Trp Fluorescence Intensity Research Articles

Classification of protein solubilizing solutes by fluorescence assay

Aromatic interaction plays a crucial role in controlling protein interaction by additives. Here we investigated the interaction of protein salting-in (solubilizing) additives with tryptophan (Trp), tyrosine (Tyr), indole, and proteins based on their fluorescence spectra. Five salting-in additives, i.e., arginine (Arg), urea, guanidine (Gdn), ethylene glycol (EG), and magnesium chloride (MgCl2), showed different effects on the fluorescence properties of Trp and Tyr. Arg significantly reduced fluorescence intensity of Trp and Tyr, as was the case for glycine to a lesser extent. MgCl2 and calcium chloride (CaCl2) showed little effect on the aromatic fluorescence spectra. Gdn also showed little effect on the aromatic fluorescence spectra of Trp and Tyr even at high concentrations. EG increased the aromatic fluorescence intensity of Trp and Tyr with blue-shifted emission wavelength. Urea enhanced fluorescence of Trp and Tyr without altering emission wavelength. These results indicate that the protein solubilizing additives interact with aromatic groups differently.

Insights from alpha-Lactoalbumin and beta-Lactoglobulin into mechanisms of nanoliposome-whey protein interactions

The complexation between nanoliposomes and two dominant monomers in whey proteins, i.e. alpha-Lactoalbumin (α-La) and beta-Lactoglobulin (β-Lg), were investigated to reveal the mechanisms of nanoliposome-whey protein interactions. The UV–vis spectra showed that nanoliposomes could significantly interact with the two monomeric proteins. The red shift phenomenon and intensity decrease were observed in the endogenous fluorescence spectra. It indicated that the exposure of hydrophobic groups in α-La/β-Lg increased, and the endogenous fluorescence was quenched. Synchronous fluorescence results showed that Trp fluorescence intensity decreased more significantly than that of Tyr, and more powerful interactions between monomeric proteins and nanoliposomes occurred in the regions around Trp residues. CD spectra showed an increase of β-sheet secondary structures of α-La and β-Lg in content at the cost of a decrease in α-helix and random coil in the two monomeric proteins after complexation. FTIR results indicated that the secondary structures of monomeric proteins altered, and the main interaction forces were hydrogen bond and hydrophobic forces. Surface hydrophobicity and free sulfhydryl content of monomeric proteins enhanced after complexation with nanoliposomes. Molecular docking theoretically demonstrated that the main interaction forces between nanoliposomes and monomeric proteins were hydrogen bond and hydrophobic forces. The interaction between nanoliposomes and α-La was more strong than that between nanoliposomes and β-Lg. The natural properties, such as amino acid composition, advanced structure and surface hydrophobicity, could play a critical role in the interaction between nanoliposomes and monomeric proteins. These findings could deepen the understanding of the interaction mechanisms between nanoliposomes and whey proteins.

Fluorine induced conformational switching and modulation in photophysical properties of 7-fluorotryptophan: Spectroscopic, quantum chemical calculation and molecular dynamics simulation studies

7-Fluorotryptophan (7F-Trp) is an important noncanonical derivative of tryptophan (Trp) amino acid which depicts high in vivo stability for the positron emission tomography imaging of Trp metabolism. Irrespective of biological importance of 7F-Trp, molecular level understanding about its fluorescence behavior and the photophysical properties in different solvents are still unknown. Hence, the photophysical properties of 7F-Trp in presence of biochemically important alcoholic solvents ethanol (ETH), 2,2,2 trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoro-propan-2-ol (HFIP) has been investigated using spectroscopic, quantum chemical calculation, molecular dynamic simulation and compared with Trp to understand the role of fluorine in its fluorescence behavior. The substitution of fluorine on Trp decreases its fluorescence intensity significantly. The presence of fluorine on tryptophan perturbs the interaction of its backbone containing COO− and NH3+ with the chromophoric indole group which probably decreases the barrier of the conical intersection leading to decreased fluorescence intensity of 7F-Trp compared to Trp. The fluorescence intensity of Trp and 7F-Trp decreases in presence of fluorinated alcohols compared to ETH. However, the decrease in fluorescence intensity of 7F-Trp is less than Trp and peak maxima are blue shifted. Moreover, the trend of excited state lifetime of 7F-Trp in fluorinated alcohols is opposite to Trp suggesting that the interaction of 7F-Trp with fluorinated solvents is different than Trp. The excited state lifetime of Trp in alcoholic solvents is controlled mainly by solvent polarity. In contrast, the weak interaction mediated by the fluorine of 7F-Trp with the fluorine and OH groups of fluorinated solvents plays an important role in modulation of its excited state lifetime. This study clearly depicts that noncanonical fluorine of 7F-Trp plays an important role in controlling its fluorescence properties in the biochemically important alcoholic solvents.

Anomalous fluorescence of white hair compared to other unpigmented keratin fibres.

To demonstrate that the tryptophan (Trp) fluorescence of natural white hair is much weaker than other unpigmented keratin fibres such as wool, cashmere, rabbit hair and mink fur, and to explore possible reasons for this behaviour. The origin of the blue visible fluorescence (~450nm) excited by UVA radiation in the range 360-380nm, often associated with Trp degradation products, is also discussed and compared to other fibrous and globular proteins. As the fluorescence spectrum of keratin fibres usually contains at least two major features, a visual comparison is more effectively demonstrated by creating a 3D contour plot of excitation versus emission wavelength, which is sometimes referred to as an excitation emission matrix (EEM). The Trp fluorescence from white hair is very much weaker than for wool, cashmere, rabbit hair and mink fur, but its visible fluorescence emission is stronger. Oxidation and reduction have little effect on the Trp intensity, which suggests quenching by cystine is not a major factor. Decuticulation of hair fibres had no effect on the Trp intensity showing that the increased number of cuticle scales surrounding the fibre cortex is not responsible. Trp fluorescence is very sensitive to exposure to UVB wavelengths, so possibly its low intensity in hair is due to greater levels of environmental exposure to sunlight than the other fibres examined. Trp fluorescence from natural white hair is either extremely weak or completely absent, in contrast to the four other keratin fibres examined. It is possible that environmental exposure to UV wavelengths presents in sunlight contributes to a reduction in the Trp fluorescence intensity of white hair. However, another explanation is that Trp is quenched, by either an unknown substance introduced into hair during keratinization or as a result of regular exposure to personal care products, which may interact with Trp or tyrosine residues and disrupt the energy transfer process involved in keratin fluorescence. Further studies will be required to definitively determine the cause.

Tyrosine mediated conformational change in bone morphogenetic protein – 2: Biophysical implications of protein – phytoestrogen interaction

The biophysical aspects of the binding interaction between a phytoestrogen (quercetin, QT) and bone morphogenetic protein – 2 (BMP – 2) was analyzed by various spectroscopic, calorimetric and molecular docking techniques. Interaction studies represented a loss in the absorbance of protein (only the amide region) along with a prominent red shift indicating ground-state complexation which was further confirmed by quenching with significant blue shift observed from steady-state fluorescence measurements. To narrow down the involvement of aromatic residues (Tyr & Trp), synchronous fluorescence spectroscopy was employed. Both Tyr and Trp fluorescence intensity was quenched, however, shifting was noticed only in case of Tyr residues; thus, confirming the alteration in confirmation was mediated upon reduction in polarity around tyrosine residues. It was further validated by quenching studies which highlighted the existence of a buried fraction of fluorophore upon interaction. The nature of fluorescence quenching was static and the binding efficiency was low (binding constant K ~ 10−2 M). Mechanistically, the involvement of van der Waals and hydrogen bonding interaction was confirmed from both van't Hoff plot and molecular docking studies. Secondary structure and thermal stability of the protein was not significantly affected by quercetin. All these investigations confirmed a significant effect on the structure and conformation of BMP – 2 in presence of quercetin which might serve as a potential therapeutic for the treatment of osteoporosis in postmenopausal women.

Anomalous Fluorescence of White Hair Compared to Other Unpigmented Keratin Fibres.

To demonstrate that the tryptophan (Trp) fluorescence of natural white hair is much weaker than other unpigmented keratin fibres such as wool, cashmere, rabbit hair and mink fur, and to explore possible reasons for this behaviour. The origin of the blue visible fluorescence (~450 nm) excited by UVA radiation in the range 360-380 nm, often associated with Trp degradation products, is also discussed and compared to other fibrous and globular proteins. As the fluorescence spectrum of keratin fibres usually contains at least two major features, a visual comparison is more effectively demonstrated by creating a 3D contour plot of excitation versus emission wavelength, which is sometimes referred to as an excitation emission matrix (EEM). The Trp fluorescence from white hair is very much weaker than for wool, cashmere, rabbit hair and mink fur, but its visible fluorescence emission is stronger. Oxidation and reduction have little effect on the Trp intensity, which suggests quenching by cystine is not a major factor. Decuticulation of hair fibres had no effect on the Trp intensity showing that the increased number of cuticle scales surrounding the fibre cortex is not responsible. Trp fluorescence is very sensitive to exposure to UVB wavelengths, so possibly its low intensity in hair is due to greater levels of environmental exposure to sunlight than the other fibres examined. Trp fluorescence from natural white hair is either extremely weak or completely absent, in contrast to the four other keratin fibres examined. It is possible that environmental exposure to UV wavelengths present in sunlight contributes to a reduction in the Trp fluorescence intensity of white hair. However another explanation is that Trp is quenched, by either an unknown substance introduced into hair during keratinisation, or as a result of regular exposure to personal care products, which may interact with Trp or tyrosine residues and disrupt the energy transfer process involved in keratin fluorescence. Further studies will be required to definitively determine the cause.

Inactivation and structural alteration of α-amylase by low-pressure carbon dioxide microbubbles

Abstract The efficiency of low-pressure carbon dioxide microbubbles (CO2MB) to inactivate α-amylase was analysed kinetically, and structural alteration of α-amylase by CO2MB was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence analysis of tryptophan (Trp) residues. Activity and Trp fluorescence intensity of α-amylase treated by CO2MB decreased with increasing temperature, pressure and exposure time, and lowering the initial buffer pH, respectively. In the kinetic analysis, it was confirmed that the decreased temperature-dependency and increased activation energy associated with the inactivation of α-amylase by CO2MB were induced by pressurizing the mixing vessel and that the decreased pressure-dependency and increased activation volume concomitant to the inactivation of α-amylase by CO2MB was induced by increasing the temperature in the heating coil. In SDS-PAGE, CO2MB was suggested to induce the structural alteration of α-amylase because the band density decreased after CO2MB treatment, although this phenomenon was not related to the inactivation efficiency. However, Trp fluorescence analysis showed that the alteration of the tertiary structure of α-amylase by CO2MB was related to the inactivation efficiency. Therefore, CO2MB was more effective than thermal treatment in inactivating α-amylase, and the inactivation efficiency was suggested to be related to the alteration of the enzyme’s tertiary structure.

A tryptophan synchronous and normal fluorescence study on bacteria inactivation mechanism

The UV photodissociation kinetics of tryptophan amino acid, Trp, attached to the membrane of bacteria, Escherichia coli and Bacillus subtilis, have been studied by means of normal and synchronous fluorescence. Our experimental data suggest that the fluorescence intensity of Trp increases during the first minute of irradiation with 250 nm to ∼ 280 nm, 7 mW/cm2 UV light, and subsequently decreases with continuous irradiation. During this short, less than a minute, period of time, 70% of the 107 cell per milliliter bacteria are inactivated. This increase in fluorescence intensity is not observed when tryptophan is in the free state, namely, not attached to a protein, but dissolved in water or saline solution. This increase in fluorescence is attributed to the additional fluorescence of tryptophan molecules formed by protein unfolding, the breakage of the bond that attaches Trp to the bacterial protein membrane, or possibly caused by the irradiation of 2 types of tryptophan residues that photolyze with different quantum yields.

Probing the Tryptophan Environment in Therapeutic Proteins: Implications for Higher Order Structure on Tryptophan Oxidation

Tryptophan (Trp) oxidation in proteins leads to a number of events, including changes in color, higher order structure (HOS), and biological activity. We describe here a number of new findings through a comprehensive characterization of 6 monoclonal antibodies (mAbs) following selective oxidation of Trp residues by 2,2'-azobis(2-amidinopropane) dihydrochloride. Fluorescence spectroscopy, in combination with second derivative analysis, demonstrates that the loss of Trp fluorescence intensity is a sensitive indicator of Trp oxidation in mAbs. Size-exclusion chromatography with UV and intrinsic Trp fluorescence detection was demonstrated to be a useful method to monitor Trp oxidation levels in mAbs. Furthermore, the Trp oxidation levels measured by size-exclusion chromatography with UV and intrinsic Trp fluorescence detection were found to be in agreement with the values obtained from tryptic peptide mapping by liquid chromatography with mass spectrometric detection and correlate with the total solvent accessible surface area of the exposed Trp residues from in silico modeling. Finally, near-UV circular dichroism and Raman spectroscopy were used to evaluate the impact of Trp oxidation on HOS and identify specific oxidation products, respectively. This work demonstrates that protein HOS is altered on Trp oxidation in mAbs and multiple spectroscopic markers can be used to monitor the molecule-dependent Trp oxidation behavior.

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2W208F, HET-C2W208A and HET-C2F149Y all retained >90% activity and 80–90% intrinsic Trp fluorescence intensity; whereas HET-C2F149A transfer activity decreased to ~55% but displayed ~120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~85–90%) originates from Trp109. This conclusion was supported by HET-C2W109Y/F149Y which displayed ~8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λmax by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~30–40%) and λmax blue-shifts (~12nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λmax blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λmax blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.

Tryptophan and Non-Tryptophan Fluorescence of the Eye Lens Proteins Provides Diagnostics of Cataract at the Molecular Level

The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of “free” Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their “free” counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age.

Conformational analysis of champedak galactose-binding lectin under different urea concentrations

Conformational analysis of champedak galactose-binding (CGB) lectin under different urea concentrations was studied in phosphate-buffered saline (pH 7.2) using far-ultraviolet circular dichroism (far-UV CD), tryptophan (Trp) fluorescence and ANS fluorescence. In all cases, CGB lectin displayed a two-step, three-state transition. The first transition (from the native state to the intermediate state) started at ∼2.0 M urea and ended at ∼4.5 M urea, while the second transition (from the intermediate state to the completely denatured state) was characterized by the start- and end-points at ∼5.75 M and ∼7.5 M urea, respectively, when analyzed by the emission maximum of Trp fluorescence. A marked increase in the Trp fluorescence, ANS fluorescence and −CD values at 218 nm (−CD218 nm) represented the first transition, whereas a decrease in these parameters defined the second transition. On the other hand, emission maximum of the Trp fluorescence showed a continuous increase throughout the urea concentration range. Transformation of tetramer into monomer represented the first transition, whereas the second transition reflected the unfolding of monomer. Far-UV CD, Trp fluorescence and ANS fluorescence spectra were used to characterize the native, the intermediate and the completely denatured states of CGB lectin, obtained at 0.0 M, 5.0 M and 9.0 M urea, respectively. The intermediate state was characterized by the presence of higher secondary structures, increased ANS binding as well as increased Trp fluorescence intensity. A gradual decrease in the hemagglutination activity of CGB lectin was observed with increasing urea concentrations, showing complete loss at 4.0 M urea.

Tryptophan-[Re6Se8I6]3− Cluster Interaction: A Computational Study

The interaction between tryptophan (Trp) and the [Re6Se8I6]3− cluster is studied here using density functional theory calculations including relativistic scalar interactions via the zero-order regular approximation and solvent effects, which we describe in terms of interaction energies, Mulliken charge analysis and molecular orbitals. In the indole functional groups of the Trp-[Re6Se8I6]3− cluster conjugates, modification of the molecular orbitals associated with Trp occurs because states associated with the [Re6Se8I6]3− cluster and the hybrid orbitals have a mixed metal–molecule character, which is attributed to the stronger than expected π interactions facilitated by the indole group in Trp. Here, we find that the energy transfer from hybrid states of Trp-[Re6Se8I6]3− to clusters could reduce intrinsic fluorescence intensity of Trp in the conjugated system.

Tryptophan Point Mutants of HET-C2, a Fungal Glycolipid Transfer Protein, Provide Insights into Glycolipid Binding/Transfer and Membrane Interaction

Fungal glycolipid transfer protein (GLTP), HET-C2, forms a GLTP-fold, but with higher binding/transfer preference for simple neutral glycosphingolipids than broad-selectivity mammalian GLTPs (Kenoth et al., 2010, J. Biol. Chem. 285, 13066-13078). In human GLTP, three Trp residues function in glycolipid binding (Trp96), membrane interaction (Trp142) and protein fold stabilization (Trp85). HET-C2 contains two Trp residues. Trp109 is structurally homologous to GLTP Trp96, consistent with function as a stacking plate that orients the ceramide-linked sugar during hydrogen bonding with Asp66, Asn70, and Lys73. HET-C2 Trp208 resides on the surface in a different location than either Trp142 or Trp85 of GLTP. In this study, we generated single-Trp mutants of HET-C2 to assess potential functional roles. Phe149, a structural homologue of GLTP Trp142, was also investigated for potential function in membrane interaction. W208F-, W208A- and F149Y-HET-C2 retained >90% activity and 80-90% intrinsic Trp intensity; whereas F149A-HET-C2 transfer activity decreased to ∼60% but displayed ∼120% intrinsic Trp intensity. W109Y/F149Y-HET-C2 was nearly inactive and displayed ∼8% intrinsic Trp intensity. Thus, neither W208 nor F149 is essential for activity and most Trp emission intensity (∼90%) originates from Trp109. With wtHET-C2, incubation with POPC vesicles containing glycolipid decreases Trp fluorescence intensity (25-30%) and blue-shifts Δmax (6-7 nm). For HET-C2 mutants involving Trp208 or F149, the intensity changes and Δmax blue-shifts induced by vesicles containing glycolipid become elevated and similar to human GLTP (∼30-40% intensity decrease and ∼12 nm Δmax blue-shift). Only W109Y/F149Y-HET-C2, which lacks Trp109, deviated from the pattern, showing that the Trp Δmax blue-shift is diagnostic for glycolipid binding in the HET-C2 GLTP-fold; whereas, the Trp intensity decrease reflects both glycolipid binding and nonspecific bilayer interaction [Support: NIH/NIGMS-GM45928, NIH/NCI-CA121493, Russian Fdn. Basic Research 012-04-00168, Hormel Fdn.]

Fluorescence quenching studies on the interaction of riboflavin with tryptophan and its analytical application

The ineraction between riboflavin (RBF) and tryptophan (Trp) was investigated using fluorescence spectroscopy and UV-vis absorption spectroscopy under physiological conditions. The fluorescence of Trp was quenched by RBF via dynamic quenching, which was analyzed using the Stern-Volmer relation. The value of the Forster distance R0 (2.31 nm) was obtained according to the Forster's theory of nonradiative energy transfer. Under physiological conditions, a linear relationship could be established between the quenched fluorescence intensity of Trp and the concentration of RBF in the range of 5.8 × 10(-7) -2.0 × 10(-5) mol/L. The detection limit was 1.8 × 10(-7) mol/L. The method was successfully applied to determine riboflavin concentrations in pharmaceutical samples.

Tryptophan Rotamer Distribution Revealed for the α-Helix in Tear Lipocalin by Site-Directed Tryptophan Fluorescence

Rotamer libraries are a valuable tool for protein structure determination, modeling, and design. Site-directed tryptophan fluorescence (SDTF) was used in combination with the rotamer model for the fluorescence intensity decays to solve α-helical conformations of proteins in solution. Single Trp mutations located in an α-helical segment of human tear lipocalin were explored for structure assignment. Along with fluorescence λ(max) values, the rotamer model assignment of fluorescence lifetimes fits the backbone conformation. Typically, Trp fluorescence in proteins shows three lifetimes. However, for the α-helix, two lifetimes assigned to t and g(-) rotamers were satisfactory to describe Trp fluorescence intensity decays. The g(+) rotamer is not feasible in the α-helix due to steric restriction. Trp rotamer distributions obtained by fluorescence were compared with the rotamer library derived from X-ray crystallography data of proteins. The Trp rotamer distributions vary for solvent exposed and buried (tertiary interaction) sites. A new strategy using the rotamer distribution with SDTF (RD-SDTF) removes the limitation of regular SDTF and other labeling techniques, in which site-specific differences, e.g., accessibility, are presumed. The RD-SDTF technique does not rely on environmental differences of side chains and is able to detect α-helical structure where all side chains are exposed to solvent. Potentially, this technique is applicable to various proteins including membrane proteins, which are rich in α-helix motif.

Simulations of Tryptophan Fluorescence Dynamics during Folding of the Villin Headpiece

Protein folding kinetics is commonly monitored by changes in tryptophan (Trp) fluorescence intensity. Considerable recent discussion has centered on whether the fluorescence of the single Trp in the much-studied, fast-folding villin headpiece C-terminal domain (HP35) accurately reflects folding kinetics, given the general view that quenching is by a histidine cation (His(+)) one turn away in an α-helix (helix III) that forms early in the folding process, according to published MD simulations. To help answer this question, we ran 1.0 μs MD simulations on HP35 (N27H) and a faster-folding variant in its folded form at 300 K and used the coordinates and force field charges with quantum calculations to simulate fluorescence quenching caused by electron transfer to the local amide and to the His(+). The simulations demonstrate that quenching by His(+) in the fully formed helix III is possible only during certain Trp and His(+) rotamer and solvent conformations, the propensity of which is a variable that can allow Trp fluorescence to report the global folding rate, as recent experiments imply.

Deciphering the role of pH in the binding of Ciprofloxacin Hydrochloride to Bovine Serum Albumin

The effect of the added fluoroquinolone, Ciprofloxacin Hydrochloride (CpH), on structural properties of Bovine Serum Albumin (BSA) was investigated by Circular Dichroism (CD), steady-state, time-resolved and Dynamic Light Scattering (DLS) spectroscopic approaches. The intrinsic fluorescence of the Tryptophan (Trp) amino acid residue in the globular protein BSA was made use of and the effect of pH at two different temperatures was thoroughly investigated. CD results indicate that CpH induces some structural changes in BSA and this has been well-supported by steady-state, lifetime and DLS data. The fluorescence intensity of Trp gradually decreases with the rise in concentration of CpH and we have conclusively proved that at pH 7.4 and 9.2, the mechanism of fluorescence quenching is mostly dynamic in nature, whereas at pH 4.5 mainly static quenching is operational. Thermodynamic parameters have been studied to rationalize the nature of binding of CpH to BSA, and we have concluded that hydrophobic and van der Waals forces play an important role in the process of drug-protein interaction at three different pH values. The lifetime of Trp was found to decrease with the rise in CpH concentration and the percentage reduction in lifetime was found to be a function of the pH of the medium under investigation.

An Effector of Hemoglobin Structure: The Guanosine 3', 5'-Triphosphate

The effect of guanosine 3', 5'-triphosphate (GTP) on the hemoglobin structure was studied by UV-visible, fluorescence and circular dichroism (CD) spectroscopies, and cyclic voltammetry. UV-visible absorption spectra showed an increase in absorbance in the regions of 420 nm and 280 nm. Fluorescence spectra showed that the Trp fluorescence intensity increased upon excitation at 280 nm, when guanosine 3', 5'-triphosphate concentration was increased in hemoglobin solution. Along with the increased fluorescence intensity, a slightly shift of λmax was also observed toward the higher wavelengths. CD spectral analysis demonstrated a significant decrease in negative ellipsity in the region of 205–235 nm. After adding guanosine 3', 5'-triphosphate to the hemoglobin solution α-helix structure decreases by 20 % while β-sheet conformation increases by 9 %. The effects of GTP on hemoglobin resulted in a 61 mV shift in the cathodic and 40 mV for anodic peak of hemoglobin in the CD. Our data showed the change of secondary and tertiary structure of hemoglobin in the presence of guanosine 3', 5'-triphosphate. (doi: 10.5562/cca1955)

Tryptophan–Gold Nanoparticle Interaction: A First-Principles Quantum Mechanical Study

The nature of interaction between tryptophan (Trp) and gold (Au) nanoparticles is studied using first-principles density functional theory calculations and described in terms of equilibrium configurations, interaction energies, density of states, molecular orbitals, and charge density. The calculated results find the binding involving both carboxyl and indole functional groups with mixed salt–bridge and charge–solvation structure to be energetically preferred and is attributed to the stronger-than-expected π interactions facilitated by the indole group in Trp. In the Trp–Au conjugates, modification of the molecular orbitals associated with Trp occurs because states associated with Au and the hybrid orbitals have mixed metal–molecule character. We find that the nonradiative energy transfer from excited states of Trp to hybrid states of Trp–Au may reduce intrinsic fluorescence intensity of Trp in the conjugated system.