Tryptophan absorptivity

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). 

Fig. 23. Comparison of the effects of A -bromosuccinimide on the disappearance of tryptophan absorption in trypsinogen and trypsin. From Viswanatha et al. (1960).

In Hartnup disease tryptophan absorption is impaired and in malignant carcinoid syndrome tryptophan metabolism is altered resulting in excess serotonin synthesis. 

In Crohn s disease, serum tryptophan is often found to be low in patients. Beeken42 reported that tryptophan absorption in patients with Crohn s disease had distinctly subnormal results (13 patients) while 19 patients had normal tryptophan absorption values (compared to 16 healthy controls). The patients with abnormal results ate less, lost more weight, and had lower serum albumin levels than those with normal absorption. 

Using the tools of molecular biology and genetics, research should define the mechanisms by which genes influence nutrient (L-tryptophan) absorption, metabolism, excretion, and other actions and also the mechanisms by which the nutrient (L-tryptophan) influences gene expression. Such studies should be productive and will clarify whether and how L-tryptophan may be primarily or secondarily involved in and during the pathogenesis of a variety of important chronic diseases. 

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. 

In this section we shall consider only the coenzyme active site the regulatory sites for purine nucleotides will be discussed later. Cross and Fisher [309] have divided the coenzyme site into two subsites, one specific for the amide portion of the nicotinamide and the other for the adenosine diphosphate moiety. Binding of coenzyme at the amide subsite causes perturbations in the tyrosine and tryptophan absorption regions. Presumably, the tryptophan involved is the one found by Cross et al. (SOI) to have a red-shifted spectrum upon coenzyme binding. 

The difference peak at 292 nm is characteristic of a red shift of the tryptophan absorption band. The dominant difference peaks at 287 and 280 nm arise from the perturbation of one or more tyrosine residues. The... 

Alpert, B. and Lopez-Delgado, R, 1976, Fluorescence lifetimes of haem proteins excited Into the tryptophan absorption band with synchrotron radiation. Nature, 263, 445 - 446. 

Excitation spectra are often used to study energy transfer. This is because energy transfer can be detected by enhanced emission from the acceptor when the excitation is centered at the donor absorption. The effects of melittin self-association are evident from the excitation spectra (Fig. 10). For these spectra the emission monochromator is centered on the NMA emission (430 nm) and the intensity recorded as the excitation monochromator is scanned through the absorption bands of the NMA label (350 nm) and the tryptophan absorption (280 nm). Increasing salt concentrations result in increased intensity of the tryptophan excitation band (280 nm). This increase in energy transfer is due to the close proximity of the three additional donors to the NMA acceptor. 

As a preliminary step in determination of the full set of the mRFPl photopysical parameters, the analysis of fluorescence decay at picoseconds excitation has been done. The excitation wavelength was 266 nm in order to match the acceptor (tryptophan) absorption band. The acceptor fluorescence decay under excitation of an ensemble of donor-acceptor pairs by pulse is described (Valeur, 2002) as ... 

Fig. 25. Absorption spectrum of L-tryptophan in the presence of NAD+.71.71-10 2 M tryptophan +56.5 mg NAD+/ml. Dashed line is tryptophan absorption. (From ref. 102)...

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... 

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... 

The intense blue color which is obtained when tryptophan, in the presence of an aldehyde, is treated with concentrated sulfuric acid containing an oxidizing agent (Adamkiewicz-Hopkins-Cole reaction) was beheved to involve formation of a tetrahydro-j8-carboline intermediate, since most l,2,3,4-tetrahydro-j8-carbohne derivatives yield a similar color with concentrated sulfuric acid containing an oxidizing agent. The two colors have now been shown to have different absorption spectra. The nature of the carboline-blue color is still obscure. 

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. 

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... 

Ru(dppz)(x) (y)3+/ Me, tryptophan AG -0.6 V detect G(-H), M, and tryptophan radical s by transient absorption, EPR Gs in intervening sequence k 107 s 1 up to 50 A CT not rate limiting up to 50 A GGox varies with sequence, base-stack pertubrations and extent of intercalation... 

Enzyme structure may be studied by fluorescence spectroscopy [238-244]. Excitation in the 280-310 nm absorption bands of proteins, usually results in fluorescence from tryptophan (Trp) residues in the 310-390 nm region. The fluorescence from the Trp residues is a convenient marker for protein denaturation and large decreases or red-shifts in fluorescence are observed when proteins are denatured. These changes are most often due to the exposure of the Trp residues that are buried in the protein and may be due to the changes in the proximities of specific residues that may act as fluorescence quenchers. Fluorescence emission characterization of the immobilized... 

Phenylalanine and tryptophan contain aromatic side chains that, like the aliphatic amino acids, are also relatively non-polar and hydrophobic (Figure 1.4). Phenylalanine is unreactive toward common derivatizing reagents, whereas the indolyl ring of tryptophan is quite reactive, if accessible. The presence of tryptophan in a protein contributes more to its total absorption at 275-280nm on a mole-per-mole basis than any other amino acid. The phenylalanine content, however, adds very little to the overall absorbance in this range. 

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... 

ROS can modify amino acid side chains, with histidine, tryptophan, cysteine, proline, arginine, and lysine among those most susceptible to attack (Brown and Kelly 1994). As a result, carbonyl groups are generated, and these carbonyl concentrations can be measured directly in plasma by using atomic absorption spectroscopy, fluorescence spectroscopy, or HPLC following reaction with 2,4-dinitrophenylhydrazine. 

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. 

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. 

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