Water-miscible Organic Solvents Research Articles

Organic solvent high-field amplified stacking for basic compounds in capillary electrophoresis

Many water-miscible organic solvents, especially acetonitrile and acetone, bring along significant degrees (∼30 times) of stacking by electroinjection through high-field amplified injection for the basic compounds compared to that for aqueous buffers or water. The relative stacking of different compounds in acetonitrile or acetone is different compared to that for water. Stacking by electroinjection in organic solvents is less stringent and easier to accomplish in practice. Acids and salts, in aqueous solutions, can ruin the stacking for both organic and aqueous solvents; however, this effect can be better tolerated by diluting the sample in acetonitrile. Thus, this stacking is termed “organic solvent high-field amplified injection”. This stacking by electroinjection is enhanced by increasing the electrophoresis buffer concentration and can be better than that by pressure injection. From the practical aspects, some cationic drugs present in serum such as amiodarone can be detected at the therapeutic levels by electroinjection on the capillary after protein precipitation by acetonitrile.

Homogeneous liquid–liquid extraction method for the selective separation and preconcentration of ultra trace molybdenum

A new simple and efficient homogeneous liquid–liquid extraction method for the selective separation and preconcentration of molybdenyl ions was developed. α-Benzoin oxime (ABO) was investigated as a complexing ligand, and perfluorooctanoate ion (PFOA −) was applied as a phase-separator agent under strongly acidic conditions. Under the optimal conditions ([ABO] = 2.1 × 10 −3 M, [PFOA −] = 1.8 × 10 −2 M, [HNO 3] = 1.7 M, [acetone] = 11.8% (v/v)), 10 μg of molybdenum in 5 ml aqueous phase could be extracted quantitatively into 40 μl of the sedimented phase. The maximum concentration factor was 125-fold. Thiocyanate was applied as a chromogenic reagent for the direct spectrophotometric determination of molybdenum in the sedimented phase. The reproducibility of the proposed method is at the most 2.4%. The influence of the type and concentration of acid solution, the concentration of ABO, the type and volume of the water-miscible organic solvent, the concentration of PFOA −, and the effect of different diverse ions on the extraction and determination of molybdenum(VI) were investigated. The proposed method was applied to the extraction and determination of molybdenum(VI) in natural water, Spinach, and Lucerne samples. A satisfactory agreement exists between the results obtained by the proposed method and those reported by GF-AAS.

Log P effect of organic solvents on a thermophilic alcohol dehydrogenase

An alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix was activated by water-miscible organic solvents. This activation was influenced by the kind and the concentration of the added organic solvents. The k cat was increased by a factor of over ten when the mole fraction of acetonitrile was 0.1. This effect was large when organic solvents with large log P values were added. In fact, the k cat showed a strong positive correlation with the log P value of the mixed solvent at a constant mole fraction of water, while it was not affected by the kind of organic solvents added. Both the activation enthalpy and the entropy decreased with an increase in log P. The contribution of the activation enthalpy to the free energy of activation was larger than that of the activation entropy, and the free energy of activation decreased with an increase in log P.

TBADH activity in water-miscible organic solvents: correlations between enzyme performance, enantioselectivity and protein structure through spectroscopic studies

The enantioselective reduction of 2-pentanone to (R)- and (S)-2-pentanol by Thermoanaerobacter (formerly Thermoanaerobium) brockii alcohol dehydrogenase (TBADH) in mixtures of water and water-miscible organic solvents was investigated. Significant enzymatic activity was retained in up to 87% methanol, ethanol and acetonitrile. The changes in enzyme activity as a function of organic solvent were correlated to structural alterations of TBADH with a series of spectroscopic studies (fluorescence, fluorescence quenching and circular dichroism (CD)). Interestingly, this study shows that the tetrameric form of TBADH is not critical for catalytic performance.

Gels of zirconium phosphate in organic solvents and their use for the preparation of polymeric nanocomposites

Gels of α-zirconium phosphate (α-ZrP) in water have been prepared by exfoliation of crystalline α-ZrP induced by intercalation–deintercalation of propylamine. Although these gels are X-ray amorphous, their Raman spectra indicate that the α-type layers of the starting material remain essentially unaltered. Replacement of gel water with water miscible organic solvents has allowed the formation of α-ZrP gels in a great variety of solvents, such as alkanols, N,N-dimethylformamide (DMF), 1-methyl-2-pyrrolidone (NMP), acetone, tetrahydrofuran (THF) and even chloroform. Particular attention has been devoted to α-ZrP gels in DMF. On the basis of the surface area of the dry gels, the average thickness of the particles of exfoliated α-ZrP has been estimated between 25 and 80 nm for α-ZrP percentages in the starting gels between 2.5 and 10 wt%, respectively, in agreement with SEM images. α-ZrP gels in THF have been used for the preparation of polystyrene/α-ZrP nanocomposites with filler loadings up to 8 wt%. The presence of exfoliated α-ZrP delays the beginning of thermal decomposition of the polymer by a maximum of 45 °C for 4 wt% filler loading. The state of filler exfoliation has been confirmed by transmission electron microscopy.

Lipase-catalyzed synthesis of O-lauroyl l-serinamide and O-lauroyl l-threoninamide

O-Lauroyl l-serinamide and O-lauroyl l-threoninamide were synthesized with an immobilized lipase using lauric acid and l-serinamide or l-threoninamide in water-miscible organic solvents, of which 2-methyl-2-propanol gave the highest conversion of 93% for O-lauroyl l-serinamide at 50 mmol- l-serinamide/L and 100 mmol-lauric acid/L. The addition of 4 Å 1/16 molecular sieves at 50 mg/mL increased the conversion to 53% for O-lauroyl l-threoninamide at 50 mmol- l-threoninamide/L and 150 mmol-lauric acid/L in 2-methyl-2-butanol. The difference of conversion between the two products was exactly proportional to the difference of solubility of aminoacid amides.

Mass spectrometric analysis of affinity-captured proteins on a dendrimer-based immunosensing surface: investigation of on-chip proteolytic digestion

The monolayer of fourth-generation poly(amidoamine) dendrimers was adopted to construct the immunoaffinity surface of an antibody layer. The antibody layer as a bait on the dendrimer monolayer was found to result in high binding capacity of antigenic proteins and a reliable detection. The affinity-captured protein at the immunosensing surface was subjected to direct on-chip tryptic digestion, and the resulting proteolytic peptides were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The performance of the on-chip digestion procedure was investigated with respect to the ratio of trypsin to protein, digestion time, composition of a reaction buffer, and the amount of affinity-captured protein on a surface. Addition of a water-miscible organic solvent to a reaction buffer had no significant effect on the digestion efficiency under the optimized digestion conditions. The on-chip digestion method identified the affinity-captured bovine serum albumin (BSA), lysozyme, and ferritin at the level of around 100 fmol. Interestingly, the detected number of peptide hits through the on-chip digestion was almost similar regardless of the amount of captured protein ranging from low- to high-femtomole levels, whereas the efficiency of in-solution digestion decreased significantly as the amount of protein decreased to low-femtomole levels. The structural alignment of the peptide fragments from on-chip-digested BSA revealed that the limited exterior of the captured protein is subjected to attack by trypsin. The established detection procedures enabled the identification of BSA in the biological mixtures at the level of 0.1 ng/mL. The use of antibodies against the proteins involved in the metabolic pathway of l-threonine in Escherichia coli also led to discrimination of the respective target proteins from cell lysates.

Simultaneous purification and immobilization of mushroom tyrosinase on an immunoaffinity support

The use of immobilized and stable enzymes has immense potential in the enzymic analysis of clinical, industrial and environmental samples. But their widespread use is limited due to the high cost of their production. In the present study, an effort has been made to immobilize tyrosinase directly from ammonium sulphate precipitated proteins of the mushroom ( Agaricus bisporus) on a polyclonal antibody bound to Seralose 4B support. Polyclonal antibodies were raised in male albino rabbits by injecting commercially available mushroom tyrosinase in the presence of Freund's adjuvants. Antibodies were purified from antisera by ammonium sulphate fractionation followed by DEAE-cellulose chromatography. Two distinct bands of light and heavy chains of purified IgG, appeared on SDS-PAGE. The homogeneity of the purified IgG was further confirmed by Ouchterlony double immunodiffusion. One milliliter of cyanogen bromide-activated Seralose 4B bound 9.0 mg purified IgG and retained nearly 573 tyrosinase units. Immunoaffinity bound tyrosinase was more stable against heat and pH inactivation compared to the soluble enzyme. Immobilized enzyme exhibited no change in temperature optima between 30–35 °C whereas soluble tyrosinase has temperature optima of 35 °C. Immunoaffinity bound tyrosinase retained greater fraction of activity on both sides of temperature-optima compared to soluble enzyme. However, the broadening in pH-optima was observed from pH 5.5 to 6.0 for immobilized enzyme. Moreover, immobilized tyrosinase preparation exhibited remarkably high resistance against denaturation induced by urea and water-miscible organic solvents.

A microtiter plate assay for the determination of the synthetic activity of protease

Proteases have several industrial applications due to their hydrolytic activity [1] and have attracted considerable interest due to their ability to synthesize peptides in organic media [2,3]. Protease-catalyzed peptide synthesis occurs either by a thermodynamically controlled process (condensation of the acid and amine groups) or by a kinetically controlled process (aminolysis of an ester). In the kinetically controlled process the rapidly formed covalent acyl enzyme can transfer the acyl moiety either to the nucleophilic amino component, resulting in the formation of the desired peptide, or to water, resulting in hydrolysis [3]. The hydrolysis step can be suppressed by performing the reaction at subzero temperature [4-6]. Serine proteases have been employed in the synthesis of many biological active oligopeptides [2], including kyotorphin, which is an analgesic dipeptide (Tyr-Arg). This dipeptide has been synthesized using L-Tyr-OEt or Bz-L-Tyr-OEt and H-Arg-OH or L-Arg-OEt as the nucleophile in organic solvent [6,7] and analyzed using high-performance liquid chromatography (HPLC). The presence of water-miscible organic solvents in enzyme-catalyzed reaction increases the solubility of hydrophobic substrates and shifts the thermodynamic equilibrium of hydrolytic reaction to synthetic reaction [8,9]. In addition water-miscible organic solvents such as dimethylformamide (DMF) when added to a subtilisin BPN-catalyzed reaction have shown preference toward aminolysis as His on the active site flips in and a strong hydrogen bond observed between His and Asp in water is disrupted [10]. However, increasing solvent concentration has led to enzyme inactivation [11,12]. Hence, techniques such as site-directed mutagenesis and directed evolution have been employed to improve the stability of enzymes in organic solvents. Directed evolution has been used as an elegant approach to generate and identify new enzyme variants [13]. A range of methods for generation of enzyme libraries is available [14], but the major problem is the lack of a rapid and reliable assay system for the screening of improved enzyme variants out of a pool of 10-10 mutants. To overcome such difficulty, hydrolase variants were identified using chromogenic esters to identify an active esterase [15], to increase the stereoselectivity of a lipase [16], and to identify active esterases using pH indicators [17]. Due to the high number of clones generated during directed evolution techniques, the use of HPLC or gas chromatography for screening is often a cumbersome and time-consuming method. It can, however, be used as the tool to finally confirm the results. In the present study, a microtiter plate (MTP) assay based on p-nitroanilide (pNA) release has been developed for the first time for rapid analysis of synthetic activity of protease.

Synthesis of levan in water-miscible organic solvents

The synthesis of levan using a levansucrase from a strain of Bacillus subtilis was studied in the presence of the water-miscible solvents: acetone, acetonitrile and 2-methyl-2-propanol (2M2P). It was found that while the enzyme activity is only slightly affected by acetone and acetonitrile, 2M2P has an activating effect increasing the total activity 35% in 40–50% (v/v) 2M2P solutions at 30 °C. The enzyme is highly stable in water at 30 °C; however, incubation in the presence of 15 and 50% (v/v) 2M2P reduced the half-life time to 23.6 and 1.8 days, respectively. This effect is reversed in 83% 2M2P, where a half-life time of 11.8 days is observed. The presence of 2M2P in the system increases the transfer/hydrolysis ratio of levansucrase. As the reaction proceeds with 10% (w/v) sucrose in 50/50 water/2M2P sucrose is converted to levan and an aqueous two-phase system (2M2P/Levan) is formed and more sucrose can be added in a fed batch mode. It is shown that high molecular weight levan is obtained as an hydrogel and may be easily recovered from the reaction medium. However, when high initial sucrose concentrations (40% (w/v) in 50/50 water/2M2P) are used, an aqueous two-phase system (2M2P/sucrose) is induce, where the synthesized levan has a similar molecular weight distribution as in water and remains in solution.

A novel thermostable esterase from the thermoacidophilic archaeonSulfolobus tokodaiistrain 7

We have characterized an esterase expressed from the putative esterase gene (ST0071) selected from the total genome analysis from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as a fusion protein in Escherichia coli. The protein was purified with heat treatment, affinity column chromatography, and size exclusion filtration. The optimum activity for ester cleavage against p-nitrophenyl esters was observed at around 70 °C and pH 7.5–8.0. The enzyme exhibited high thermostability and also showed activity in a mixture of a buffer and water-miscible organic solvents, such as acetonitrile and dimethyl sulfoxide. From the kinetic analysis, p-nitrophenyl butyrate was found to be a better substrate than caproate and caprylate.

A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7

We have characterized an esterase expressed from the putative esterase gene ( ST0071) selected from the total genome analysis from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as a fusion protein in Escherichia coli. The protein was purified with heat treatment, affinity column chromatography, and size exclusion filtration. The optimum activity for ester cleavage against p-nitrophenyl esters was observed at around 70 °C and pH 7.5–8.0. The enzyme exhibited high thermostability and also showed activity in a mixture of a buffer and water-miscible organic solvents, such as acetonitrile and dimethyl sulfoxide. From the kinetic analysis, p-nitrophenyl butyrate was found to be a better substrate than caproate and caprylate.

Cyclosporin Nanoparticulate Lipospheres for Oral Administration

Cyclosporin is a first line immunosuppressive drug used to prevent transplant rejection and to treat autoimmune diseases. It is a hydrophobic cyclic peptide built from nonmammalian amino acids with low oral bioavailability. The aim of this study was to develop an oral delivery system for cyclosporin A (CyA) and investigate the effect of composition and particle size of the CyA lipid nanoparticles (lipospheres) on the oral bioavailability of this drug. Dispersible concentrated oil formulations that upon mixing in water spontaneously form a nanodispersion were developed. The concentrated oil formulations were clear solutions composed of the drug, a solid triglyceride, a water miscible organic solvent, and a mixture of surfactants and emulsifiers. The activity of the formulated cyclosporin was determined in vitro following the effect on the proliferation of T cells. The oral bioavailability was determined on humans following the cyclosporin blood levels after oral intake of formulated cyclosporin. Cyclosporin dispersion systems resulting in particle size of 25 to 400nm were prepared from acceptable pharmceutical components. The composition of the surfactants and emulsifiers, the lipid core component, and the amount and type of the water miscible organic solvent N-methylpyrrolidone (NMP) and alcohols had a strong effect on the particle size of the dispersions. All formulations were reproducible and stable at room temperature for at least 6 months, with full activity of cyclosporin retained. Human oral bioavaiability study indicated a correlation between the AUC and Cmax and the particle size of the dispersion. A Cmax of ∼1300 ng/mL was found after 2h of oral intake of four capsules, each loaded with 50mg cyclosporin. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1264–1270, 2004

Biotechnological applications of peroxidases

Peroxidases are widely distributed in nature. Reduction of peroxides at the expense of electron donating substrates, make peroxidases useful in a number of biotechnological applications. Enzymes such as lignin peroxidase and manganese peroxidase, both associated with lignin degradation, may be successfully used for biopulping and biobleaching in the paper industry, and can produce oxidative breakdown of synthetic azo dyes. Oxidative polymerization of phenols and aromatic amines conducted by horseradish peroxidase (HRP) in water and water-miscible organic solvents, may lead to new types of aromatic polymers. Site directed mutagenesis of HRP has been used to improve the enantioselectivity of arylmethylsulfide oxidations. Peroxidase has a potential for soil detoxification, while HRP as well as soybean and turnip peroxidases have been applied for the bioremediation of wastewater contaminated with phenols, cresols, and chlorinated phenols. Peroxidase based biosensors have found use in analytical systems for determination of hydrogen peroxide and organic hydroperoxides, while co-immobilized with a hydrogen peroxide producing enzyme, they can be used for determination of glucose, alcohols, glutamate and choline. Peroxidase has also been used for practical analytical applications in diagnostic kits, such as quantitation of uric acid, glucose, cholesterol, lactose, and so on. Enzyme linked immunorbent assay (ELISA) tests on which peroxidase is probably the most common enzyme used for labeling an antibody, are a simple and reliable way of detecting toxins, pathogens, cancer risk in bladder and prostate, and many other analytes. Directed evolution methods, appear to be a valuable alternative to engineer new catalyst forms of plant peroxidases from different sources to overcome problems of stability and to increase thermal resistance.

Determination of lower sub ppt levels of environmental analytes using high-powered concentration system and high-performance liquid chromatography with fluorescence detection.

The proposed method was successful in the determination and separation of lower sub ppt levels of polycyclic aromatic hydrocarbons (PAHs). In this study, a new phase separation phenomenon (i.e., homogeneous liquid-liquid extraction) was developed that rapidly and simply separated an immiscible liquid phase from water-miscible organic solvents. One liter of sample solution was preconcentrated by a solid phase extraction method, then the obtained eluate (5 mL) was further preconcentrated to just 20 microL by this homogeneous liquid-liquid extraction. The 20 [micro sign]L sedimented phase was directly injected into the high-performance liquid chromatography with fluorescence detection (FL-HPLC). The entire preconcentration factor was 50,000-fold. Six kinds of PAHs were determined in the range of 3.0 x 10(-18) approximately 4.5 x 10(-11) mol L(-1). These chemicals were also satisfactorily separated.

Swelling behavior of PMMA- g-PEO microgel particles by organic solvents

Aqueous dispersions of cross-linked poly(methylmethacrylate)- g-poly(ethylene oxide) [PMMA- g-PEO] microgel particles have been prepared from mixtures of methylmethacrylate [MMA] and MMA–PEO macromonomer, with ethylene glycol dimethacrylate [EGDM] as the cross-linking monomer (0.2–0.5% wt%). The hydrodynamic radius of these (unswollen) microgel particles ranged from 73 to 85 nm, and the particles were essentially monodisperse with regard to their size distribution. Their swelling behavior has been investigated in the presence of both water-miscible and water-immiscible organic solvents. In general, with the addition of a water-miscible solvent, deswelling behavior was observed. However, the microgel particles were swollen on addition of 1,4-dioxan, which is a good solvent for PMMA. With water-immiscible organic solvents, the extent of swelling depended on the solvency properties of the organic liquid for PMMA. In the presence of benzene, the somewhat large increases in particle size have been attributed to weak flocculation. This has been assumed from an estimate of the van der Waals attraction energy between the swollen microgel particles.

유화제로서 PEG-PPG 블록 공중합체를 이용한 Poly(DL-Lactide-co-Glycolide) 나노입자: 제조 및 지용성 약물의 로딩

In this study, poly(DL-lactide-co-glycolide) nanoparticles loaded with water-insoluble vitamins such as vitamin A (Retinol) and vitamin E acetate were prepared by the emulsification diffusion method. Polymer solution was prepared by the two water-miscible organic solvent, such as ethanol and acetone. Because of its biocompatible property, polyethyleneglycol-polypropyleneglycol diblock copolymer was used as surfactant and stabilizer. The influence of some preparative variables on the nanoparticle formation and on the loading efficiency of active agents, such as the type and concentration of stabilizing agent, the stirring methods, the water/oil phase ratio and the polymer concentration were investigated in order to control and optimize the process. After preparation of nanoparticles loaded with active agent, par- ticle size and distribution were evaluated by the light scattering particle analyzer. The loading efficiency of active agents was evaluated by the UV-visible spectroscopy. As the results, particle size were 50-200 nm and dispersibility was mon- odisperse. The optimum loading efficiency of active agents was observed 50-60%. It was found that the appropriate of selections of binary solvent mixtures and polymeric concentrations in both organic and aqueous phases could provide good yield and favorable physical properties of PLGA nanoparticles.

Unique crystal morphology of hydrophobic CaCO 3 composite by sodium trisilanolate in a mixture of a water-miscible organic solvent and water

We describe unique crystal morphologies of hydrophobic CaCO 3 crystals in a mixture of water and tetrahydrofuran (THF). After addition of sodium trisilanolate [(c–C 5H 9) 7Si 7O 9(ONa) 3] ( 1 ̄ ) into the mixture of water and THF, calcium reactants (0.1 M CaCl 2 and 0.1 M (NH 4) 2CO 3) were added to the reaction mixture at 30°C. FT-IR and SEM images showed that concave terraced calcite was obtained in 80% THF solution. In the absence of 1 ̄ , the crystalline product was a mixture of rhombohedral calcite with aggregated vaterite particles. The hydrophobic modified surface of the concave terraced calcite was confirmed by the contact angle and dispersability in organic solvents.

Core−Shell Structure of PLA−PEG Nanoparticles Used for Drug Delivery

Small-angle neutron scattering (SANS) has been used to study the internal structure of poly(lactic acid)−poly(ethylene glycol) (PLA(d)−PEG) block copolymer assemblies, which are being investigated as particulate drug carriers. Three PLA(d)−PEG copolymers with a fixed PEG of 5 kDa and a fully deuterated PLA(d) block of either 3, 15, or 45 kDa were synthesized by the ring opening polymerization of d8-d,l-lactide, using stannous octoate as a catalyst. These copolymers assembled to form nanoparticles in aqueous media, following precipitation from a water miscible organic solvent. The hydrodynamic radius of the PLA(d)−PEG nanoparticles increased with the molecular weight of the PLA(d) block. SANS data obtained at three different solvent contrasts were analyzed simultaneously using core−shell models, which assumed a homogeneous core of uniform scattering length density and a simple functional form for the scattering length density profile of the shell. The thickness and structure of the stabilizing PEG layer were found to depend on the molecular weight of the PLA(d) block. The splayed PEG chains of the PLA(d)−PEG 3:5 assemblies were characteristic of those found in polymeric micelles. However, as the molecular weight of the PLA(d) block was increased, the PEG brush became more radially homogeneous, in accord with recent Scheutjens−Fleer mean-field theory predictions.

Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in a Ca 2+-independent two-step mechanism: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I-1-P). Moderate amounts of water-miscible organic solvents have previously been shown to dramatically enhance the cyclic phosphodiesterase activity, that is, hydrolysis of cIP. Cosolvents [isopropanol (iPrOH), dimethylsufoxide (DMSO), and dimethylformamide (DMF)] also enhance the phosphotransferase activity of PI-PLC toward PI initially presented in vesicles, monomers, or micelles. Although these water-miscible organic cosolvents caused large changes in PI particle size and distribution (monitored with pyrene-labeled PI fluorescence, 31P NMR spectroscopy, gel filtration, and electron microscopy) that differed with the activating solvent, the change in PI substrate structure in different cosolvents was not correlated with the enhanced catalytic efficiency of PI-PLC toward its substrates. PI-PLC stability was decreased in water/organic cosolvent mixtures (e.g., the T m for PI-PLC thermal denaturation decreased linearly with added iPrOH). However, the addition of myo-inositol, a water-soluble inhibitor of PI-PLC, helped stabilize the protein. At 30% iPrOH and 4 °C (well below the T m for PI-PLC in the presence of iPrOH), cosolvent-induced changes in protein secondary structure were minimal. iPrOH and diheptanoylphosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed with PI as substrate. This behavior is consistent with a mechanism for cosolvent activation that involves changes in active site polarity along with small conformational changes involving the barrel rim tryptophan side chains that have little effect on protein secondary structure.