scholarly journals Exploring the possibility of early cataract diagnostics based on tryptophan fluorescence

2011 ◽  
Vol 8 (64) ◽  
pp. 1616-1621 ◽  
Author(s):  
Dmitry M. Gakamsky ◽  
Bal Dhillon ◽  
John Babraj ◽  
Matthew Shelton ◽  
S. Desmond Smith
Keyword(s):  
Structural Changes ◽  
Tryptophan Fluorescence ◽  
Aqueous Environment ◽  
Wavelength Selection ◽  
Optical Wavelength ◽  
Red Edge ◽  
Tryptophan Residues ◽  
Depth Control ◽  
Replacement Decisions ◽  
Tryptophan Emission

A novel route for early cataract diagnostics is investigated based on the excitation of tryptophan fluorescence (TF) at the red edge of its absorption band at 317 nm. This allows penetration through the cornea and aqueous humour to provide excitation of the ocular lens. The steepness of the red edge gives the potential of depth control of the lens excitation. Such wavelength selection targets the population of tryptophan residues, side chains of which are exposed to the polar aqueous environment. The TF emissions around 350 nm of a series of UV-irradiated as well as control lenses were observed. TF spectra of the UV cases were red-shifted and the intensity decreased with the radiation dose. In contrast, intensity of non-tryptophan emission with maximum at 435 nm exhibited an increase suggesting photochemical conversion of the tryptophan population to 435 nm emitting molecules. We demonstrate that the ratio of intensities at 435 nm to that around 350 nm can be used as a measure of early structural changes caused by UV irradiation in the lens by comparison with images from a conventional slit-lamp, which can only detect defects of optical wavelength size. Such diagnostics at a molecular level could aid research on cataract risk investigation and possible pharmacological research as well as assisting surgical lens replacement decisions.

scholarly journals Study of Organization and Dynamics of Multi-Tryptophan Protein Molecules Utilizing Red Edge Excitation Shift Approach

2020 ◽  
Vol 32 (10) ◽  
pp. 2416-2422
Author(s):  
Anisur R. Molla ◽  
Pritha Mandal
Keyword(s):  
Fluorescence Probe ◽  
Protein Structures ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Excitation Wavelength ◽  
Denatured State ◽  
Red Edge ◽  
Tryptophan Residues ◽  
Red Edge Excitation Shift ◽  
Red Edge Effect

A shift in the fluorescence emission maxima with gradual increase in excitation wavelength is termed as red edge excitation shift (REES). Tryptophan residues are widely utilized as intrinsic fluorescence probe to investigate the protein structures. Wavelength selective tryptophan fluorescence can explore the dynamics of surrounded water molecules, the ubiquitous biological solvent. Thus REES experiment of various protein conformational states can provide significant input to the study of protein folding pathway and it can also be useful to study interaction of proteins with others. In this review article, we shall focus on red edge effect of various multi-tryptophan proteins in their respective native, intermediate and denatured state.

Synthesis and solution conformation of [Trp1, Val5]-angiotensin II

1977 ◽  
Vol 55 (1) ◽  
pp. 75-82 ◽  
Author(s):  
Peter W. Schiller
Keyword(s):  
Angiotensin Ii ◽  
Solid Phase ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
Tryptophan Fluorescence ◽  
Exchange Chromatography ◽  
Phase Method ◽  
Aqueous Environment ◽  
Solution Conformation ◽  
Intramolecular Distance

[Trp1, Val5]-Angiotensin II was synthesized by the solid-phase method and purified by partition chromatography on Sephadex G-25 and by ion-exchange chromatography on Sephadex SP-25. Relative to [Val5]-angiotensin II, the analog displayed 36% smooth muscle activity in a preparation of the superior mesenteric artery. Conformational aspects of the analog were revealed by fluorescence techniques. The fluorescence emission maximum at 350 nm suggests a completely aqueous environment for the tryptophanyl residue in [Trp1, Val5]-angiotensin II. Singlet–singlet resonance energy transfer between Tyr in position 4 and Trp in position 1 was evaluated for a calculation of the intramolecular distance between these two residues on the basis of the Förster equation. From the relative increase of tryptophan fluorescence a transfer efficiency of > 0.9 was obtained, which is compatible with the observed complete quenching of tyrosine fluorescence in the analog. The computed average intramolecular distance of < 8 Å precludes an extended conformation for the N-terminal sequence encompassing residues 1 through 4 at neutral pH and suggests the existence of a loop in this part of the molecule. The results are discussed in relation to the various models proposed for the solution conformation of angiotensin II.

Fluorescent Properties of Firefly Luciferases and Their Complexes with Luciferin

2000 ◽  
Vol 20 (1) ◽  
pp. 21-30 ◽  
Author(s):  
Ekaterina I. Dementieva ◽  
Elena A. Fedorchuk ◽  
Lubov Yu. Brovko ◽  
Alexander P. Savitskii ◽  
Natalya N. Ugarova
Keyword(s):  
Energy Transfer ◽  
Tryptophan Fluorescence ◽  
Tryptophan Residue ◽  
Amino Acid Residues ◽  
Photinus Pyralis ◽  
Fluorescent Properties ◽  
Effective Energy Transfer ◽  
Tryptophan Residues ◽  
Luciola Mingrelica

Fluorescence of luciferases from Luciola mingrelica (single tryptophanresidue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417,Trp-426) was studied. Analysis of quenching of tryptophan fluorescenceshowed that the tryptophan residue conserved in all luciferases is notaccessible for charged quenchers, which is explained by the presence ofpositively and negatively charged amino acid residues in the close vicinityto it. An effective energy transfer from tryptophan to luciferin wasobserved during quenching of tryptophan fluorescence of both luciferaseswith luciferin. From the data on the energy transfer, the distance betweenthe luciferin molecule and Trp-417 (419) in the luciferin–luciferasecomplex was calculated: 11–15 Å for P. pyralis and 12–17Å for L. mingrelica luciferases. The role of the conserved Trp residuein the catalysis is discussed.

scholarly journals Aggregation equilibria of tyrosinase of Harding-Passey mouse melanoma

1985 ◽  
Vol 228 (1) ◽  
pp. 95-101 ◽  
Author(s):  
J C Garcia-Borron ◽  
F Solano ◽  
J L Iborra ◽  
J A Lozano
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Aqueous Environment ◽  
Fluorescence Studies ◽  
Mouse Melanoma ◽  
Low Protein ◽  
Tryptophan Residues ◽  
Sample Dilution

The purification of two isoenzymes of tyrosinase has been carried out in Harding-Passey mouse melanoma. One is found in the cytosol and the other one bound to melanosomes. Both migrate as single bands on sodium dodecyl sulphate/polyacrylamide gels, having an apparent Mr of 58 000. Solubilized particulate tyrosinase showed an aggregation equilibrium involving a monomer, tetramer, octamer and a high-Mr micellar form with Brij 35, the solubilizing agent. H.p.l.c. studies indicated a interconversion between those species, the monomer contribution increasing with the sample dilution. The tetramer and the octamer probably represent the predominant forms in vivo. Soluble tyrosinase showed a simpler aggregation equilibrium, involving two forms, monomer and tetramer, with the same interconversion pattern. Fluorescence studies suggested that tryptophan residues were exposed to the aqueous environment when tyrosinase was dissociated by dilution. Tyrosinase shows a tendency to aggregate, at low protein concentration, and a resistance to dissociation by urea or SDS so remarkable that gel-permeation chromatography in 4M-urea does not affect the equilibrium, and the band obtained on SDS/polyacrylamide-gel electrophoresis is a dimer.

scholarly journals The conformational changes of α2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes

1987 ◽  
Vol 243 (1) ◽  
pp. 47-54 ◽  
Author(s):  
L J Larsson ◽  
P Lindahl ◽  
C Hallén-Sandgren ◽  
I Björk
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Cross Linking ◽  
Bait Region ◽  
Close Proximity ◽  
Tryptophan Residues ◽  
Thioester Bond ◽  
Bond Region

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent ‘bait’ region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].

The Steady-State and Decay Characteristics of Protein Tryptophan Fluorescence from Algae

1988 ◽  
Vol 42 (8) ◽  
pp. 1405-1412 ◽  
Author(s):  
M. Baek ◽  
W. H. Nelson ◽  
P. E. Hargraves ◽  
J. F. Tanguay ◽  
S. L. Suib
Keyword(s):  
Steady State ◽  
Tryptophan Fluorescence ◽  
Fluorescence Decay ◽  
Direct Application ◽  
Fluorescence Lifetimes ◽  
Bacterial Protein ◽  
Steady State Fluorescence ◽  
Tryptophan Residues ◽  
Rapid Characterization

The intrinsic steady-state fluorescence due to tryptophan has been obtained from monospecific cultures of fourteen plankton algae of various genera. Fluorescence decay profiles of protein tryptophan residues were obtained for eight marine plankton algae. Each organism exhibits a strong maximum in its emission spectrum at 320–340 nm when excited at 290 nm. Iodide quenching and denaturization experiments with 8 M urea provide strong evidence for the assignment of the 320–340 nm fluorescence to protein tryptophan. Most importantly, the decay of this bacterial protein tryptophan fluorescence has been described. The observation that characteristic protein-tryptophan fluorescence lifetimes have been obtained for each organism suggests that measurements of fluorescence lifetimes may be helpful in the rapid characterization of algae. Direct application will likely be found in combination with the measurement of other luminescence parameters.

Binding Studies of Bile Acids using the Native Fluorescence of the Tryptophan Residue Of Bax Protein

2006 ◽  
Vol 26 (3) ◽  
pp. 245-250 ◽  
Author(s):  
Wei Zhang ◽  
Clifford J. Steer ◽  
Kenneth T. Douglas ◽  
Cecilia M. P. Rodrigues
Keyword(s):  
Bile Acids ◽  
Direct Interaction ◽  
Tryptophan Fluorescence ◽  
Potential Interaction ◽  
Tryptophan Residue ◽  
Binding Studies ◽  
Native Fluorescence ◽  
Bax Protein ◽  
Tryptophan Residues ◽  
Direct Binding

Ursodeoxycholic acid (UDCA) and its taurine-conjugate, tauroursodeoxycholic acid (TUDCA), play a unique role in modulating the apoptotic threshold in cells. The mechanism is thought to involve, in part, inhibition of translocation for Bax from the cytosol to mitochondria. Here, we attempted to use the native fluorescence of the tryptophan residues of Bax to determine whether bile acids bind directly to recombinant Bax protein. The results showed that UDCA had no effect on the tryptophan fluorescence of Bax. Similarly, there was no evidence of direct binding between Bax protein and the more hydrophobic bile acid, deoxycholic acid (DCA). In contrast, the fluorescence change detected for Bax solution titrated against TUDCA in dimethylsulfoxide was greater than that observed with solvent alone. In conclusion, data from fluorescence spectroscopy does not support a direct interaction of UDCA or DCA with Bax protein, whereas it suggests that there may be some potential interaction with TUDCA.

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