Stereoselective binding of flurbiprofen enantiomers and their methyl esters to human serum albumin studied by time-resolved phosphorescence.

The interaction of the nonsteroidal anti-inflammatory drug flurbiprofen (FBP) with human serum albumin (HSA) hardly influences the fluorescence of the protein's single tryptophan (Trp). Therefore, in addition to fluorescence, heavy atom-induced room-temperature phosphorescence is used to study the stereoselective binding of FBP enantiomers and their methyl esters to HSA. Maximal HSA phosphorescence intensities were obtained at a KI concentration of 0.2 M. The quenching of the Trp phosphorescence by FBP is mainly dynamic and based on Dexter energy transfer. The Stern-Volmer plots based on the phosphorescence lifetimes indicate that (R)-FBP causes a stronger Trp quenching than (S)-FBP. For the methyl esters of FBP, the opposite is observed: (S)-(FBPMe) quenches more than (R)-FBPMe. The Stern-Volmer plots of (R)-FBP and (R)-FBPMe are similar although their high-affinity binding sites are different. The methylation of (S)-FBP causes a large change in its effect on the HSA phosphorescence lifetime. Furthermore, the quenching constants of 3.0 × 10(7) M(-1) s(-1) of the R-enantiomers and 2.5 × 10(7) M(-1) s(-1) for the S-enantiomers are not influenced by the methylation and indicate a stereoselectivity in the accessibility of the HSA Trp to these drugs.

Noninvasive detection of concealed explosives: depth profiling through opaque plastics by time-resolved Raman spectroscopy.

The detection of explosives concealed behind opaque, diffusely scattering materials is a challenge that requires noninvasive analytical techniques for identification without having to manipulate the package. In this context, this study focuses on the application of time-resolved Raman spectroscopy (TRRS) with a picosecond pulsed laser and an intensified charge-coupled device (ICCD) detector for the noninvasive identification of explosive materials through several millimeters of opaque polymers or plastic packaging materials. By means of a short (250 ps) gate which can be delayed several hundred picoseconds after the laser pulse, the ICCD detector allows for the temporal discrimination between photons from the surface of a sample and those from deeper layers. TRRS was applied for the detection of the two main isomers of dinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene as well as for various other components of explosive mixtures, including akardite II, diphenylamine, and ethyl centralite. Spectra were obtained through different diffuse scattering white polymer materials: polytetrafluoroethylene (PTFE), polyoxymethylene (POM), and polyethylene (PE). Common packaging materials of various thicknesses were also selected, including polystyrene (PS) and polyvinyl chloride (PVC). With the demonstration of the ability to detect concealed, explosives-related compounds through an opaque first layer, this study may have important applications in the security and forensic fields.

Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography.

Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-ß-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of L-MTX and its enantiomeric impurity D-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10(-7) M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of L-MTX. Furthermore, L-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples.

Computational study on the anomalous fluorescence behavior of isoflavones.

Isoflavones are known to show fluorescence with intensities that depend strongly on the solvent properties and exhibit Stokes' shifts as large as 1.4 eV. While some of this behavior can be explained by (excited state) deprotonation, this mechanism does not apply for all isoflavones. The aim of this study is to computationally and experimentally investigate the reasons for this anomalous behavior of neutral isoflavones, taking the daidzein molecule as a model compound. We find that the absence in fluorescence in aprotic solvents and the weak fluorescence in protic solvents can be explained by a change of order of the lowest singlet states in which a fluorescent charge-transfer state lies below the nonfluorescent locally excited state in water but not in acetonitrile. The large Stokes' shift is partly due to a significant rotation among the chromone-phenyl bond in the excited state.

Sensitized enantioselective laser-induced phosphorescence detection in chiral capillary electrophoresis.

The sensitivity of enantioselective cyclodextrin-induced room-temperature phosphorescence detection of camphorquinone (CQ) is enhanced using sensitization via a donor with a high extinction coefficient. The enantiomeric distinction is based on the different phosphorescence lifetimes of (+)-CQ and (-)-CQ after their complexation with α-cyclodextrin (α-CD). The collisional Dexter energy transfer from the selected donor 2,6-naphthalenedisulfonic acid (2,6-NS) to the acceptor CQ is still very efficient despite the inclusion of the acceptor into CD. For coupling to the chiral separation of (±)-CQ in cyclodextrin-based electrokinetic chromatography, the donor was added to the deoxygenated background electrolyte that consisted of 20 mM α-CD, 10 mM carboxymethyl-ß-CD, and 25 mM borate buffer at pH 9.0. Time-resolved batch studies on sensitized phosphorescence show a significant enantioselectivity for (+)- and (-)-CQ in the presence of both α-CD and CM-ß-CD although the lifetime difference is somewhat reduced with respect to direct excitation. The enantiomers were distinguished after their separation using an online time-resolved detection system. Excitation was performed at 266 nm with a pulsed, small-sized, quadrupled Nd:YAG laser. With 1 × 10(-5) M 2,6-NS, limits of detection of 4.1 × 10(-8) M and 5.2 × 10(-8) M were found for (+)-CQ and (-)-CQ, respectively. The online measured lifetimes were 238 ± 8 µs for (+)-CQ and 126 ± 10 µs for (-)-CQ. The method was used to determine the concentration of (±)-CQ leaching from a cured dental resin into water. The extracts contained 4.7 ± 0.1 × 10(-7) M of both (+)-CQ and (-)-CQ.

Sensitized phosphorescence as detection method for the enantioseparation of bupropion by capillary electrophoresis.

A new CE detection method was developed for the chiral drug bupropion (a second-generation antidepressant), based on phosphorescence both in the direct and in the sensitized mode using pulsed laser excitation at 266 nm. Electrokinetic chromatography using 5 mM sulfated-α-CD as chiral selector in 25 mM phosphate buffer at pH 3 allowed the separation of bupropion enantiomers with a high chiral resolution (Rs>3). In the sensitized phosphorescence detection mode, excitation energy is transferred from the analyte to an acceptor (1-bromo-4-napthhalenesulfonic acid or biacetyl) followed by time-resolved phosphorescence detection under deoxygenated buffer conditions. Using 2 × 10(-4) M biacetyl as the acceptor an LOD of 2 × 10(-7) M was obtained for each enantiomer, about 40 times better than in the direct mode. Under these separation conditions, no significantly different phosphorescence lifetimes (measured on-line) were obtained for the two bupropion enantiomers. The suitability of the method was demonstrated with the quantification of bupropion in a pharmaceutical formulation and its determination in a spiked urine sample.

The impact of urea-induced unfolding on the redox process of immobilised cytochrome c.

We have studied the effect of urea-induced unfolding on the electron transfer process of yeast iso-1-cytochrome c and its mutant K72AK73AK79A adsorbed on electrodes coated by mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol self-assembled monolayers. Electrochemical measurements, complemented by surface enhanced resonance Raman studies, indicate two distinct states of the adsorbed proteins that mainly differ with respect to the ligation pattern of the haem. The native state, in which the haem is axially coordinated by Met80 and His18, displays a reduction potential that slightly shifts to negative values with increasing urea concentration. At urea concentrations higher than 6 M, a second state prevails in which the Met80 ligand is replaced by an additional histidine residue. This structural change in the haem pocket is associated with an approximately 0.4 V shift of the reduction potential to negative values. These two states were found for both the wild-type protein and the mutant in which lysine residues 72, 73 and 79 had been substituted by alanines. The analysis of the reduction potentials, the reaction enthalpies and entropies as well as the rate constants indicates that these three lysine residues have an important effect on stabilising the protein structure in the adsorbed state and facilitating the electron transfer dynamics.

Time-resolved spatially offset Raman spectroscopy for depth analysis of diffusely scattering layers.

The objective of this study is to use time-resolved (TR) Raman spectroscopy, spatially offset Raman spectroscopy (SORS), and a combination of these approaches to obtain high quality Raman spectra from materials hidden underneath an opaque layer. Both TR Raman and SORS are advanced techniques that allow for an increased relative selectivity of photons from deeper layers within a sample. Time-resolved detection reduces fluorescence background, and the selectivity for the second layer is improved. By combining this with spatially offset excitation we additionally increased selectivity for deeper layers. Test samples were opaque white polymer blocks of several mm thicknesses. Excitation was carried out with a frequency-doubled Ti:sapphire laser at 460 nm, 3 ps pulse width and 76 MHz repetition rate. Detection was either with a continuous-wave CCD camera or in time-resolved mode using an intensified CCD camera with a 250 ps gate width. The Raman photons were collected in backscatter mode, with or without lateral offset. By measuring the delay of the Raman signal from the second layer (polyethylene terephthalate/PET/Arnite), the net photon migration speeds through Teflon, polythene, Delrin and Nylon were determined. Raman spectra could be obtained from a second layer of PET through Teflon layers up to 7 mm of thickness. The ability to obtain chemical information through layers of diffusely scattering materials has powerful potential for biomedical applications.

Direct spectroscopic evidence of 8- and 9-fold coordinated europium(III) species in H(2)O and D(2)O.

In the present paper a detailed analysis of high-resolution luminescence spectra of Eu(III)-H(2)O species in frozen aqueous solution (T = 5 K) is presented. From the total luminescence spectra (TLS, excitation vs emission) and the luminescence decay matrixes (time vs emission), fundamental species-selective spectroscopic parameters are determined: excitation wavelength λ(exc), decay time τ, crystal field energy splitting ΔE (crystal field strength parameter N(ν)(B(2q))), crystal field parameters B(20) and B(22), asymmetry ratio r, and point symmetry group. The spectroscopic findings clearly show the presence of two distinct Eu(III) aquo species. Samples prepared with different counterions (Cl(-), ClO(4)(-)) and at different pH values (2 and 5) yielded comparable results. Furthermore, in D(2)O solutions the same two species were found, with similar spectral properties but much longer decay times. On the basis of the spectroscopic analysis, the two species were attributed to 8- and 9-fold coordinated Eu(III) aquo ions.

Excited state processes of 2-butylamino-6-methyl-4-nitropyridine N-oxide in nonpolar solvents. A transient absorption spectroscopy study.

Earlier steady-state fluorescence studies showed that 2-butylamino-6-methyl-4-nitropyridine N-oxide (2B6M) can undergo fast excited-state intramolecular proton transfer (ESIPT). In a nonpolar solvent such as n-octane, both normal and tautomeric fluorescence was observed. Strikingly, the relative ratio of those two emission bands and the fluorescence quantum yield of the normal emission were found to depend on the excitation wavelength in violation of the Kasha-Vavilov rule. In this work, the system was investigated further by means of transient absorption spectroscopy, followed by global and target analysis. Upon excitation at 420 nm, a normal excited singlet state S(1)(N) is reached, which decays in about 12 ps via fluorescence and ESIPT (minor pathways) and to a long-lived "dark" state (major pathway) that is most probably the triplet T(1)(N). Upon 330 nm excitation, however, a more complex pattern emerges and additional decay channels are opened. A set of four excited-state species is required to model the data, including a hot state S(1)(N)* that decays in about 3 ps to the tautomer, to the long-lived "dark" state and to the relaxed S(1)(N) state. A kinetic scheme is presented that can explain the observed transient absorption results as well as the earlier fluorescence data.

Phosphorescence for sensitive enantioselective detection in chiral capillary electrophoresis.

Enantioselective phosphorescence lifetime detection was combined with chiral cyclodextrin-based electrokinetic chromatography for the analysis of camphorquinone (CQ). A time-gated detection system based on a pulsed light-emitting diode for excitation at 465 nm was developed for the online lifetime determination. The background electrolyte for the chiral separation consisted of 20 mM alpha-cyclodextrin (alpha-CD), 10 mM carboxymethyl-beta-CD, and 25 mM borate buffer at pH 9.0. The separation of (+)-CQ and (-)-CQ is caused by a difference in association constants of these enantiomers with alpha-CD. Under the separation conditions, different phosphorescence lifetimes were obtained for (+)-CQ and (-)-CQ (tau = 384 +/- 8 and 143 +/- 5 micros, respectively), which could be used to distinguish the enantiomers. This selectivity in detection is based on a difference in protection of the enantiomers against phosphorescence quenching after their complexation with alpha-CD. Concentration detection limits were 2 x 10(-7) and 1 x 10(-6) M for (+)-CQ and (-)-CQ, respectively. After correction for the lifetime shortening by triplet-triplet annihilation at higher CQ concentrations, a linear dynamic range was obtained from the detection limit up to 2 mM. The system was used to determine the enantiomeric impurity levels of commercial samples of (+)-CQ and (-)-CQ; 0.2% and 0.1%, respectively.

Effectiveness of charged noncovalent polymer coatings against protein adsorption to silica surfaces studied by evanescent-wave cavity ring-down spectroscopy and capillary electrophoresis.

Protein adsorption to silica surfaces is a notorious problem in analytical separations. Evanescent-wave cavity ring-down spectroscopy (EW-CRDS) and capillary electrophoresis (CE) were employed to investigate the capability of positively charged polymer coatings to minimize the adsorption of basic proteins. Adsorption of cytochrome c (cyt c) to silica coated with a single layer of polybrene (PB), or a triple layer of PB, dextran sulfate (DS), and PB, was studied and compared to bare silica. Direct analysis of silica surfaces by EW-CRDS revealed that both coatings effectively reduce irreversible protein adsorption. Significant adsorption was observed only for protein concentrations above 400 microM, whereas the PB-DS-PB coating was shown to be most effective and stable. CE analyses of cyt c were performed with and without the respective coatings applied to the fused-silica capillary wall. Monitoring of the electroosmotic flow and protein peak areas indicated a strong reduction of irreversible protein adsorption by the positively charged coatings. Determination of the electrophoretic mobility and peak width of cyt c revealed reversible protein adsorption to the PB coating. It is concluded that the combination of results from EW-CRDS and CE provides highly useful information on the adsorptive characteristics of bare and coated silica surfaces toward basic proteins.

Anomalous photophysics of H1 antihistamines in aqueous solution.

Electronic absorption, emission, and excitation spectra, and fluorescence lifetimes of two H1 antihistamines--tripelennamine and mepyramine--are investigated in detail to ascertain their usefulness as fluorescent probes for ligand binding to G-protein coupled receptors. The photophysical behavior of these compounds in aqueous solution is complex due to the presence of three protonable nitrogens, intramolecular hydrogen bonding, quenching due to the formation of a charge transfer state, and intramolecular fluorescence resonance energy transfer. At physiological pH values, anomalous photophysical behavior is observed: the compounds are found to be in a ground-state equilibrium mixture of two species, one with the alkylamine tail involved in an intramolecular hydrogen bond and a second without such a bond. This internal hydrogen-bonded tail has a profound effect on the ground and excited-state properties of both tripelennamine and mepyramine, which is further elucidated by comparing them to the reference compounds 2-aminopyridine and 2-(N,N-dimethylamino)pyridine.

Picosecond Raman spectroscopy with a fast intensified CCD camera for depth analysis of diffusely scattering media.

A spectroscopic depth profiling approach is demonstrated for layers of non-transparent, diffusely scattering materials. The technique is based on the temporal discrimination between Raman photons emitted from the surface and Raman photons originating from a deeper layer. Excitation was carried out with a frequency-doubled, 3 ps Ti:sapphire laser system (398 nm; 76 MHz repetition rate). Time-resolved detection was carried out with an intensified CCD camera that can be gated with a 250 ps gate width. The performance of the system was assessed using 1 mm and 2 mm pathlength cuvettes with powdered PMMA and trans-stilbene (TS) crystals, respectively, or solid white polymer blocks: Arnite (polyethylene terephthalate), Delrin (polyoxymethylene), polythene (polyethylene) and Teflon (polytetrafluoroethylene). These samples were pressed together in different configurations and Raman photons were collected in backscatter mode in order to study the time difference in such media corresponding with several mm of extra net photon migration distance. We also studied the lateral contrast between two different second layers. The results demonstrate that by means of a picosecond laser system and the time discrimination of a gated intensified CCD camera, molecular spectroscopic information can be obtained through a turbid surface layer. In the case of the PMMA/TS two-layer system, time-resolved detection with a 400 ps delay improved the relative intensity of the Raman bands of the second layer with a factor of 124 in comparison with the spectrum recorded with a 100 ps delay (which is more selective for the first layer) and with a factor of 14 in comparison with a non-gated setup. Possible applications will be discussed, as well as advantages/disadvantages over other Raman techniques for diffusely scattering media.

Excited-state double proton transfer in 1H-pyrazolo[3,4-b]quinoline dimers.

Pyrazoloquinolines are highly fluorescent, both in liquid solutions and in the solid state, which makes them good candidates for various optical devices. The aim of the current work is to understand the photochemical behavior of pyrazolo[3,4-b]quinoline (PQ), which is quite complicated since in n-alkane solvents PQ tends to form strong complexes with protic solvent constituents (often present as minor impurities), as well as dimers. Both types of H-bond complexes were studied systematically by temperature-dependent conventional absorption and fluorescence spectroscopy; the effect of protic solvent constituents was mimicked by varying the ethanol concentration in n-octane in the range from 0.0 to 0.8%. At room temperature the PQ:ethanol association constant was estimated at 80 M(-1) and the dimerization constant at 2 x 10(3) M(-1). Dimer formation is enhanced upon lowering the temperature in pure n-alkane down to 220 K, and the fluorescence is strongly reduced since the dimer is nonfluorescent. Surprisingly, when irradiating a frozen sample for several minutes at very low temperatures (<40 K), a narrow-banded Shpol'skii-type fluorescence spectrum gradually appears. To explain this unusual photochemical behavior, PQ and its deuterated analogue were studied using low-temperature absorption and fluorescence spectroscopy over the 300-5 K temperature range. In the case of normal (protonated) PQ, very fast excited-state intermolecular double proton transfer is responsible for the efficient quenching of PQ dimer fluorescence. Deuteration significantly slows down this proton transfer process, and in that case under cryogenic conditions a fluorescent dimer is observed. Photoirradiation under cryogenic conditions leads to molecular rearrangement of the dimers and the appearance of monomer spectra. For both H-PQ and D-PQ, these processes were found to be reversible. A simplified reaction scheme, in which the excited tautomeric dimer plays a crucial role, is presented to explain the observations.

Cryogenic fluorescence and absorption spectroscopy studies on monomeric and dimeric species of 2-butylamino-6-methyl-4-nitropyridine N-oxide.

2-Butylamino-6-methyl-4-nitropyridine-N-oxide (2B6M) belongs to a group of compounds that can undergo not only excited-state intra-, but also intermolecular proton transfer. The latter of course requires the presence of dimeric species. Previously, we have shown that for 2B6M in aprotic non-polar solvents in the liquid state such dimers play no role. Under these conditions, only one single monomeric species exists, exhibiting anomalous fluorescence behavior, i.e. proton transfer not only starting from the lowest excited electronic singlet state, but also from higher excited states. However, we also noted that under frozen, crystalline matrix conditions more species show up in the spectra. In order to study this multi-species system in more detail, we present absorption and fluorescence experiments on 2B6M, recorded in n-octane at various temperatures between 293 and 5 K. High-resolution spectra are included, not only in fluorescence but also in absorption. We demonstrate that under cryogenic conditions three species can be discerned, two of these providing high-resolution spectra with their main 0-0 lines around 452 and 465 nm, respectively. A detailed vibrational analysis of their emission spectra is included. The third species gives broad-banded spectra, in absorption extending to about 520 nm with its long-wavelength maximum around 460 nm, in emission with a maximum around 535 nm. We tentatively assign the three species to a monomer, a H-bonded dimer and a strongly interacting (pi-pi-stacked) dimer, respectively. We conclude from the excitation spectra that (anomalous) intramolecular proton transfer at higher excited states is still operative under cryogenic conditions. Indications for excited-state intermolecular proton transfer in the stacked dimeric species were not found.

Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3.

Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt-Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455-461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV-VIS spectroscopy, as well as coupling efficiency and substrate-binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3.

Fluorescence rejection in resonance Raman spectroscopy using a picosecond-gated intensified charge-coupled device camera.

A Raman instrument was assembled and tested that rejects typically 98-99% of background fluorescence. Use is made of short (picosecond) laser pulses and time-gated detection in order to record the Raman signals during the pulse while blocking most of the fluorescence. Our approach uses an ultrafast-gated intensified charge-coupled device (ICCD) camera as a simple and straightforward alternative to ps Kerr gating. The fluorescence rejection efficiency depends mainly on the fluorescence lifetime and on the closing speed of the gate (which is about 80 ps in our setup). A formula to calculate this rejection factor is presented. The gated intensifier can be operated at 80 MHz, so high repetition rates and low pulse energies can be used, thus minimizing photodegradation. For excitation we use a frequency-tripled or -doubled Ti : sapphire laser with a pulse width of 3 ps; it should not be shorter in view of the required spectral resolution. Other critical aspects tested include intensifier efficiency as a function of gate width, uniformity of the gate pulse across the spectrum, and spectral resolution in comparison with ungated detection. The total instrumental resolution is 7 cm(-1) in the blue and 15 cm(-1) in the ultraviolet (UV) region. The setup allows one to use resonance Raman spectroscopy (RRS) for extra sensitivity and selectivity, even in the case of strong background fluorescence. Excitation wavelengths in the visible or UV range no longer have to be avoided. The effectiveness of this setup is demonstrated on a test system: pyrene in the presence of toluene fluorescence (lambda(exc) = 257 nm). Furthermore, good time-gated RRS spectra are shown for a strongly fluorescent flavoprotein (lambda(exc) = 405 nm). Advantages and disadvantages of this approach for RRS are discussed.

Binding of 7-methoxy-4-(aminomethyl)-coumarin to wild-type and W128F mutant cytochrome P450 2D6 studied by time-resolved fluorescence spectroscopy.

Enzyme structure and dynamics may play a main role in substrate binding and the subsequent steps in the CYP (cytochrome P450) catalytic cycle. In the present study, changes in the structure of human CYP2D6 upon binding of the substrate are studied using steady-state and time-resolved fluorescence methods, focusing not only on the emission of the tryptophan residues, but also on emission of the substrate. As a substrate, MAMC [7-methoxy-4-(aminomethyl)-coumarin] was selected, a compound exhibiting native fluorescence. As well as the wild-type, the W128F (Trp128-->Phe) mutant of CYP2D6 was studied. After binding, a variety of energy transfer possibilities exist, and molecular dynamics simulations were performed to calculate distances and relative orientations of donors and acceptors. Energy transfer from Trp128 to haem appeared to be important; its emission was related to the shortest of the three average tryptophan fluorescence lifetimes observed for CYP2D6. MAMC to haem energy transfer was very efficient as well: when bound in the active site, the emission of MAMC was fully quenched. Steady-state anisotropy revealed that besides the MAMC in the active site, another 2.4% of MAMC was bound outside of the active site to wild-type CYP2D6. The tryptophan residues in CYP2D6 appeared to be less accessible for the external quenchers iodide and acrylamide in presence of MAMC, indicating a tightening of the enzyme structure upon substrate binding. However, the changes in the overall enzyme structure were not very large, since the emission characteristics of the enzyme were not very different in the presence of MAMC.

Hexamerization of the bacteriophage T4 capsid protein gp23 and its W13V mutant studied by time-resolved tryptophan fluorescence.

The bacteriophage T4 capsid protein gp23 was studied using time-resolved and steady-state fluorescence of the intrinsic protein fluorophore tryptophan. In-vitro gp23 consists mostly of monomers at low temperature but forms hexamers at room temperature. To extend our knowledge of the structure and hexamerization characteristics of gp23, the temperature-dependent fluorescence properties of a tryptophan mutant (W13V) were compared to those of wild-type gp23. The W13V mutation is located in the N-terminal part of the protein, which is cleaved off after prohead formation in the live bacteriophage. Results show that W13 plays a role in the hexamerization process but is not needed to stabilize the hexamer once it is formed. Furthermore, besides the monomer-to-hexamer temperature transition (15-23 degrees C and 12-43 degrees C for wild-type and W13V gp23, respectively), we were able to observe denaturation of the N-terminus in hexameric wild-type gp23 around 40 degrees C. In addition, with the aid of a recently published homology model of gp23, the lifetimes obtained from time-resolved fluorescence measurements could tentatively be assigned to specific tryptophan residues.