Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study

Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Carbohydr. Res., 318 (1999) 193-200]. Alpha1-acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to alpha1-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated alpha1-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation (one Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of alpha1-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor White and the carbohydrate residues of alpha1-acid glycoprotein are also performed at saturating concentrations of Calcofluor using the red-edge excitation spectra and steady-state anisotropy studies. The red-edge excitation spectra experiments show an important shift (13 nm) of the fluorescence emission maximum of the probe. This reveals that emission of Calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that the microenvironment of bound Calcofluor is rigid, inducing in this way the rigidity of the fluorophore itself, a result confirmed by anisotropy studies.     

Interaction between carbohydrate residues of alpha(1)-acid glycoprotein (orosomucoid) and progesterone. A fluorescence study

Interaction between progesterone and the carbohydrate residues of alpha(1)-acid glycoprotein was followed by fluorescence studies using calcofluor white. The fluorophore interacts with polysaccharides and is commonly used in clinical studies. Binding of progesterone to the protein induces a decrease in the fluorescence intensity of calcofluor white, accompanied by a shift to the short wavelengths of its emission maximum. The dissociation constant of the complex was found equal to 8.62 microM. Interaction between progesterone and free calcofluor in solution induces a low decrease in the fluorescence intensity of the fluorophore without any shift of the emission maximum. These results show that in alpha(1)-acid glycoprotein, the binding site of progesterone is very close to the carbohydrate residues. Fluorescence intensity quenching of free calcofluor in solution with cesium ion gives a bimolecular diffusion constant (k(q)) of 2.23 x 10(9) M(-1) s(-1). This value decreases to 0.19 x 10(9) M(-1) s(-1) when calcofluor white is bound to alpha(1)-acid glycoprotein. Binding of progesterone does not modify the value of k(q) of the cesium. Previous studies have shown that the terminal sialic acid residue is mobile, while the other glycannes are rigid [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94]. Red-edge excitation spectra and Perrin plot experiments performed on sialylated and asialylated alpha(1)-acid glycoprotein show that binding of progesterone to alpha(1)-acid glycoprotein does not modify the local dynamics of the carbohydrate residues of the protein.     

Relation between the secondary structure of carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and the fluorescence of the protein

We studied in this work the relation that exists between the secondary structure of the glycans of alpha(1)-acid glycoprotein and the fluorescence of the Trp residues of the protein. We calculated for that the efficiency of quenching and the radiative and non-radiative constants. Our results indicate that the glycans display a spatial structure that is modified upon asialylation. The asialylated conformation is closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence parameters of the Trp residues. In fact, the mean quantum yield of Trp residues in sialylated and asialylated alpha(1)-acid glycoprotein are 0.0645 and 0.0385, respectively. Analysis of the fluorescence emission of alpha(1)-acid glycoprotein as the result of two contributions (surface and hydrophobic domains) indicates that quantum yields of both classes of Trp residues are lower when the protein is in the asialylated form. Also, the mean fluorescence lifetime of Trp residues decreases from 2.285 ns in the sialylated protein to 1.948 ns in the asialylated one. The radiative rate constant k(r) of the Trp residues in the sialylated alpha(1)-acid glycoprotein is higher than that in the asialylated protein. Thus, the carbohydrate residues are closer to the Trp residues in the absence of sialic acid. The modification of the spatial conformation of the glycans upon asialylation is confirmed by the decrease of the fluorescence lifetimes of Calcofluor, a fluorophore that binds to the carbohydrate residues. Finally, thermal intensity quenching of Calcofluor bound to alpha(1)-acid glycoprotein shows that the carbohydrate residues have slower residual motions in the absence of sialic acid residues.     

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.     

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.     

Effect of the secondary structure of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) on the local dynamics of Trp residues

We studied in this work the relation between the secondary structure of the carbohydrate residues of alpha1-acid glycoprotein and the local motions of Trp residues of the protein. We measured for this purpose the fluorescence emission intensity and anisotropy of the Trp residues between -46 and +30 degrees of the sialylated and asialylated protein. Our results indicate that, in both forms, the global profile of the emission intensity with temperature shows that Trp residues display static and collisional interaction with the neighboring amino acids. However, the profile of the asialylated form is more structured than that observed for the sialylated protein. The Y-plot analysis of the emission-anisotropy results indicated that the frictional resistance to rotation of the surface Trp residue is less important in the sialylated protein than in the asialylated form. This result is in good agreement with the fact that, in the asialylated conformation, the carbohydrate residues are closer to the protein surface than in the sialylated form, thereby increasing the contact of the surface Trp residue with the neighboring amino acids. Also, the interaction between the carbohydrate residues and the surface Trp residue contributes to the modification of the frictional resistance to rotation of the fluorophore.     

Motions studies of the human alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and polarization of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues.

Dynamics studies on tryptophan residues of human alpha 1-acid glycoprotein (orosomucoid) and of 2-p-toluidinylnaphthalene-6-sulfonate bound to the protein are performed. Excitation at the red edge of the absorption spectrum of the tryptophan does not lead to a shift of the fluorescence emission maximum of the fluorophore. This reveals that Trp residues present motions with respect to their microenvironment. This is confirmed by polarization studies as a function of temperature. Excitation at the red edge of the absorption spectrum of TNS leads to an important shift (15 nm) of the fluorescence emission maximum of the probe. This reveals that emission of TNS occurs before relaxation of the amino-acids dipole occurs. Emission from a non-relaxed state means that TNS molecules are bound tightly to the protein, a result confirmed by polarization studies.     

Effect of binding of Calcofluor White on the carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) on the structure and dynamics of the protein moiety. A fluorescence study

Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously studied at low and high concentrations of Calcofluor compared to that of the protein. alpha1-Acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. At equimolar concentrations of Calcofluor and alpha1-acid glycoprotein, the fluorophore displays free motions [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94], while at high concentration of Calcofluor, its surrounding microenvironment is rigid, inducing the rigidity of the fluorophore itself [Albani, J. R.; Sillen, A.; Plancke, Y. D.; Coddeville, B.; Engelborghs, Y. Carbohydr. Res. 2000, 327, 333-340]. In the present work, red-edge excitation spectra and steady-state anisotropy studies performed on Trp residues in the presence of Calcofluor, showed that the apparent dynamics of Trp residues are not modified. However, deconvoluting the emission spectra with two different methods into different components, reveals that the structure of the protein matrix has been disrupted in the presence of high Calcofluor concentrations.     

Potential charge transfer probe induced conformational changes of model plasma protein human serum albumin: Spectroscopic, molecular docking, and molecular dynamics simulation study

The nature of binding of specially designed charge transfer (CT) fluorophore at the hydrophobic protein interior of human serum albumin (HSA) has been explored by massive blue-shift (82 nm) of the polarity sensitive probe emission accompanying increase in emission intensity, fluorescence anisotropy, red edge excitation shift, and average fluorescence lifetimes. Thermal unfolding of the intramolecular CT probe bound HSA produces almost opposite spectral changes. The spectral responses of the molecule reveal that it can be used as an extrinsic fluorescent reporter for similar biological systems. Circular dichrosim spectra, molecular docking, and molecular dynamics simulation studies scrutinize this binding process and stability of the protein probe complex more closely.     

Studies on incorporation of mannose into rat alpha 1-acid glycoprotein: evidence for precursor forms in rough membranes

Rats were given pulse injections of D-[14C]mannose and were killed at various times up to 60 min after injection. Rough, smooth, and Golgi fractions were prepared from liver, and alpha 1-acid glycoprotein was isolated from Lubrol extracts of the fractions. The kinetics of incorporation of D-[14C]mannose into total protein, Lubrol protein, and alpha 1-acid glycoprotein showed that proteins associated with rough fractions had particularly high specific radioactivities at early times of incorporation. One explanation for the kinetic data is that glycoproteins contain a high mannose content at early times of assembly of oligosaccharide chains. This idea was confirmed in the case of alpha 1-acid glycoprotein by isolation of a high mannose containing precursor species of alpha 1-acid glycoprotein from rough fractions of liver. This species contained 56 residues of hexose (mainly mannose) compared with 35 residues of hexose (roughly equal amounts of mannose and galactose) which are found in the native protein. It is proposed that the high mannose precursor is a form of alpha 1-acid glycoprotein that exists at an early stage in assembly of the glycoprotein and which contains largely unprocessed carbohydrate chains. In addition, evidence is presented from amino acid analyses and gel electrophoresis of the high mannose precursor and another fraction from which it is formed by limited tryptic treatment, that pro-forms of alpha 1-acid glycoprotein with extensions of the polypeptide chain may also exist.     

Exploring structural change of protein bovine serum albumin by external perturbation using extrinsic fluorescence probe: spectroscopic measurement, molecular docking and molecular dynamics simulation

Structural modification through binding interaction of plasma protein bovine serum albumin (BSA) with an extrinsic charge transfer fluorophore 5-(4-dimethylamino-phenyl)-penta-2,4-dienoic acid (DMAPPDA) and its response to external perturbation due to interactions with surfactant sodium dodecyl sulphate (SDS) have been explored at physiological pH by steady state absorption, emission, fluorescence anisotropy, red edge excitation shift, far-UV circular dichroism and time resolved spectral measurements in combination with Molecular Docking and Molecular Dynamics (MD) simulation. Interaction of the probe with BSA is reflected by a small change in protein secondary structure with fluorescence enhancement and blue shift of probe emission. Molecular docking studies revealed that the probe binds to the hydrophobic cavity of sub-domain IIA of BSA. The distance for energy transfer from the tryptophan of BSA to the bound DMAPPDA measured by Fluorescence Resonance Energy Transfer is in good agreement with the molecular docking results. MD simulation predicts stabilization of the complex with respect to the bare molecule. Interaction of BSA and SDS with DMAPPDA supports the movement of the probe from hydrophilic free water region to a more restricted hydrophobic zone inside the protein.     

Use of probes with fluorescence indicator distributed throughout the pharmacophore to examine the peptide agonist-binding environment of the family B G protein-coupled secretin receptor

Fluorescence techniques can provide insight into the environment of fluorescence indicators situated at distinct sites within a ligand as it is bound to its receptor. Here, we have developed a series of analogues of the 27-amino acid hormone, secretin, that incorporate a fluorescent Alexa Fluor 488 into the amino terminus, the carboxyl terminus, and positions 13 and 22. Each probe bound with high affinity and was biologically active, stimulating full cAMP responses in receptor-bearing Chinese hamster ovary-SecR cells. Treatment with 10 mum guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp) shifted the agonist-bound receptor into a G protein-uncoupled low affinity state. Fluorescence spectra for the probes in solution and bound to the receptor demonstrated maximal emission at 521 nm after excitation at 481 nm. Collisional quenching of fluorescence with potassium iodide revealed that Alexa at the amino terminus of secretin was more accessible than at the other three positions within the probes. Of note, quenching constants for each probe were higher when bound in the active state than in the G protein-uncoupled, low affinity state of the receptor, with the most marked changes occurring for the two midregion probes. Anisotropy values and fluorescence lifetimes confirmed this, with higher anisotropy and longer lifetimes observed for position 13 and 22 probes bound to the receptor in its uncoupled state than in its active state. These observations suggest that the amino terminus of secretin as docked to the receptor is most exposed to the hydrophilic aqueous milieu, and that the major changes in conformation and exposure to the medium occur in the midregion of secretin. Photoaffinity labeling studies have demonstrated approximation of each of these ligand residues with distinct receptor residues. Combining the fluorescence data with photoaffinity labeling data provides insights into the conformation and dynamics of a natural peptide ligand docked to a Family B G protein-coupled receptor.     

Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket

Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the F?rster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.     

Factors influencing the fluorescence spectra of the formaldehyde-induced reaction product of 5-hydroxytryptamine

Summary Fluorescence excitation and emission spectra of the formaldehyde-induced fluorophore of 5-hydroxytryptamine in a Sephadex model have been examined following exposure to hydrochloric acid or ammonia vapour. Exposure to hydrochloric acid vapour produced excitation spectra with broad maxima centred around 400 nm, whilst exposure to ammonia vapour intensified the maximum normally seen at approximately 450 nm relative to that seen at 400 nm. The emission maximum was generally broad and poorly defined following exposure to hydrochloric acid vapour; exposure to ammonia vapour had little effect on its location. Exposure of the formaldehyde-induced fluorophore in models containing 5-hydroxytryptamine to 300 nm irradiation caused a substantial shift in the position of the emission maximum; a concomitant increase in the fluorescence intensity was also observed. When the fluorescence present in duodenal enterochromaffin cells was examined after similar treatments, a number of differences in the response of the fluorophore were noted.     

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.     

Dynamic fluorescence of extrinsic fluorophores as a tool for studying protein conformational substates

Summary The fluorescence lifetime distribution of 2-p-toluidinyl-6-naphthalene sulfonic acids (TNS) bound to the heme site of apomyoglobin has been examined. The results were compared to those observed for the free fluorophore in isotropic nonviscous solvent. Two different excitation wavelengths were used, i.e. 290 and 350 nm. The results showed that the distribution of TNS bound to apomyoglobin is wider than that of the free fluorophore, thus indicating the existence of a large number of conformational substates originating from the interaction between TNS and the protein matrix. The comparison of the distribution obtained at two different excitation wavelengths allowed the emission arising from conformational substates, in which the excited state of fluorophore moiety has a higher probability to be populated by Forster energy transfer mechanism, to be distinguished.     

Porcine pancreatic alpha amylase and its isoforms--effect of deglycosylation by peptide-N-glycosidase F

Porcine pancreatic alpha-amylase (PPA) and its isoforms (PPA-I and PPA-II) were deglycosylated by peptide-N-glycosidase F (PNGase F) to investigate the role of bound carbohydrate. On deglycosylation, the effect on thermal stability was less pronounced. Deglycosylation resulted in a shift of the mid-point of thermal transition by 1-2 degrees C towards lower temperature. The fluorescence emission maxima of PPA, PPA-I and PPA-II were found to be 340nm indicating the presence of tryptophan residues in a fairly hydrophilic environment. A red shift in emission spectra accompanied by an increase in fluorescence intensity was observed upon deglycosylation.     

Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA

The potent RNA polymerase inhibitors actinomycin D and 7-aminoactinomycin D are shown to bind to single-stranded DNAs. The binding occurs with particular DNA sequences containing guanine residues and is characterized by hypochromic UV absorption changes similar to those observed in interactions of the drugs with double-stranded duplex DNAs. The most striking feature of the binding is the dramatic (ca. 37-fold) enhancement in fluorescence that occurs when the 7-aminoactinomycin is bound to certain single-stranded DNAs. This fluorescence of the complex is also characterized by a 40-nm hypsochromic shift in the emission spectrum of the drug and an increase in the emission anisotropy relative to the free drug or the drug bound to calf thymus DNA. The fluorescence lifetimes change in the presence of the single-stranded DNA in a manner compatible with the intensity difference. Thus, there is an increase in the fraction of the emission corresponding to a 2-ns lifetime component compared to the predominant approximately 0.5-ns lifetime of the free drug. The 7-aminoactinomycin D comigrates in polyacrylamide gels with the single-stranded DNAs, and the fluorescence of the bound drug can be visualized by excitation with 540-nm light. The binding interactions are characterized by association constants of 2.0 x 10(6) to 1.1 x 10(7) M-1.     

Exploring membrane organization and dynamics by the wavelength-selective fluorescence approach

Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy which can be used to directly monitor the environment and dynamics around a fluorophore in a complex biological system. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of absorption band, is termed red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore which is a function of the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise 'optically silent' water molecules. This makes REES and related techniques extremely useful since hydration plays a crucial modulatory role in a large number of important cellular events, including lipid-protein interactions and ion transport. The interfacial region in membranes, characterized by unique motional and dielectric characteristics, represents an appropriate environment for displaying wavelength-selective fluorescence effects. The application of REES and related techniques (wavelength-selective fluorescence approach) as a powerful tool to monitor the organization and dynamics of probes and peptides bound to membranes, micelles, and reverse micelles is discussed.     

Förster energy-transfer studies between Trp residues of alpha1-acid glycoprotein (orosomucoid) and the glycosylation site of the protein

Energy-transfer studies between Trp residues of alpha(1)-acid glycoprotein and the fluorescent probe Calcofluor White were performed. Calcofluor White interacts with carbohydrate residues of the protein, while the three Trp residues are located at the surface (Trp-160) and in hydrophobic domains of the protein (Trp-25 and Trp-122). Binding of Calcofluor to the protein induces a decrease in the fluorescence intensity of the Trp residues accompanied by an increase of that of Calcofluor White. Efficiency (E) of Trp fluorescence quenching was determined to be equal to 45%, and the F?rster distance R(o), at which the efficiency of energy transfer is 50%, was calculated to be 18.13 A. This low distance and the value of the efficiency clearly indicate that energy transfer between Trp residues and Calcofluor White is weak.