Characterization of subdomain IIA binding site of human serum albumin in its native, unfolded, and refolded states using small molecular probes

Subdomain IIA binding site of human serum albumin (HSA) was characterized by examining the change in HSA fluorescence in the native, unfolded, and refolded states. The study was carried out in the absence and presence of small molecular probes using steady-state and time-resolved fluorescence measurements. 2-Pyridone, 3-pyridone, and 4-pyridone bear similar molecular structures to those found in many drugs and are used here as probes. They are found to specifically bind in subdomain IIA and cause a reduction in the fluorescence intensity and lifetime of the Trp-214 residue in native HSA which is located in the same subdomain. The efficiency of energy transfer from Trp-214 fluorescence to the probes was found to depend on the degree of the spectral overlap between the donor's fluorescence and the acceptor's absorption. After probe binding in subdomain IIA, the distance between the donor and acceptor was calculated using Forster theory. The calculated quenching rate constants and binding constants were also shown to depend on the degree of spectral overlap. The results point to a static quenching mechanism operating in the complexes. Denaturation of HSA in the presence of guanidine hydrochloride (GdnHCl) starts at [GdnHCl] > 1.0 M and is complete at [GdnHCl] > or = 6.0 M. Upon unfolding, two fluorescence peaks were observed. One peak was assigned to the fluorescence of Trp-214 in a polar environment, and the other peak was assigned to tyrosine fluorescence. A reduction of the fluorescence intensity of the two peaks upon binding of the probes to the denatured HSA indicates that Tyr-263 in subdomain IIA is one of the tyrosine residues responsible for the second fluorescence peak. The results were confirmed by measuring the fluorescence spectra and lifetimes of denatured HSA at different excitation wavelengths, and of L-tryptophan and L-tyrosine free in buffer. The measured lifetimes of denatured HSA are typical of tryptophan in a polar environment and are slightly reduced upon probe binding. Dilution of the denatured HSA by buffer results in a partial refolding of subdomain IIA. This partial refolding is attributed to some swelling of the binding site caused by water. The swelling prevents a full recovery from the denatured state.     

Solvation dynamics of a protein in the pre molten globule state

The nature of solvent molecules around proteins in native and different non-native states is crucial for understanding the protein folding problem. We have characterized two compact denatured states of glutaminyl-tRNA synthetase (GlnRS) under equilibrium conditions in the presence of a naturally occurring osmolyte, l-glutamate. The solvation dynamics of the compact denatured states and the fully unfolded state has been studied using a covalently attached probe, acrylodan, near the active site. The solvation dynamics progressively becomes faster as the protein goes from the native to the molten globule to the pre molten globule to the fully unfolded state. Anisotropy decay measurements suggest that the pre-molten-globule intermediate is more flexible than the molten globule although the secondary structure is largely similar. Dynamic light scattering studies reveal that both the compact denatured states are aggregated under the measurement conditions. The implications of solvation dynamics in aggregated compact denatured states have been discussed.     

Binding of Organic Dyes with Human Serum Albumin: A Single‐Molecule Study

Kinetics of binding of dyes at different sites of human serum albumin (HSA) has been studied by single‐molecule spectroscopy. The protein was immobilized on a glass surface. To probe different binding sites (hydrophobic and hydrophilic) two dyes, coumarin 153 ( C153 , neutral) and rhodamine 6G ( R6G , cationic) were chosen. For both the dyes, a major (ca. 96‐98 %) and minor (ca. 3 %) binding site were detected. Rate constants of association and dissociation were simultaneously determined from directly measuring fluctuations in fluorescence intensity (τ_off and τ_on) and from this the equilibrium (binding) constants were calculated. Fluorescence lifetimes at individual sites were obtained from burst‐integrated lifetime analysis. Distributions of lifetime histograms for both the probes ( C153 and R6G ) exhibit two maxima, which indicates the presence of two binding domains in the protein. Unfolding of the protein has been studied by adding guanidinium hydrochloride (GdnHCl) to the solution. It is observed that addition of GdnHCl affects the dissociation and association kinetics and hence, binding equilibrium of the association of C153 . However, the effect of binding of R6G is not affected much. It is proposed that GdnHCl affects the hydrophobic binding sites more than the hydrophilic site.     

Probing Deuterium Isotope Effect on Structure and Solvation Dynamics of Human Serum Albumin

The deuterium isotopic effect on the structure and solvation dynamics of the protein, human serum albumin (HSA), has been studied by using circular dichroism (CD), femtosecond up‐conversion, FRET, and single‐molecule spectroscopy. The CD spectra suggest that D₂O affects the structure of HSA, leading to a 20 % decrease in the helical structure. The FRET study indicates that the distance of C153 from the lone tryptophan residue of HSA is quite similar (≈21 Å) in H₂O and D₂O, and hence, the location of the probe in the protein remains the same in the two solvents. The single‐molecule study suggests that coumarin 153 (C153) binds almost exclusively (>96 %) to one site of HSA. Solvation dynamics of C153 in HSA is found to be markedly retarded in D₂O compared with H₂O. In H₂O, the solvation of C153 bound to HSA is found to be biexponential with one component of 7 ps (30 %) and a long component of 350 ps (70 %). In D₂O, we detected a short component of 4 ps (41 %) and a long component of 950 ps (59 %). Thus, the ultraslow component of the solvation dynamics of C153 bound to HSA in D₂O (950 ps) is 2.5‐fold slower than that in H₂O (350 ps). The marked deuterium isotope effect has been ascribed to water molecules confined in the protein environment and to a lesser extent to the structural modification of protein by D₂O.     

Interaction of ionic liquid with water with variation of water content in 1-butyl-3-methyl-imidazolium hexafluorophosphate ([bmim][PF6])/TX-100/water ternary microemulsions monitored by solvent and rotational relaxation of coumarin 153 and coumarin 490

The interaction of water with room temperature ionic liquid (RTIL) [bmim][PF6] has been studied in [bmim][PF6]/TX-100/water ternary microemulsions by solvent and rotational relaxation of coumarin 153 (C-153) and coumarin 490 (C-490). The rotational relaxation and average solvation time of C-153 and C-490 gradually decrease with increase in water content of the microemulsions. The gradual increase in the size of the microemulsion with increase in w0 (w0=[water]/[surfactant]) is evident from dynamic light scattering measurements. Consequently the mobility of the water molecules also increases. In comparison to pure water the retardation of solvation time in the RTIL containing ternary microemulsions is very less. The authors have also reported the solvation time of C-490 in neat [bmim][PF6]. The solvation time of C-490 in neat [bmim][PF6] is bimodal with time constants of 400 ps and 1.10 ns.     

Dynamics of solvent and rotational relaxation of Coumarin 153 in a room temperature ionic liquid, 1-butyl-3-methylimidazolium octyl sulfate, forming micellar structure

The solvent and rotational relaxation of Coumarin 153 (C-153) was investigated by picosecond time-resolved fluorescence spectroscopy in a room temperature ionic liquid (RTIL), 1-butyl-3-methylimidazolium octyl sulfate ([C4mim][C8SO4]). This is a typical RTIL, which form micellar structure above certain concentration of the RTIL (0.031 M). Dynamic light scattering (DLS) measurements show that the average hydrodynamic diameter ( Dh) of a [C4mim][C8SO4]-water micelle is 2.8 (+/-0.2) nm. Both the solvent and rotational relaxation of C-153 are retarded in this micelle compared to the solvation time of a similar type of dye in neat water. However, the solvent relaxation in this ionic liquid surfactant is different from that of a conventional ionic surfactant. The slow component of the solvation dynamics in C8H17SO4Na or TX-100 micelle is on the nanoseconds time scale, whereas in [C4mim][C8SO4] micelle the same component is on the subnanoseconds time scale. The different molecular motions with different time scale is the main reason behind this difference in the solvation time in micelles composed of RTIL with other conventional micelles.     

Ultrafast surface solvation dynamics and functionality of an enzyme alpha-chymotrypsin upon interfacial binding to a cationic micelle

In this contribution we report studies on enzymatic activity of alpha-chymotrypsin (CHT) upon complexation with cationic cetyltrimethylammonium bromide (CTAB) micelle. With picosecond time resolution, we examined solvation dynamics at the interface of CHT-micelle complex, and rigidity of the binding. We have used 5-(dimethyl amino) naphthalene-1-sulfonyl chloride (dansyl chloride; DC) that is covalently attached to the enzyme at the surface sites. The solvation processes at the surface of CHT in buffer solution are found to be mostly in the sub-50 ps time scale. However, at the interface the solvation correlation function decays with time constant 150 ps (65%) and 500 ps (35%), which is significantly different from those found at the enzyme and micellar surfaces. The binding structure of the enzyme-micelle complex was examined by local orientational motion of the probe DC and compared with the case without micelle. The orientational dynamics of the probe DC in the complex reveals a structural perturbation at the surface sites of CHT upon complexation, consistent with other reported structural studies. We also found possible entanglement of charge transfer dynamics of the probe DC on the measured solvation processes by using time-resolved area normalized emission spectroscopic technique. The interfacial solvation process and complex rigidity elucidate the strong recognition mechanism between CHT and the micelle, which is important to understand the biological function of CHT upon complexation with the micelle.     

Solvation dynamics in ionic liquid swollen p123 triblock copolymer micelle: a femtosecond excitation wavelength dependence study

Femtosecond solvation dynamics of coumarin 480 (C480) in a mixed micelle is reported. The mixed micelle consists of a triblock copolymer (PEO)20-(PPO) 70-(PEO)20 (Pluronic P123) and an ionic liquid (IL), 1-pentyl-3-methylimidazolium tetrafluoroborate ([pmim][BF4]). At a low concentration (0.3 M), the sparingly water soluble IL ([pmim][BF4]) penetrates the hydrophobic PPO core of the P123 micelles. Thus emission maximum of C480 in the core (accessed at lambdaex=375 nm) in 0.3 M IL is red-shifted by 8 nm from that in its absence and the red edge excitation shift (REES) is large (19+/-1 nm). At a high concentration (0.9 M), the ionic liquid [pmim][BF4] invades both the core and corona region and the mixed micelle exhibits very small REES (3+/-1 nm). Anisotropy decay and solvation dynamics in different regions of the mixed micelle are studied by variation of excitation wavelength (lambda ex). In P123 micelle, the average rotational time () is 2800 ps in the core (at lambdaex=375 nm) and 1350 ps in the corona region (at lambdaex=435 nm). In 0.3 M [pmim][BF4], tau rot at the core of the mixed micelle decreases to 1950 ps while that in the corona remains unaffected. In 0.9 M IL, both the core and corona (lambda ex=375 and 435 nm) exhibit similar and short approximately 600 ps. In 0.3 M IL, solvation dynamics in the core region (lambdaex=375 nm) of P123 micelle is about 2 times faster than in its absence. In 0.3 M IL, solvation dynamics in the corona region (lambdaex=435 nm) is approximately 100 times faster than that in the core. In 0.9 M IL, the solvation dynamics in the core and in the corona is, respectively, approximately 9 times and 4 times faster than that in 0.3 M IL.     

Femtosecond solvation dynamics in different regions of a bile salt aggregate: excitation wavelength dependence

Solvation dynamics of coumarin 480 (C480) in the secondary aggregate of a bile salt (sodium deoxycholate, NaDC) is studied using femtosecond up-conversion. The secondary aggregate resembles a long (approximately 40 A) hollow cylinder with a central water-filled tunnel. Different regions of the aggregate are probed by variation of the excitation wavelength (lambdaex) from 375 to 435 nm. The emission maximum of C480 displays an 8 nm red shift as the lambdaex increases from 345 to 435 nm. The 8 nm red edge excitation shift (REES) suggests that the probe (C480) is distributed over regions of varied polarity. Excitation at a short wavelength (375 nm) preferentially selects the probe molecule in the buried locations and exhibits slow dynamics with a major (84%) slow component (3500 ps) and a small (16%) contribution of the ultrafast component (2.5 ps). Excitation at lambdaex=435 nm (red end) corresponds to the exposed sites where solvation dynamics is very fast with a major (73%) ultrafast component (相似文献   

Femtosecond to second studies of a water-soluble porphyrin derivative in chemical and biological nanocavities

The interactions of 5,10,15,20-tetrakis(4-sulfonatophenyl)-porphyrin (TSPP) with a quaternary ammonium modified β-cyclodextrin (QA-β-CD) and human serum albumin (HSA) protein in aqueous solutions at pH 7 were studied using steady-state, stopped-flow, and femtosecond to millisecond spectroscopy. TSPP forms 1:1 and 1:2 complexes with QA-β-CD (K(1) = 1.9 × 10(5) M(-1) and K(2) = 7 × 10(3) M(-1)) at 293 K, whereas with the HSA protein only 1:1 complex (K(1) = 1.7 × 10(6) M(-1)) has been found. The chemical and biological nanocavities have notable effects on the fluorescence lifetimes of the Q(x) state (from 9.3 to 11.1 ns in QA-β-CD and 11.6 ns in HSA). Furthermore, the rotational times (400 ps for the free TSPP, 1.6 and 19 ns for QA-β-CD and HSA protein complexes, respectively) clearly indicate the robustness of the formed entities. The confined environment does not affect much the fs dynamics (0.1-0.2 ps) of the encapsulated molecule. However, it clearly affect the ps one (1-2 ps (H(2)O) and 5-10 ps (QA-β-CD and HSA)). The effect of O(2) on the relaxation of the triplet state of the free and encapsulated TSPP is also studied and the obtained results are discussed in light of the shielding effect provided by the chemical and biological cavities. The observed difference, longer triplet lifetime upon encapsulation, might be relevant to the efficiency of this porphyrin in photodynamic therapy. The presteady-state kinetics of the TSPP:HSA has been studied by the stopped-flow spectrometer, and a two-step model was proposed for the complexation processes. The results show the importance of the initial association step for the overall ligand recognition process. This first step occurs with rate constant of ~4 × 10(5) M(-1) s(-1), which is about 5 orders of magnitude larger than the rate constant of the consecutive relaxation processes. We believe that our observations of molecular interaction between TSPP, QA-β-CD, and HSA protein from femtosecond to second at both ground and electronically first excited state give detailed information to improve our understanding of this kind of system and thus for a better design of drug delivery nanocarriers.     

A femtosecond study of excitation wavelength dependence of a triblock copolymer-surfactant supramolecular assembly: (PEO)20-(PPO)70-(PEO)20 and CTAC

Solvation dynamics and anisotropy decay of coumarin 480 (C480) in a supramolecular assembly containing a triblock copolymer, PEO20-PPO70-PEO20 (Pluronic P123) and a surfactant, CTAC (cetyl trimethylammonium chloride) are studied by femtosecond up-conversion. In a P123-CTAC complex, C480 displays a significant (22 nm) red edge excitation shift (REES) in the emission maximum as lambda ex increases from 335 to 445 nm. This suggests that the P123-CTAC aggregate is quite heterogeneous. The average rotational relaxation time (tau rot) of C480 in a P123-CTAC complex decreases by a factor of 2 from 2500 ps at lambda ex = 375 nm to 1200 ps at lambda ex = 435 nm. For lambda ex = 375 nm, the probe molecules in the buried core region of P123-CTAC are excited and the solvation dynamics displays three components, 2, 60, and 4000 ps. It is argued that insertion of CTAC in P123 micelle affects the polymer chain dynamics, and this leads to reduction of the 130 ps component of P123 micelle to 60 ps in P123-CTAC. For lambda ex = 435 nm, which selects the peripheral highly polar corona region, solvation dynamics in P123-CTAC and P123 are extremely fast with a major component of <0.3 ps ( approximately 80%) and a 2 ps ( approximately 20%) component.     

The dynamics of water at DNA interfaces: computational studies of Hoechst 33258 bound to DNA

Together, spectroscopy combined with computational studies that relate directly to the experimental measurements have the potential to provide unprecedented insight into the dynamics of important biological processes. Recent time-resolved fluorescence experiments have shown that the time scales for collective reorganization at the interface of proteins and DNA with water are more than an order of magnitude slower than in bulk aqueous solution. The molecular interpretation of this change in the collective response is somewhat controversial some attribute the slower reorganization to dramatically retarded water motion, while others describe rapid water dynamics combined with a slower biomolecular response. To connect directly to solvation dynamics experiments of the fluorescent probe Hoechst 33258 (H33258) bound to DNA, we have generated 770 ns of molecular dynamics (MD) simulations and calculated the equilibrium and nonequilibrium solvation response to excitation of the probe. The calculated time scales for the solvation response of H33258 free in solution (0.17 and 1.4 ps) and bound to DNA (1.5 and 20 ps) are highly consistent with experiment (0.2 and 1.2 ps, 1.4 and 19 ps, respectively). Decomposition of the calculated response revealed that water solvating the probe bound to DNA was still relatively mobile, only slowing by a factor of 2-3, while DNA motion was responsible for the long-time component (approximately 20 ps).     

Temperature dependence of solvation dynamics and anisotropy decay in a protein: ANS in bovine serum albumin

Temperature dependence of solvation dynamics and fluorescence anisotropy decay of 8-anilino-1-naphthalenesulfonate (ANS) bound to a protein, bovine serum albumin (BSA), are studied. Solvation dynamics of ANS bound to BSA displays a component (300 ps) which is independent of temperature in the range of 278-318 K and a long component which decreases from 5800 ps at 278 K to 3600 ps at 318 K. The temperature independent part is ascribed to a dynamic exchange of bound to free water with a low barrier. The temperature variation of the long component of solvation dynamics corresponds to an activation energy of 2.1 kcal mol(-1). The activation energy is ascribed to local segmental motion of the protein along with the associated water molecules and polar residues. The time scale of solvation dynamics is found to be very different from the time scale of anisotropy decay. The anisotropy decays are analyzed in terms of the wobbling motion of the probe (ANS) and the overall tumbling of the protein.     

Denaturing Effects of Urea and Guanidine Hydrochloride on Hyperthermophilic Esterase from Aeropyrum pernix K1

Introduction Esterases(EC3.1.1.x)representadiversegroup ofhydrolasescatalyzingthecleavageandformationof esterbonds.Theyarewidelydistributedinanimals,plantsandmicroorganisms.Becauseoftheiractivities inbothaqueousandnonaqueoussolventsystems,ester aseshavebe…     

Pyrene excimer fluorescence of yeast alcohol dehydrogenase: a sensitive probe to investigate ligand binding and unfolding pathway of the enzyme

The cysteine residues of yeast alcohol dehydrogenase (YADH) were covalently modified by N-(1-pyrenyl) maleimide (PM). A maximum of 3.4 cysteines per YADH monomer could be modified by PM. The secondary structure of PM-YADH was found to be similar to that of the native YADH using far-UV circular dichroism. The covalent modification of YADH by PM inhibited the enzymatic activity indicating that the active site of the enzyme was altered. PM-YADH displayed maximum excimer fluorescence at an incorporation ratio of 2.6 mol of PM per monomeric subunit of YADH. Nucleotide adenine dinucleotide (NAD) divalent zinc and ethanol reduced the excimer fluorescence of PM-YADH indicating that these agents induce conformational changes in the enzyme. Guanidinium hydrochloride (GdnHCl)-induced unfolding of YADH was analyzed using tryptophan fluorescence, pyrene excimer fluorescence and enzymatic activity. The unfolding of YADH was found to occur in a stepwise manner. The loss of enzymatic activity preceded the global unfolding of the protein. Further, changes in tryptophan fluorescence with increasing GdnHCl suggested that YADH was completely unfolded by 2.5 M GdnHCl. Interestingly, residual structures of YADH were detected even in the presence of 5 M GdnHCl using the excimer fluorescence of PM-YADH.     

Femtosecond/picosecond time-resolved fluorescence study of hydrophilic polymer fine particles

Femtosecond/picosecond time-resolved fluorescence study of hydrophilic polymer fine particles (polyacrylamide, PAAm) was reported. Ultrafast fluorescence dynamics of polymer/water solution was monitored using a fluorescent probe molecule (C153). In the femtosecond time-resolved fluorescence measurement at 480 nm, slowly decay components having lifetimes of tau(1) approximately 53 ps and tau(2) approximately 5 ns were observed in addition to rapid fluorescence decay. Picosecond time-resolved fluorescence spectra of C153/PAAm/H2O solution were also measured. In the time-resolved fluorescence spectra of C153/PAAm/H2O, a peak shift from 490 to 515 nm was measured, which can be assigned to the solvation dynamics of polymer fine particles. The fluorescence peak shift was related to the solvation response function and two time constants were determined (tau(3) approximately 50 ps and tau(4) approximately 467 ps). Therefore, the tau(1) component observed in the femtosecond time-resolved fluorescence measurement was assigned to the solvation dynamics that was observed only in the presence of polymer fine particles. Rotational diffusion measurements were also carried out on the basis of the picosecond time-resolved fluorescence spectra. In the C153/PAAm/H2O solution, anisotropy decay having two different time constants was also derived (tau(6) approximately 76 ps and tau(7) approximately 676 ps), indicating the presence of two different microscopic molecular environments around the polymer surface. Using the Stokes-Einstein-Debye (SED) equation, microscopic viscosity around the polymer surface was evaluated. For the area that gave a rotational diffusion time of tau(6) approximately 76 ps, the calculated viscosity is approximately 1.1 cP and for tau(7) approximately 676 ps, it is approximately 10 cP. The calculated viscosity values clearly revealed that there are two different molecular environments around the polyacrylamide fine particles.     

Medium decoupling of dynamics at temperatures ~100 K above glass-transition temperature: a case study with (acetamide + lithium bromide/nitrate) melts

Time-resolved fluorescence Stokes shift and anisotropy measurements using a solvation probe in [0.78CH(3)CONH(2) + 0.22{f LiBr + (1-f) LiNO(3)}] melts reveal a strong decoupling of medium dynamics from viscosity. Interestingly, this decoupling has been found to occur at temperatures ～50-100 K above the glass transition temperatures of the above melt at various anion concentrations (f(LiBr)). The decoupling is reflected via the following fractional viscosity dependence (η) of the measured average solvation and rotation times (<τ(s)> and <τ(r)>, respectively): <τ(x)> ∝ (η∕T)(p) (x being solvation or rotation), with p covering the range, 0.20 < p < 0.70. Although this is very similar to what is known for deeply supercooled liquids, it is very surprising because of the temperature range at which the above decoupling occurs for these molten mixtures. The kinship to the supercooled liquids is further exhibited via p which is always larger for <τ(r)> than for <τ(s)>, indicating a sort of translation-rotation decoupling. Multiple probes have been used in steady state fluorescence measurements to explore the extent of static heterogeneity. Estimated experimental dynamic Stokes shift for coumarin 153 in these mixtures lies in the range, 1000 < Δν(t)/cm(-1) < 1700, and is in semi-quantitative agreement with predictions from our semi-molecular theory. The participation of the fluctuating density modes at various length-scales to the observed solvation times has also been investigated.     

Solvation dynamics of Hoechst 33258 in water: an equilibrium and nonequilibrium molecular dynamics study

Integrated within an appropriate theoretical framework, molecular dynamics (MD) simulations are a powerful tool to complement experimental studies of solvation dynamics. Together, experiment, theory, and simulation have provided substantial insight into the dynamic behavior of polar solvents. MD investigations of solvation dynamics are especially valuable when applied to the heterogeneous environments found in biological systems, where the calculated response of the environment to the electrostatic perturbation of the probe molecule can easily be decomposed by component (e.g., aqueous solvent, biomolecule, ions), greatly aiding the molecular-level interpretation of experiments. A comprehensive equilibrium and nonequilibrium MD study of the solvation dynamics of the fluorescent dye Hoechst 33258 (H33258) in aqueous solution is presented. Many fluorescent probes employed in experimental studies of solvation dynamics in biological systems, such as the DNA minor groove binder H33258, have inherently more conformational flexibility than prototypical fused-ring chromophores. The role of solute flexibility was investigated by developing a fully flexible force-field for the H33258 molecule and by simulating its solvation response. While the timescales for the total solvation response calculated using both rigid (0.16 and 1.3 ps) and flexible (0.17 and 1.4 ps) models of the probe closely matched the experimentally measured solvation response (0.2 and 1.2 ps), there were subtle differences in the response profiles, including the presence of significant oscillations for the flexible probe. A decomposition of the total response of the flexible probe revealed that the aqueous solvent was responsible for the overall decay, while the oscillations result from fluctuations in the electrostatic terms in the solute intramolecular potential energy. A comparison of equilibrium and nonequilibrium approaches for the calculation of the solvation response confirmed that the solvation dynamics of H33258 in water is well-described by linear response theory for both rigid and flexible models of the probe.     

Fluorescence studies in a pyrrolidinium ionic liquid: polarity of the medium and solvation dynamics

While the imidazolium ionic liquids have been studied for some time, little is known about the pyrrolidinium ionic liquids. In this work, steady-state and picosecond time-resolved fluorescence behavior of three electron donor-acceptor molecules, coumarin-153 (C153), 4-aminophthalimide (AP), and 6-propionyl-2-dimethylaminonaphthalene (PRODAN), has been studied in a pyrrolidinium ionic liquid, N-butyl-N-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide, abbreviated here as [bmpy][Tf2N]. The steady-state fluorescence data of the systems suggest that the microenvironment around these probe molecules, which is measured in terms of the solvent polarity parameter, E(T)(30), is similar to that in 1-decanol and that the polarity of this ionic liquid is comparable to that of the imidazolium ionic liquids. All three systems exhibit wavelength-dependent fluorescence decay behavior, and the time-resolved fluorescence spectra show a progressive shift of the fluorescence maximum toward the longer wavelength with time. This behavior is attributed to solvent-mediated relaxation of the fluorescent state of these systems. The dynamics of solvation, which is studied from the time-dependent shift of the fluorescence spectra, suggests that approximately 45% of the relaxation is too rapid to be measured in the present setup having a time resolution of 25 ps. The remaining observable components of the dynamics consist of a short component of 115-440 ps (with smaller amplitude) and a long component of 610-1395 ps (with higher amplitude). The average solvation time is consistent with the viscosity of this ionic liquid. The dynamics of solvation is dependent on the probe molecule, and nearly 2-fold variation of the solvation time depending on the probe molecule could be observed. No correlation of the solvation time with the probe molecule could, however, be observed.     

On-column capture of a specific protein separated by SDS-CGE using an immunological reaction on magnetic beads

The on-column capture of a specific protein using magnetic beads was applied to SDS-CGE. In a preliminary study, an immunological reaction in the presence of SDS, using a batch method, was attempted. Carbonic anhydrase (CA), alpha-lactalbumin (LA), and HSA were denatured by heating in the presence of SDS and 2-mercaptoethanol, and then reacted with anti-CA that had been immobilized on magnetic beads. Not only native CA, but also the denatured CA reacted with anti-CA, even in the presence of SDS. Therefore, the on-column capture of denatured CA separated by SDS-CGE was attempted using a two-point detection technique. A mixture of proteins, containing LA, CA, and HSA, were separated by SDS-CGE according to their Mr. The CA was then specifically captured on anti-CA-immobilized magnetic beads, which were packed between two detection windows in the capillary column, during the electrophoresis. The results show that the technique leads to information similar to that obtained by Western blotting, i.e., a protein can be identified by its Mr and reaction with its antibody.