Absorption spectra tryptophan

Subsequently, Cross et al. (SOI) demonstrated that formation of the E-NADPH binary complex, and the abortive ternary complexes E-NADPH-L-glutamate and E-NADP-a-ketoglutarate are all characterized by a red shift in the tryptophan absorption spectrum. It appears likely, therefore, that a tryptophan residue is located in or near the coenzyme binding site. 

Set the excitation wavelength at 300 nm (the red edge of the tryptophan absorption spectrum). Excitation at 300 nm will additionally decrease the inner filter effect. Set... 

Fluorescent probes are divided in two categories, i.e., intrinsic and extrinsic probes. Tryptophan is the most widely used intrinsic probe. The absorption spectrum, centered at 280 nm, displays two overlapping absorbance transitions. In contrast, the fluorescence emission spectrum is broad and is characterized by a large Stokes shift, which varies with the polarity of the environment. The fluorescence emission peak is at about 350 nm in water but the peak shifts to about 315 nm in nonpolar media, such as within the hydrophobic core of folded proteins. Vitamin A, located in milk fat globules, may be used as an intrinsic probe to follow, for example, the changes of triglyceride physical state as a function of temperature [20]. Extrinsic probes are used to characterize molecular events when intrinsic fluorophores are absent or are so numerous that the interpretation of the data becomes ambiguous. Extrinsic probes may also be used to obtain additional or complementary information from a specific macromolecular domain or from an oil water interface. 

The case of indole and tryptophan is peculiar because the low-lying absorption bands overlap. Box 5.2 shows how the indole absorption spectrum can be resolved into two bands from the combined measurement of the excitation spectrum and the exdtation polarization spectrum. 

Figure 2.11. The dependence of the position of the fluorescence spectrum maximum on excitation wavelength for tryptophan in a model medium (glycerol) at different temperatures (a) and singletryptophan proteins (b). 1, Whiting parvalbumin, pH 6.S in the presence of Ca2+ ions 2, ribonuclease Th pH 6.5 3, ribonuclease C2, pH 6.5 4, human serum albumin, pH 7.0, +10"4 M sodium dodecyl sulfate 5, human serum albumin, pH 3.2 6, melittin, pH 7.5, +0.15 M NaCl 7, protease inhibitor IT-AJ from Actinomyces janthinus, pH 2.9 8, human serum albumin, pH 7.0 9, -casein, pH 7.5 10, protease inhibitor IT-AJ, pH 7.0 11, basic myelin protein, pH 7.0 12, melittin in water. The dashed line is the absorption spectrum of tryptophan.

The appearance and disappearance of the triplet state can be measured by light emission or by absorption change. The absorption change arises because the ground and triplet states have different absorption spectra. The absorption spectrum of tryptophan in the triplet state is red shifted in com-... 

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-... 

Most proteins have a broad characteristic absorption spectrum centered at about 280 nm. The major absorption is due to the presence of aromatic moieties in the amino acids phenylalanine, tyrosine, and tryptophan. During the a-lactalbumin purification described in this experiment, you will monitor the process by measuring the absorption at 280 nm (A2S0) of column fractions to be sure the experiment is proceeding correctly. You must recognize that you are measuring not the concentration or presence of a-lactalbumin specifically but the total amount of all proteins present. 

UV-Visible Absorption Spectrum Absorption spectroscopy Tyrosine-Tryptophan environments, presence of absorbing ligands or impurities. ... 

Figure B3.5.2 The two near-UV absorbance transitions for tryptophan. The near-UV absorption spectrum for A/-stearyl tryptophan n-hexyl ester dissolved in methylcyclohexane at 24°C (solid line) has been resolved into the 1La and 1Lb bands. (Drawn from Strickland, 1974.)...

Ribonuclease contains no tryptophan. The absorption near 280 nm is almost entirely resulting from the 6 tyrosine residues. The ionization of tyrosine produces a marked shift to longer wavelengths in the absorption spectrum. The ionization can be monitored near 295 nm. Shugar (293) was the first to point out the abnormal behavior of 3 of the tyrosine residues on alkaline titration. Three titrate normally with apparent pK values near 10, but three do not titrate until much more alkaline pH values have been reached and irreversible alkaline de-naturation has set in. Some typical spectra and difference spectra are... 

Absorption spectrum is the plot of light intensity as a function of wavelength. Figure 1.2 shows the absorption spectra of tryptophan, tyrosine, and phenylalanine in water. A strong band at 210-220 nm and a weaker band at 260-280 nm can be seen. 

Absorption spectrum of ai-acid glycoprotein displays two peaks at 225 and 278 nm (Figure 2.3). This feature is characteristic for all proteins. The peak at 278 nm originates from the three aromatic amino acids of the proteins, tyrosine, tryptophan, and phenylalanine. The e for a protein is generally calculated at 278 nm. 

Figure 8.1 Normalized fluorescence emission spectrum (>.ex = 295 nm with emission peak = 355 nm) (c), excitation spectrum obtained at >.em = 340 nm (b), and absorption spectrum (a) of L-tryptophan in phosphate buffer, pH 7.

Since ANS dissolved in a polar medium does not fluoresce, one cannot record its fluorescence excitation spectrum. For tryptophan, ethidium bromide, and riboflavin, one can see that for each molecule, the absorption spectrum looks like the fluorescence... 

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). 

The part of a molecule that absorbs the light and is, therefore, responsible for its colour (whether in the visible or UV region) is called the chromophore, and the wavelength dependence of the absorption defines its absorption spectrum. Figure 7-3 illustrates the absorption spectrum of the three aromatic amino acids tryptophan, tyrosine and phenylalanine. 

The absorption spectrum of protein is maximum at 280 nm due to the presence of tyrosine and tryptophan, which are the strongest chromophores in that region. Hence the absorbance of the protein at this wavelength is adapted for its determination. 

The visible absorption spectrum of a solution containing a known concentration of nitrated protein is measured in a solution buffered at pH 9.0, and the absorbance at the maximum (near 428 nm) used to calculate the nitrotyrosine content ( 428nm for the nitrophenoxide ion is 4200). The tyrosine and nitrotyrosine content of the modified protein should also be determined by amino acid analysis. If the sum of these values does not add up to the tyrosine content of the unmodified protein, intra- or intermolecular cross-linking may have occurred. The amino acid analysis may also reveal whether other side-reactions have taken place. Particular attention should be paid to the half-cystine, cysteine, methionine, histidine and tryptophan contents of the modified proteins. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate offers a rapid and highly sensitive way of detecting products of intermolecular cross-linking. Such products are readily removed by gel filtration. 

Tryptophan fluorescence spectrum. The emission spectrum appears at longer wavelengths as compared to the absorption spectrum. 

Figure 34. A, Circular dichroism spectra of the Gramicidin A transmembrane channel in phospholipid bilayers (curve a) and of hydrogenated Gramicidin A in trifluoroethanol at high concentration ( 100 mg/ml), which is likely a double-stranded j3-helix. See text for discussion. Mean residue ellipticities are given. B, Absorption spectrum of the Gramicidin A transmembrane channel in phospholipid bilayers. The absorption spectrum is dominated by the four tryptophan residues per pentadecapeptide. The extinction coefficient is given on a per residue basis.

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