Binding of naproxen enantiomers to human serum albumin studied by fluorescence and room-temperature phosphorescence.

The interaction of the enantiomers of the non-steroidal anti-inflammatory drug naproxen (NPX) with human serum albumin (HSA) has been investigated using fluorescence and phosphorescence spectroscopy in the steady-state and time-resolved mode. The absorption, fluorescence excitation, and fluorescence emission spectra of (S)-NPX and (R)-NPX differ in shape in the presence of HSA, indicating that these enantiomers experience a different environment when bound. In solutions containing 0.2M KI, complexation with HSA results in a strongly increased NPX fluorescence intensity and a decreased NPX phosphorescence intensity due to the inhibition of the collisional interaction with the heavy atom iodide. Fluorescence intensity curves obtained upon selective excitation of NPX show 8-fold different slopes for bound and free NPX. No significant difference in the binding constants of (3.8±0.6)×10(5) M(-1) for (S)-NPX and (3.9±0.6)×10(5) M(-1) for (R)-NPX was found. Furthermore, the addition of NPX quenches the phosphorescence of the single tryptophan in HSA (Trp-214) based on Dexter energy transfer. The short-range nature of this mechanism explains the upward curvature of the Stern-Volmer plot observed for HSA: At low concentrations NPX binds to HSA at a distance from Trp-214 and no quenching occurs, whereas at high NPX concentrations the phosphorescence intensity decreases due to dynamic quenching by NPX diffusing into site I from the bulk solution. The dynamic quenching observed in the Stern-Volmer plots based on the longest phosphorescence lifetime indicates an overall binding constant to HSA of about 3×10(5) M(-1) for both enantiomers.

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.

Complementary fluorescence and phosphorescence study of the interaction of brompheniramine with human serum albumin.

Binding of the antihistamine drug brompheniramine (BPA) to human serum albumin (HSA) is studied by measuring quenching of the fluorescence and room temperature phosphorescence (RTP) of tryptophan. The modified Stern-Volmer equation was used to derive association constants and accessible fractions from the steady-state fluorescence data. Decay associated spectra (DAS) revealed three tryptophan fluorescence lifetimes, indicating the presence of three HSA conformations. BPA causes mainly static quenching of the long-living, solvent-exposed conformer. RTP spectra and lifetimes, recorded under deoxygenated conditions in the presence of 0.2 M KI, provided additional kinetic information about the HSA-BPA interactions. Fluorescence DAS that were also recorded in the presence of 0.2 M KI revealed that the solvent-exposed conformer is the major contributor to the RTP signal. The phosphorescence quenching is mostly dynamic at pH 7 and mostly static at pH 9, presumably related to the protonation state of the alkylamino chain of BPA. This provides direct insight into the binding mode of the antihistamine drug, as well as kinetic information at both the nanosecond and the millisecond time scales.

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.

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.

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.

The tandem zinc-finger region of human ZHX adopts a novel C2H2 zinc finger structure with a C-terminal extension.

Binding of the nuclear factor-Y complex (NF-Y) to the inverted CCAAT-box interferes with transcription activation through nucleosome reorganization. The three homologous proteins forming the zinc-fingers and homeoboxes (ZHX) family interact with the activation domain of NF-Ya to repress transcription. Each ZHX-protein contains two generic C2H2 zinc-fingers (ZNF1 and ZNF2) followed by five homeodomains. Although the proteins have been related to the occurrence of certain cancers, the function and structure of the individual ZHX domains are still unknown. Here, we determined the structure of the tandem zinc-finger region of human ZHX1. Folding and secondary structure predictions combined with expression screening revealed that the C-terminal extension (E) to ZNF2 could form a single domain with the two hZHX1 zinc-fingers. We therefore decided to determine the solution structure of the zinc-fingers followed by this extension. We show that both zinc-fingers adopt canonical betabetaalpha-folds in which a zinc ion is coordinated by two cysteine and two histidine residues. The C-terminal extension to ZNF2 forms two beta-strands to make a beta-sheet with the beta-strands of this zinc-finger. The ZNF1 and ZNF2-E domains do not show evident contacts and their mutual orientation seems variable. The high degree of sequence conservation among ZHX family members permitted us to prepare homology models for ZHX2 and ZHX3, revealing distinct surface characteristics for each family member. Implications of these structural features for ZHX-functioning in transcription regulation are discussed.

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.