Tryptophan absorbance

Amino acids do not absorb visible hght and thus are colorless. However, tyrosine, phenylalanine, and especially tryptophan absorb high-wavelength (250—290 nm) ultraviolet light. Tryptophan therefore makes the major contribution to the abihty of most proteins to absorb hght in the region of 280 nm. 

Detection and quantification of protein by measuring absorbency at 280 nm is perhaps the simplest such method. This approach is based on the fact that the side chains of the amino acids tyrosine and tryptophan absorb at this wavelength. The method is popular, as it is fast, easy to perform and is non-destructive to the sample. However, it is a relatively insensitive technique, and identical concentrations of different proteins will yield different absorbance values if their content of tyrosine and tryptophan vary to any significant extent. Hence, this method is rarely used to determine the protein concentration of the final product, but it is routinely used during downstream processing to detect protein elution off chromatographic columns, and hence track the purification process. 

The side chain of selected amino acids (particularly tyrosine and tryptophan) absorbs UV at 280 nm Peptide bonds absorb UV at 190-220 nm... 

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 chromophores tyrosine and tryptophan absorb radiation in the 280 nm region, re-emitting a proportion of it as fluorescence. The quantum yields for tryptophan, tyrosine, and phenylalanine are 0.2,0.1, and 0.04, respectively. Coupled with their relative absorption coefficients (unit bi.3), it can be seen why most protein fluorescence spectra are dominated by tryptophan. 

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. 

Although thermostability experiments using tryptophan absorbance as the probe give fairly reproducible Tm values, the thermodynamic parameters such as free energy, enthalpy, entropy, and heat capacity cannot be obtained directly. Calorimetric measurement provides the most complete picture of the thermodynamic process of protein unfolding. This technique, however, requires more material and is more time consuming. 

Cystine and the aromatic amino acids, e.g., tyrosine, phenylalanine, and tryptophan, absorb radiation at wavelengths greater than 254 nm (124). Tyrosine and tryptophan inhibit the photodecomposition of pyridoxine (84). 

The eluant is passed through an optical detector to monitor the elution of the sample components. Again, observation wavelengths of 214-230 nm are useful in that they detect the carbonyl group of the amide bond. Tyrosine and tryptophan absorb at 280 nm, so this wavelength is useful for peptides having these residues. 

Phe) is dominated by the tryptophan absorbance, whereas ribonuclease (RNase 0 Trp, 6 Tyr, 3 Phe), which (unusually) contains no tryptophan residues, is more characteristic of the tyrosine side chains... 

Absorption studies of amino acids in the UV region go back to the mid-thirties [15]. An overview on this subject appeared as early as 1952 [16]. Only the aromatic amino acids, phenylalanine, tyrosine and tryptophan, absorb in a conveniently observed region (Fig. 6). The weakest of them is phenylalanine, revealing at least 6 absorption bands it has a molecular absorption coefBcient of fimoi 0.195 X 10 at 257 nm. The UV-spectrum of tyrosine is strongly dependent on pH in the presence of 0.1 N HCl (protonated hydroxyl group) Sj oi = 1-34 x 10 at the maximum, (l x 275 nm) in 0.1 N NaOH (phenolate) = 2.33 x 10 at 293 nm. The absorption of tryptophan, is nearly independent of the pH it shows a maximum at 280 nm with = 5.55 x 10 and a second maximum (shoulder) at 288 nm of = 4.55 x 10. These parameters have often been used in the quantitative analysis of peptides. The sulfur-containing side chains show weak absorption below 250 nm and are of interest only in rare special cases. 

Ultraviolet (UV) detectors are, by far, the most commonly used detectors for peptide analysis. Peptide bonds absorb light strongly in the far ultraviolet (<220nm Fig. 1 [1]), providing a convenient means of detection (usually at 210-220 nm). In addition, the aromatic side chains of tyrosine, phenylalanine, and tryptophan absorb light in the 250- to 290-nm ultraviolet range it should be noted, however, that these aromatic residues arc not present in all peptides. 

Aromatic amino acids such as phenylalanine, tyrosine and tryptophan absorb in the UV-range of the spectrum with absorption maxima at 200-230 nm and 250-290 nm (Fig. 1.4). Dissociation of the phenolic HO-group of tyrosine shifts the absorption curve by about 20 nm towards longer wavelengths (Fig. 1.5). 

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