absorption bands arise from the interaction of radiation with electrons in the aromatic rings. The near-... 

Figure 4. Ultrafast hydration correlation function c(t) of tryptophan. The c(t) can be fit with a stretched biexponential model as shown. The inset shows the fluorescence anisotropy dynamics of tryptophan after ultrafast ultraviolet (UV) absorption. The internal conversion between La and Lb states occurs within 80 fs. The free rotation time of tryptophan in bulk water is 46 ps.

Among the properties of amino adds that are most pertinent to the biomedical scientist are their optical rotations, already discussed, which are listed for each amino acid in Table 4.1. Note the dramatic differences between optical rotations in the zwitterionic (water) and fully protonated (HC1) forms. Further, all amino acids absorb ultraviolet light in the range 190-220 nm. The C=0 bond in carboxyl residues is largely responsible. Moreover, aromatic amino acids, especially tryptophan, absorb in the 260-285 nm range. Protein concentrations in solutions are often determined via absorption at 210 or 280 nm. 

The ultraviolet spectra of many proteins exhibit small shifts of absorption maxima to shorter wavelengths and small decreases in extinction coefficient at these maxima when the native conformations are disrupted in aqueous solution (see Beaven and Holiday, 1952 Beaven, 1961 and the article by Wetlaufer in this volume). These effects are presumably due to the special environments in which chromophoric side chains, particularly those of tyrosine and tryptophan, find themselves within the native protein molecule as compared to the free chromophores in aqueous solutions. The special properties of these native environments have been attributed variously to hydrogen bonding, particularly of tyrosine residues (Scheraga and Laskowski, 1957), vicinal electrical effects, including ion-... 

The absorption of ultraviolet light by proteins at wavelength 280 nm is caused mostly by the amino acids tyrosine and tryptophan along the protein molecular chains. The molecular absorption coefficients for these two amino acids are ... 

Direct Photometric Methods. Absorption of ultraviolet (UV) light at 200 to 225 nm and 270 to 290 nm is used to measure the protein content of biological samples. Absorption of UV light at 280 nm depends chiefly on the aromatic rings of tyrosine and tiyptophan at pH 8. Accuracy and specificity suffer from an uneven distribution of these amino acids among individual proteins in a mixture and from the presence in body fluids of free tyrosine and tryptophan, uric... 

From the standpoint of utilizing this effect it is fortunate that many coenzymes, prosthetic groups of enzymes, and substrates contain identifiable absorptions in the near ultraviolet as do the aromatic side chains of amino acids. Thus, if one could titrate a coenzyme or substrate onto an enzyme and note this type of reciprocal relations involving a transition in the enzyme, for example in a tryptophan or tyrosine group, and an absorption band in the group being added, then one would have identified an interaction at the active... 

Apart from this the interest and application of ultraviolet spectra of proteins are analytical. On a microscale the absorption spectrum may be the simplest and best evidence for the recognition of a protein. It is possible that, with care, it will be the best means of obtaining an estimate of tyrosine and tryptophan in a protein. The instability of tryptophan under the conditions required for protein hydrolysis gives weight in favor of a method such as the spectrophotometric which allows a direct determination of tryptophan to be made (on a protein) without hydrolysis. 

The main features of the near- and far-ultraviolet spectra of the proteins are related to the absorption properties of the aromatic amino acids phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), and histidine (His) [113,114]. The peaks observed in the absorption spectra up to 185 nm (6.70 eV) can be assigned to the excited states of the chromo-phore-acting molecules benzene, phenol, indole, and imidazole, respectively. In the present section we focus on the theoretical description of the most representative valence singlet excited states of the aromatic amino acid chromophores. As the results for benzene and phenol have been recently described [13, 46], only the results for indole, [(4) in Fig. 3] and imidazole [(5) in Fig. 3] are reviewed here [115,116]. The theoretical results support the assignment of four valence singlet states as... 

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