Tryptophan polarity

The pH dependence of tryptophan polarization is not easily interpretable. The increase between pH 4 and 5 could be related to the pK of 4.3 of the tryptophyl cation radical (22). At low pH t e radical would not deprotonate and a fast exchange reaction with the parent molecule could lead to partial cancellation of polarization. Again, this cancellation is less effective in the case of proteins and... 

The back reaction (a) could in principle explain the observed results but for the initial growth of Ijj(t) expected from F-pair polarization. In addition this mechanism fails to explain the small time dependence of the tryptophan polarization. It is reasonable to expect that Tj for a ring proton in tryptophan would be somewhat smaller than for a ring-methyl proton in flavin simply because of the r dependence of the electron-proton dipolar interaction. Therefore as shown in Figure 7 for short Tj, a predominantly increasing Ijj(t) would be predicted for tryptophan in contrast to the observed results. 

Water-soluble globular proteins usually have an interior composed almost entirely of non polar, hydrophobic amino acids such as phenylalanine, tryptophan, valine and leucine witl polar and charged amino acids such as lysine and arginine located on the surface of thi molecule. This packing of hydrophobic residues is a consequence of the hydrophobic effeci which is the most important factor that contributes to protein stability. The molecula basis for the hydrophobic effect continues to be the subject of some debate but is general considered to be entropic in origin. Moreover, it is the entropy change of the solvent that i... 

A number of studies have recently been devoted to membrane applications [8, 100-102], Yoshikawa and co-workers developed an imprinting technique by casting membranes from a mixture of a Merrifield resin containing a grafted tetrapeptide and of linear co-polymers of acrylonitrile and styrene in the presence of amino acid derivatives as templates [103], The membranes were cast from a tetrahydrofuran (THF) solution and the template, usually N-protected d- or 1-tryptophan, removed by washing in more polar nonsolvents for the polymer (Fig. 6-17). Membrane applications using free amino acids revealed that only the imprinted membranes showed detectable permeation. Enantioselective electrodialysis with a maximum selectivity factor of ca. 7 could be reached, although this factor depended inversely on the flux rate [7]. Also, the transport mechanism in imprinted membranes is still poorly understood. 

Additional evidence for conformational changes in the transporter has come from measurement of the intrinsic fluorescence of the protein tryptophan residues, of which there are six, in the presence of substrates and inhibitors of transport. The fluorescence emission spectrum of the transporter has a maximum at about 336 nm, indicating the presence of tryptophan residues in both non-polar environments (which would emit maximally at about 330 nm) and in polar environments (which would emit at 340-350 nm) [154], The extent of quenching by the hydrophilic quencher KI indicates that more than 75% of the fluorescence is not available for quenching, and so probably stems from tryptophan residues buried within the hydrophobic interior of the protein or lipid bilayer [155]. Fluorescence is quenched... 

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 determination of theophylline in plasma can also be accomplished by various immunoassay techniques.66-67 Theophylline was also determined by a polarization fluoroimmunoassays but found to have a caffeine interference.88. In a more research oriented application, the interaction of caffeine with L-tryptophan was studied using h NMR with the results indicating that caffeine interacted with tryptophan in a 1 1 molar ratio through parallel stacking.69... 

In polar solvents, the structure of the acridine 13 involves some zwitterionic character 13 a [Eq. (7)] and the interior of the cleft becomes an intensely polar microenvironment. On the periphery of the molecule a heavy lipophilic coating is provided by the hydrocarbon skeleton and methyl groups. A third domain, the large, flat aromatic surface is exposed by the acridine spacer unit. This unusual combination of ionic, hydrophobic and stacking opportunities endows these molecules with the ability to interact with the zwitterionic forms of amino acids which exist at neutral pH 24). For example, the acridine diacids can extract zwitterionic phenylalanine from water into chloroform, andNMR evidence indicates the formation of 2 1 complexes 39 such as were previously described for other P-phenyl-ethylammonium salts. Similar behavior is seen with tryptophan 40 and tyrosine methyl ether 41. The structures lacking well-placed aromatics such as leucine or methionine are not extracted to measureable degrees under these conditions. 

Rachel, K., Asuncionpunzalan, E. and London, E. (1995) Anchoring of tryptophan and tyrosine analogs at the hydrocarbon polar boundary in model membrane-vesicles - paralax analysis of fluorescence quenching induced by nitroxide-labelled phospholipids. Biochemistry 34,15475-15479. 

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. 

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. 

Weber G. (1960) Fluorescence Polarization Spectrum and Electronic Energy Transfer in Tyrosine, Tryptophan and Related Compounds, Biochem. J. 75, 335-345. 

Plastocyanin from parsley, a copper protein of the chloroplast involved in electron transport during photosynthesis, has been reported to have a fluorescence emission maximum at 315 nm on excitation at 275 nm at pH 7 6 (2°8) gjncc the protein does not contain tryptophan, but does have three tyrosines, and since the maximum wavelength shifts back to 304 nm on lowering the pH to below 2, the fluorescence was attributed to the emission of the phenolate anion in a low-polarity environment. From this, one would have to assume that all three tyrosines are ionized. A closer examination of the reported emission spectrum, however, indicates that two emission bands seem to be present. If a difference emission spectrum is estimated (spectrum at neutral pH minus that at pH 2 in Figure 5 of Ref. 207), a tyrosinate-like emission should be obtained. 

K. K. Turoverov and I. M. Kuznetsova, Polarization of intrinsic fluorescence of proteins. 2. The studies of intramolecular dynamics of tryptophan residues, Mol. Biol. (Moscow) 17, 468-475 (1983). 

When tryptophan is dissolved in water, it shows the fluorescence characteristics illustrated in O Figure 5-2. However, one especially useful property of tryptophan fluorescence is that its emission spectrum is highly sensitive to the polarity of its environment. In less polar solvents (alcohols, alkanes, etc.), the emission... 

Synthesized tyrosine-, phenylalanine-, tetrahydroisoquinoline-, tetralin- and tryptophan analogs Chirobiotic R Reversed-phase mode H20/Me0H Polar organic mode MeOH/HOAc/TEA 269... 

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