Structural Insights into the Trp‐Cage Folding Intermediate Formation

The 20 residue long Trp‐cage is the smallest protein known, and thus has been the subject of several in vitro and in silico folding studies. Here, we report the multistate folding scenario of the miniprotein in atomic detail. We detected and characterized different intermediate states by temperature dependent NMR measurements of the ¹⁵N and ¹³C/¹⁵N labeled protein, both at neutral and acidic pH values. We developed a deconvolution technique to characterize the invisible—fully folded, unfolded and intermediate—fast exchanging states. Using nonlinear fitting methods we can obtain both the thermodynamic parameters (ΔH^F–I, T_m^F–I, ΔC_p^F–I and ΔH^I–U, T_m^I–U, ΔC_p^I–U) and the NMR chemical shifts of the conformers of the multistate unfolding process. During the unfolding of Trp‐cage distinct intermediates evolve: a fast‐exchanging intermediate is present under neutral conditions, whereas a slow‐exchanging intermediate‐pair emerges at acidic pH. The fast‐exchanging intermediate has a native‐like structure with a short α‐helix in the G¹¹–G¹⁵ segment, whereas the slow‐exchanging intermediate‐pair presents elevated dynamics, with no detectable native‐like residue contacts in which the G¹¹? P¹² peptide bond has either cis or trans conformation. Heteronuclear relaxation studies combined with MD simulations revealed the source of backbone mobility and the nature of structural rearrangements during these transitions. The ability to detect structural and dynamic information about folding intermediates in vitro provides an excellent opportunity to gain new insights into the energetic aspects of the energy landscape of protein folding. Our new experimental data offer exceptional testing ground for further computational simulations.     

On the Stability of the Schiff Bases of Pyridoxal 5′-Phosphate with Polypeptides Containing L-Lysine

We determined the apparent equilibrium constant of formation, K_pH, of the Schiff bases of pyridoxal 5′-phosphate (PLP) and poly- and copolymers containing L -lysine, as a function of pH at 25° and a constant ionic strength of 0.1 M . The K_pH values obtained at acidic and neutral pH were larger that those reported for Schiff bases of PLP and hexylamine. We determined calorimetrically ΔH of formation of Schiff bases of PLP and poly(L -lysine) (?4.5′kcal/mol), and PLP and hexylamine (?3.4 kcal/mol) at pH 7.00. Semi-empirical theoretical calculations (INDO and AMI methods) of a model compound of Schiff base of PLP and polypeptide containing L -lysine show the capability of specific interactions between groups of PLP and the peptide skeleton.     

Evidence for a pterin-derivative associated with the molybdenum cofactor of Neurospora crassa nitrate reductase

Abstract— Following the method of Johnson and Rajagopalan (1982) for obtaining Form B of molybdopterin cofactors, we observed a prominent fluorescence band at480–482 nm in purified NR of Neurospora crassa mutant albino-band after boiling the enzyme at acidic pH and readjusting the sample to alkaline pH. This fluorescence band is maximally excited at 410 nm and maximally emitting at pH 11 (“F-480_pH11”); at pH4–7 only a featureless fluorescence band of low intensity remains (“F-480_pH5”). The fluorescence ΔF-480 = F-480_pH11 - F-480_pHS is examined here. ΔF-480 is associated specifically with NADPH-dependent and MVH-dependent nitrate reduction activities and with cytochrome b-557 absorption. In a protease-digested preparation lacking NADPH-dependent NR activity, ΔF-480 is associated with MVH-dependent nitrate reduction. The ΔF-480 signal is followed during the course of purification of NR. Its size increases with increasing purity of the enzyme. In partially purified NR preparations and especially in aqueous extracts from mycelia of N. crassa, a second, strong fluorescence signal with a pH-dependent emission maximum at around 450 nm (maximally excited at350–370 nm) was found beside ΔF-480. This “unspecific” signal was lost during NR purification. A procedure is developed to demonstrate AF-480 also in presence of the unspecific (350-370/450 nm) signal as well as flavins. We deduce that the ΔF-480 component is part of the Mo cofactor of N. crassa NR and that the signal is caused by a pterin derivative. From calculations of total content of the AF-480 component in mycelia it is likely that in vivo it is shared also by other enzymes.     

Kinetic and thermodynamic properties of the aerial oxidation of hydroquinone in developer solutions

The aerial oxidation kinetics of hydroquinone in a freshly prepared developer solution at different temperatures and pHs has been studied. The activation parameters, Ea, ΔG# , ΔS# , ΔH# and enthalpy of formation of activated complex, ΔHfo(X# ), are determined. The large negative value of free energy of activation ΔG# proves that hydroquinone extremely tends to be oxidized by air at optimum temperature (20℃) and optimum pH (10.5) and converts to the activated complex semiquinone. It was also found that if the pH of the developer solution is increased from 9.3 to 10.5 the reaction rate will increase by a factor of 2.     

Adsorption of methyl orange on nanoparticles of a synthetic zeolite NaA/CuO

CuO supported on an NaA zeolite (CuO/NaA) was prepared with an NaA zeolite through the ion-exchange (CuO/NaA) method. The morphology and the physicochemical properties of the prepared samples were investigated by XRD, MEB, and EDS. The various parameters, such as contact time, catalyst dose, initial dye concentration, initial pH, and temperature, influencing the adsorption of methyl orange (MO) were optimized. The MO adsorption equilibrium was reached after 240 min of contact time. Removal of MO is better at neutral pH than in acidic and alkaline solutions. Among the tested models, the equilibrium adsorption data are well fitted by the Langmuir isotherm. The adsorption kinetics is best described by the pseudo-second-order model. The evaluation of the thermodynamic parameters, i.e. ΔG^o, ΔH^o, and ΔS^o, revealed that MO adsorption was spontaneous, while the activation energy (20.98 kJ/mol) indicates a physical adsorption. The photodegradation of MO decreased from 100 mg/L down to 2 mg/L when the solution is exposed to visible light.     

ATP Sensing with Anthryl‐Functionalized Open‐Chain Polyaza‐alkanes

The anthryl‐functionalized open‐chain polyaza‐alkanes L ¹ , L ² , and L ³ have been synthesized, and their activity as fluorescent chemosensors has been studied in MeCN/H₂O 70 : 30 (v/v) and H₂O at 25° against the anions bromide, phosphate, sulfate, ATP, ADP, and GMP. The crystal structure of L ³ has been solved by single‐crystal X‐ray‐diffraction techniques. The emission intensity of L ¹ and L ² is selectively quenched in the presence of ATP at acidic pH in MeCN/H₂O 70 : 30 (v/v). In H₂O, the emission intensity of L ¹ and L ² is enhanced at neutral pH in the presence of ADP and ATP. The sensing behavior is discussed in terms of H‐bonding or electrostatic anion‐cation interactions. Receptor L ³ does not show any significant change in fluorescence emission upon addition of anions. Protonation constants of the three ligands and stability constants of L ² with phosphate and sulfate were determined by potentiometric titration in MeCN/H₂O. The stability constants obtained are compared with those obtained for the interaction of these anions with related open‐chain polyamines.     

New environmentally responsive fluorescent N‐isopropylacrylamide copolymer and its application to DNA sensing

We report two novel multifunctional copolymers consisting of a temperature‐responsive poly(N‐isopropylacrylamide) (PNIPAA) segment and a fluorescent fluorene‐containing acrylic polymer segment with pH responsiveness and/or DNA‐sensing ability. The functional acrylic monomer with a fluorene dimer side group substituted with amino units was synthesized first. Then, it was copolymerized with N‐isopropylacrylamide to result in a new water‐soluble, fluorescent PNIPAA copolymer ( P1 ). The fluorescent properties of P1 under neutral and acidic conditions did not change with the temperature. However, significant variation was observed under basic conditions. The protonation of the amino moiety at a low pH improved the solubility and prevented aggregation for fluorescence quenching, but not under the basic conditions. Although aggregation of the fluorene units was significant at room temperature under basic conditions, the aggregation was resolved at a temperature above the lower critical solution temperature. These findings indicated the pH‐ and temperature‐responsive characteristics of P1 . Moreover, after the amino groups were quaternized, the obtained polymer could be used as a biosensor because the fluorescence intensity was quenched with the addition of DNA. This study demonstrates that multifunctional materials with pH‐ and temperature‐sensing characteristics and biological molecules could be realized by the incorporation of a functional fluorene‐containing moiety with PNIPAA. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5495–5504, 2006     

Aggregate Formed by a Cationic Fluorescence Probe

The aggregation behavior of a cationic fluorescence probe 10-（4,7,10,13,16-pentaoxa- 1-azacyclooctadecylmethyl）anthracen-9-ylmethyl dodecanoate （1） was observed and studied by a fluorescence methodology in acidic and neutral conditions. By using the Py scale, differences between simple aggregates and micelles have been discussed. The stability of simple aggregates was discussed in terms of hydrophobic interaction and electrostatic repulsion. The absence of excimer emission of the anthrancene moiety of probe 1 in neutral condition was attributed to the photoinduced electron transfer mechanism instead of photodimerization.     

A new oral microsphere drug delivery system

A new oral proteinoid microsphere drug delivery system is discussed with respect to its unique dependence on pH. Extensive evidence indicate that this technique not only can be used to deliver the protein and polar macromolecular drugs which are currently administered via injection, but also can be used to administer antigens and vaccines. Future work has been proposed to study the physical interaction of specific drugs with specifically designed oligopeptides structures.     

An experimental and computational approach to pH‐dependent self‐aggregation of poly(2‐isopropyl‐2‐oxazoline)

Besides temperature, self‐aggregation of poly(2‐isopropyl‐2‐oxazoline) (PIPOX) can also be triggered via pH in aqueous solution (25 °C, pH > 5). Lowest energy structures and interaction energies of PIPOX with H₃O⁺, OH^?, and H₂O were calculated by DFT methods showed that, in addition to their ability to protonate PIPOX, H₃O⁺ ions had strong interaction with both water and PIPOX in acidic conditions. H₃O⁺ ions acted as compatibilizer between PIPOX and water and increased the solubility of PIPOX. OH^? ions were found to have stronger interaction with water compared to PIPOX resulting in desorption of water molecules from PIPOX phase and decreased solubility, leading to enhanced hydrophobic interactions among isopropyl groups of PIPOX and formation of aggregates at high pH. Results concerning the effect of end‐groups on aggregate size were in good agreement with statistical mechanics calculations. Moreover, the effect of polymer concentration on the aggregate size was examined. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2019 , 57, 210–221     

Dansylated Polyamines as Fluorescent Sensors for Metal Ions: Photophysical Properties and Stability of Copper(II) Complexes in Solution

Protonation and the Cu^II complexation constants of the dansylated polyamines N‐dansylethylenediamine ( 1 ), N‐dansyldiethylenetriamine ( 2 ), N‐dansyltriethylenetetramine ( 3 ), N′‐[2‐(dansylamino)ethyl]diethylenetriamine ( 4 ), and tris(2‐dansylaminoethyl)amine ( 5 ) were determined by glass‐electrode potentiometry in MeOH/H₂O 9 : 1 (v/v) solution. For ligands 3 and 4 , the determinations were also performed in aqueous solution. The complexes formed by these ligands in neutral form correspond to those observed for the analogous unsubstituted monoprotonated amines, whereas, when the ligands are deprotonated at the sulfonamide moiety, the species parallel those of the corresponding amines. The molecular structures of the complexes were deduced from the VIS absorption spectra. The crystal structure of the [CuL₂H₋₂] complex 6 of ligand 1 (L) was determined by X‐ray diffraction. The study of the photophysical properties of the ligands 3 – 5 showed that they are good fluorescent sensors for copper(II), which induced fluorescence quenching. Time‐resolved fluorescence measurements allowed us to clarify the sensing mechanism. The pH dependence of the quenching effect demonstrated that it occurs for all Cu²⁺ complexes, even for species in which the sulfonamide moiety is not deprotonated. Sensing of Cu²⁺ was compared with that of other metal ions (Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Hg²⁺), and selectivity was studied as a function of pH. Ligands 3 and 4 were found to be selective chemosensors for Cu²⁺ in weakly acidic solution (pH ca. 4 – 5).     

UNIQUE ORAL DRUG DELIVERY SYSTEM

An oral drug delivery system using proteinoid microspheres is discussed with respect to itsunique dependence on pH. It has been found that certain drugs such as insulin and heparin canbe encapsulated in proteinoid spheres at stomach pH's (1--3). These spheres also dissemble atintestinal pH's (6--7) releasing the drug for absorption. Using this technique low molecularweight heparin and human growth hormone have been orally delivered successfully to severalanimal species. Future work has been proposed to study the interaction and binding of thespecific drugs with synthesized oligopeptides.     

Amphiphilic Polymer Nanoparticles: Characterization and Assessment as New Drug Carriers

An amino‐acid‐based hydrophobically modified biocompatible copolymer, poly[(sodium N‐acryloyl‐L ‐valinate)‐co‐(N‐octylacrylamide)] was synthesized and characterized. Techniques such as fluorescence probes, DLS, and TEM were used to investigate its aggregation behavior in aqueous solution. The copolymer was observed to form micellar aggregates having diameters in the nanometer range in aqueous solution (pH = 8) through inter‐chain hydrophobic association. This behavior was found to be similar to that of poly[(sodium N‐acryloyl‐L ‐valinate)‐co‐(N‐dodecylacrylamide)]. The compact micellar nanostructures were observed to be stable with respect to changes of pH and temperature. The encapsulation and release of griseofulvin, a hydrophobic model drug, was studied.

     

Separation of peptide diastereomers using CEC and a hydrophobic monolithic column

A neutral hydrophobic monolith prepared by radical in situ copolymerization of lauryl methacrylate and ethylene dimethacylate has been evaluated for the CEC separation of diastereomers of small peptides using acidic mobile phases containing ACN as organic modifier. Using an acidic mobile phase, the peptides migrated due to their own electrophoretic mobility. Hydrophobic interactions with the stationary phase contributed to the separation. Peptide mobility and resolution increased with increasing the ACN content. Retention times increased with the pH of the mobile phase. Peak resolution increased with buffer pH and concentration. Di‐ and tripeptides composed only of L ‐configured amino acids migrated faster than peptides containing D ‐amino acids. A mixture of isomeric Asp tripeptides that could not be completely resolved by either CZE or HPLC as well as the 24mer peptides tetracosactide and ¹⁶[D ‐Lys]‐tetracosactide could also be separated by CEC on the hydrophobic monolith.     

Surface and colloid properties of cyclic amides III. Surface activity and micellization of N-butyl-2-pyrrolidone in water

Abstract

The aggregation and surface activity of N-butyl-2-pyrrolidone (BP) in water is investigated using surface tension, solubilization, fluorescence and NMR methods. The micelle point is high (?1.0 M) as expected for the butyl chain length, and the aggregation number is approximately 5, with pre-association below the c.m.c. Despite the low aggregation number, the fluorescence data indicate that the interior of the aggregate is similar to pure liquid BP. The adsorption of BP at the solution/air interface goes to a maxmum near the c.m.c, corresponding to an area of 45 ± 1 Ų/molecule.     

Simultaneous determination of acidic and basic drugs using dual hollow fibre electromembrane extraction combined with CE

The simultaneous extraction of acidic and basic drugs from biological samples is a significant challenge for sample preparation. A novel and efficient method named dual hollow fibre electromembrane extraction combined with CE was applied for the simultaneous extraction and preconcentration of acidic and basic drugs in a single step. Under applied potential of 40 V during the extraction, ibuprofen as an acidic drug and thebaine as a basic drug migrated from a 4 mL aqueous sample solution at neutral pH into 20 μL of each basic (pH 12.5) and acidic (pH 2.0) acceptor phase, respectively; 1‐octanol and 2‐nitrophenyl octyl ether were immobilised in the pores of anodic and cathodic hollow fibres as supported liquid membranes, respectively. A Box–Behnken design and the response surface methodology were used for the optimisation of different parameters on the extraction efficiency. Under the optimised conditions, the enrichment factors were between 150 and 170 and also the LODs ranged from 3 to 7 ng/mL in different samples. The method was reproducible so that intra‐ and inter‐day RSDs% (n = 5) were less than 5.9%. Finally, the method was successfully applied for the simultaneous extraction and determination of acidic and basic drugs from plasma and urine samples.     

Spectroscopic Studies of water-soluble porphyrins with protein encapsulated in bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles: aggregation versus complexation

We have investigated the interaction of two water-soluble free-base porphyrins (negatively charged meso-tetrakis(p-sulfonatophenyl)porphyrin sodium salt (TSPP) and positively charged meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMpyP)) with two drug-carrier proteins (human serum albumin (HSA) and beta-lactoglobulin (betaLG)) in bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/water reverse micelles (RM) by using steady-state and transient-state fluorescence spectroscopy. TSPP exhibited a complex pattern of aggregation on varying the RM size and pH in the absence of the protein: at low omega0 (the ratio of water concentration to AOT concentration, the emission of H-aggregates prevails under acidic or neutral "pH(ext)" conditions. Upon formation of the water-pool, J-aggregates and monomeric diacid species dominate at low "pH(ext)" but only monomer is detected at neutral "pH(ext)". The aggregation number increases with omega0 and the presence of the protein does not seem to contribute to further growth of the aggregate. The presence of protein leads to H-deaggregation but promotes J-aggregation up to a certain protein/porphyrin ratio above which, complexation with the monomer bound to a hydrophobic site of the protein prevails. The effective complex binding constants are smaller than in free aqueous solution; this indicates a weaker binding in these RM probably due to some conformational changes imposed by encapsulation. Only a weak quenching of TMpyP fluorescence is detected due to the presence of protein in contrast to the negative porphyrin.     

Synthesis and spectral properties of 7-(N-arylsulfonyl)aminocoumarins,a new class of fluorescent pH indicators

7-Arylsulfonylamino-coumarins 3 and 5 , with electron-delocalizing substituents in position 3 were obtained by condensation of 4-arylsulfonylamino-2-hydroxybenzaldehyde with various (hetero)arylacetic esters and by other methods. Oxidative cyanation using potassium cyanide and elemental bromine gave the corresponding 4-cyano derivatives 6a,b. The absorption and emission maxima of coumarins 3 and 5 in organic solvents and in water of different pH were determined. All showed bright blue fluorescence in organic or acidic aqueous solution, which is shifted to the green in alkaline solution. The 4-cyano derivatives 6a, b exhibit green fluorescences, which change to yellow in alkaline solution. Simultaneously the color changes from yellow to orange. The pKa values of compounds 3 and 5 were found to lie between 6.33 and 7.06, whereas for the 4-cyano derivatives they were distinctly lower (5.66-5.95). The spectral changes observed in the near neutral pH range may render the new compounds useful indicators for the measurement of physiological pH's.     

牛血清蛋白在盐酸胍和尿素体系中变性的微量热研究

在30 ℃时用恒温微量热法研究了不同pH值下盐酸胍、尿素诱导牛血清蛋白变性的过程. 并用Privalov提出的简单键合模型对量热数据进行了分析, 计算了表观键合常数K, 简单键合的单个表观键合自由能ΔG和总吉布斯能ΔG(a), 用变性中点的直线外推方法求出了表观变性焓ΔH_d. 实验结果表明, 牛血清蛋白与盐酸胍的键合在碱性条件下更易进行, 牛血清蛋白在盐酸胍溶液中的变性焓ΔH_d在牛血清蛋白的pH＝6.97和7.05时为350 kJ•mol^－1, 在pH＝9.30时为275 kJ•mol^－1, 表明牛血清蛋白在接近中性时较稳定. 而牛血清蛋白与尿素的键合在酸性条件下更易进行, 此变性焓ΔH_d在牛血清蛋白的pH＝6.97时为295 kJ•mol^－1, 在pH＝7.05和9.30时为230 kJ•mol^－1. 此结果说明牛血清蛋白在两种变性剂溶液中的展开程度是不同的.     

Glucose-Responsive Monoolein Cubic Phase Containing Glucose Oxidase

Glucose-responsive monoolein (MO) cubic phase was prepared by immobilizing proteinoid composed of Asp and Leu (PAL) and hydrophobically modified glucose oxidase (HmGOD) onto the MO bilayers. The hydrodynamic mean diameter of PAL aggregate in aqueous solution decreased with increasing the pH value. The number of pamitic acid residue per one molecule of HmGOD was determined to be 6.3 by a calorimetric method. HmGOD could acidify glucose solution in a few hours, possibly because it converted glucose to gluconic acid. PAL- and HmGOD-immobilized MO cubic phase was prepared by hydrating MO melt with the mixture aqueous solution of PAL and HmGOD. The cubic phase exhibited its phase transition around 62.5°C, determined by polarizing microscopy. The release of carboxylic fluorescein (CF) from the cubic phase was suppressed when the pH value of release medium decreased, possibly because PAL can aggregate more at a lower pH value. The release was suppressed when glucose concentration increased, possibly because the release medium can be more acidified and PAL will be more aggregated at a higher glucose concentration. The cubic phase could be used as a drug carrier which releases its content in a sustained manner when the glucose concentration is abnormally high.