Tryptophan fine structure

Comparison of these results with the data for the fine-structure band positions in proteins (Table II) indicates that peptide combination alone is not sufficient to account for the longwave shifts found for proteins, which in some cases, e.g. pepsin, may exceed 30 A. for the tryptophan fine structure maximum. There is some evidence that the protein macromolecule itself constitutes an environment which also influences the position of the fine-structure features. The observations in this connection for proteins are discussed below, but the following results (Beaven et al., 1950) for the free amino acids and a peptide are also suggestive. 

Position of tryptophan fine-structure band in mixtures of adult and fetal hemoglobin. 

In studies of the recombination of heme with globin Jope,. lope and O Brien (1949) have used the concept of bound tyrosine hydroxyl groups as a criterion of native character in globin, based on the spectropho-tometric study of the alkaline ionization process. They were able to show that globin preparations which were native according to this criterion could be classified as denatured on the basis of the position of their tryptophan fine-structure bands when recombined with heme. These results led them to suggest that the process of denaturation could be separated into several stages, even by spectroscopic techniques alone (see also Jope, 1949). 

Inspection of the absorption curves of proteins in alkali shows that the estimate of shift should be possible. In Fig. 11 are shown for comparison curves of various mixtures of tyrosine and tryptophan in V/10 alkali together with curves of some proteins under the same conditions. It can be seen at once that a fair estimate of the tyrosine/tryptophan ratio may be obtained by inspection of the head of the band. By the moving-plate method the position of the tryptophan fine-structure band, which forms one maximum of the curve can be estimated to +1 A. By standard photoelectric spectrophotometry it can be estimated to +2-5 A. 

The two transitions for tryptophan exhibit distinct features, which lead to quite different CD bands. The La transition is broad, relatively featureless, and intense (Fig. B3.5.2). The Lb transition is weaker but exhibits fine structure (Fig. B3.5.2) similar to that of tyrosine. It can be masked by or superimposed on the La transition. Interactions similar to those listed for tyrosine strongly affect the peak wavelengths and intensities. The presence of bands above 285 nm is diagnostic for tryptophan in a specific environment interactions that cause the Lb transition to be shifted to the red (or high-wavelength) end of the spectrum result in bands as high as 310 nm which are highly conformation-specific. The two transitions may be affected quite independently by interactions, and their combination can thereby result in four distinct types of spectrum (Strickland, 1974). 

Disulfide bonds may exhibit a broad band of ellipticity in the range of 240 to 350 nm. This can be confused with the band associated with the La transition of tryptophan but, in cases where a disulfide makes a significant contribution to the CD, it can be recognized by its ellipticity above 320 nm. It can augment or diminish the apparent intensity of tryptophan and tyrosine contributions without being recognized as such and without fine structure necessarily being lost. 

Some of the above features can be seen in the spectra of cathepsin D (Fig. B3.5.10), where the intact single-chain bovine enzyme is compared with the same material cleaved at an exposed loop but without dissociation. The latter has 50% of the specific activity of the intact molecule. Phenylalanine residues can be seen to be present in specific environments in both forms. The fine structure of the Lb transition of tryptophan is superimposed on the broad peak of the La transition, which is apparently more intense in the intact enzyme. Alternatively, there could be a greater contribution from disulfide bonds, but the absence of ellipticity above 320 nm favors the former assignment and the CD is therefore consistent with a limited increase in dynamics of the molecule as a result of the chain... 

The coupling of electronic and vibrational modes of excitation gives rise to discrete narrow absorption bands, which are exemplified in the spectrum of phenylalanine (Fig. 1), where at least six saw-toothed projections appear to emerge from the otherwise smooth contour of the absorption envelope. Such a system of maxima within an absorption band is collectively called the fine structure of the band. Tryptophan... 

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). 

It may be readily detected spectrographically in proteins, even in presence of tyrosine and tryptophan, by means such as the moving-plate method (Holiday, 1937, 1950a). A source giving a continuous spectrum is essential to show up the fine structure bands which reveal its presence. 

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. 

Further investigation of this subject has confirmed dope s prediction and provides an example of the value of fine-structure measurements in protein biochemistry. Beaven et al. (1951) found that in fetal human hemoglobin the tryptophan longwave fine-structure feature is a resolved maximum while in the adult type it is an unresolved inflection, irrespective of the spectral band width used in obtaining the absorption curves. It was also found that the position of this feature, as measured by the... 

From the fact that in adult-type hemoglobin the fine-structure feature is an unresolved inflection while in the fetal type it is a resolved maximum, it was suggested that the differences between these two forms of human hemoglobin may arise from slightly different environmental influences of the globin molecule as a whole in the two types. In the fetal type the majority of the tryptophan residues may be considered to... 

The emission spectra from powders and crystal suspensions of the aromatic amino acids, however, are quite different from those shown in Figs. 3 and 4. The fluorescence and phosphorescence maxima of the powders are red shifted in each case by up to 50 nm and the phosphorescence lifetimes at 77 K are found to be 1.5 sec for tryptophan, 0.4 sec for t5Tosine and 0.5 sec for phenylalanine The phosphorescence lifetimes decrease markedly as the sample temperature increases In the powders the phosphorescence to fluorescence ratios are considerably smaller than in frozen aqueous solutions. The crystal suspensions at room temperature show even greater red shifts in the phosphorescence maxima, with no fine structure observed and with lifetimes on the order of 0.2 sec for each aromatic amino acid >. Although these results along with the phosphorescence excitation spectra bear little resemblence to those expected for the aromatic amino acids, Bogach et al. point out the similarities to phosphorescence and excitation spectra of photoproducts formed in solutions of the aromatic amino acids at low temperatures... 

Fine structure observed in the CT band with tryptophan attributed to tryptophan singlet to triplet transition. 

Many biologically active polypeptides have been shown to possess CD bands in the wavelength region above 270 nm. In peptide hormones and antibiotics and in non-conjugate proteins these bands are due to tryptophan and tyrosine residues, the only chromophores which absorb in this spectral region. Because of the weakness and spectral overlap of these Cotton effects, their structure is usually imperfectly resolved. However, in recent years some progress in analyzing the fine structure of the CD spectra of tryptophan and derivatives has been reported (7 9). 

The near-UV CD fine structure of tryptophan and seven of its derivatives were examined by employing high-resolution spectra recorded at 77° K 389). The spectra may be grouped into four classes i) Lb bands intense, ii) La bands intense, iii) both La and Lb bands intense, iv) fine structure whose origin was not readily identified. Both the 0-0 and 0- - 850 cm Lb transitions occur together (near 290 and 283 nm, respectively) and have the same CD sign. A number of La transitions were identified, but their relative intensities varied greatly. 

Strickland, E. H., J. Horwitz, and C. Billups Fine Structure in the Near-Ultraviolet Circular Dichroism and Absorption Spectra of Tryptophan Derivatives and Chymotrypsinogen A at IT K. Biochemistry 8, 3205-3213 (1969). 

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