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Due to the lipophilicity of the metal-ion receptor, previously reported Cu(I)-selective fluorescent probes form colloidal aggregates as revealed by dynamic light scattering. To address this problem, we have developed a hydrophilic triarylpyrazoline-based fluorescent probe, CTAP-2, that dissolves directly in water and shows a rapid, reversible, and highly selective 65-fold fluorescence turn-on response to Cu(I) in aqueous solution. CTAP-2 proved to be sufficiently sensitive for direct in-gel detection of Cu(I) bound to the metallochaperone Atox1, thus demonstrating the potential for cation selective fluorescent probes to serve as tools in metalloproteomics for identifying proteins with readily accessible metal-binding sites.

E-mail: dylim@sejong.ac.krReceived January 14, 2011, Accepted February 8, 2011Synthesis of halicylindramide A mimetics via solid-phase peptide synthesis is described. The N-Me-Gly-Thrresidues in halicyclindramide A has been replaced by N-Me-Gly-Dpr, Lys, or N-Me-Gly-allo-Thr. Solutionstructures of the mimetics were compared by CD spectroscopy in an aqueous buffer and in TFE.Key Words : Synthesis, Halicylindramide A, Mimetic, DepsipeptideIntroductionHalicylindramides A-D

An accurate, reliable, and reproducible analytical method using HPLC/MS/MS for the determination of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment (CP-60,300) was developed and validated. Briefly, the method involved an initial acid treatment of intact tissues, which yielded the common fragment (CP-60,300). A portion of the acid hydrolyzate was purified by SPE using a strong cation exchange cartridge. Evaporation of the purified extract was followed by reconstitution in aqueous buffer and analysis by HPLC/MS/MS under isocratic conditions. The developed method provided acceptable sensitivity for determinative surveillance of tulathromycin in porcine kidney and bovine liver with an LOQ of 7.50 and 2.75 g/g, respectively. The overall recovery and precision (CV) of 45 determinations of each tissue were 97.8 (5.3) for porcine kidney and 96.9 (7.9) for bovine liver. Accuracy, precision, linearity, specificity, and ruggedness were demonstrated. An HPLC/MS/MS method was also developed for use in these tissues as a confirmatory assay following modifications to the MS detection parameters. The confirmatory method demonstrated acceptable sensitivity for confirmatory evaluation of tulathromycin in porcine kidney and bovine liver at tolerances of 15 and 5.5 g/g, respectively.

MEEKC is an electrodriven separation technique that utilises the unique properties of a microemulsion (ME) as a background electrolyte to achieve separation of a diverse range of solutes. MEs are composed of nanometre-sized oil droplets suspended in aqueous buffer, which is commonly referred to as oil-in-water ME. The droplets are stabilised by the presence of both a surfactant and co-surfactant. The use of water-in-oil MEs in MEEKC has also been investigated. This review details the advances in MEEKC-based separations from the period June 2008 - June 2010. Areas covered include online sample concentration, suppressed electroosmosis MEEKC, chiral separations, MEEKC-MS, MEEKC-ICP-MS and ME structure characterisation. The review also includes a fundamental introduction to MEEKC, along with a review of recent applications.

Metal-containing polymer networks are widespread in biology, particularly for load-bearing exoskeletal biomaterials. Mytilus byssal cuticle is an especially interesting case containing moderate levels of Fe 3+ and cuticle protein—mussel foot protein-1 (mfp-1), which has a peculiar combination of high hardness and high extensibility. Mfp-1, containing 13 mol % of dopa (3, 4-dihydroxyphenylalanine) side-chains, is highly positively charged polyelectrolyte (pI ∼ 10) and didn’t show any cohesive tendencies in previous surface forces apparatus (SFA) studies. Here, we show that Fe 3+ ions can mediate unusually strong interactions between the positively charged proteins. Using an SFA, Fe 3+ was observed to impart robust bridging ( W ad ≈ 4.3 mJ/m 2 ) between two noninteracting mfp-1 films in aqueous buffer approaching the ionic strength of seawater. The Fe 3+ bridging between the mfp-1-coated surfaces is fully reversible in water, increasing with contact time and iron concentration up to 10 μM; at 100 μM, Fe 3+ bridging adhesion is abolished. Bridging is apparently due to the formation of multivalent dopa-iron complexes. Similar Fe-mediated bridging ( W ad ≈ 5.7 mJ/m 2 ) by a smaller recombinant dopa-containing analogue indicates that bridging is largely independent of molecular weight and posttranslational modifications other than dopa. The results suggest that dopa-metal interactions may provide an energetic new paradigm for engineering strong, self-healing interactions between polymers under water.

MEEKC is an electrodriven separation technique, which utilises the unique properties of a microemulsion (ME) as a background electrolyte to achieve separation of a diverse range of solutes. MEs are composed of nanometre-sized oil droplets suspended in aqueous buffer, which is commonly referred to as an oil-in-water ME. The droplets are stabilised by the presence of a surfactant and co-surfactant. The use of water-in-oil MEs in MEEKC has also been investigated. This review details the advances in MEEKC-based separations from the period 2008 to 2009. Areas covered include online sample concentration, suppressed electroosmosis MEEKC, chiral separation, MEEKC-MS, and structure-migration relationships. The review also includes a fundamental introduction to MEEKC, along with the presentation of recent applications.

The objective of this study was to determine whether a relationship exists between ice nucleation temperature and recovery of activity of a model protein, lactate dehydrogenase, after freeze drying. Aqueous buffer systems containing 50 µg/mL of protein were frozen in vials with externally mounted thermocouples on the shelf of a freeze dryer, then freeze dried. Various methods were used to establish a wide range of ice nucleation temperatures. An inverse relationship was found between the extent of supercooling during freezing and recovery of activity in the reconstituted solution. The data are consistent with a mechanism of inactivation resulting from adsorption of protein at the ice/freeze–concentrate interface during the freezing process. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3495–3498, 2009

In this paper, we describe the apparent production of enzymatically synthesized amylose in DMSO by means of the calcium alginate hydrogel beads/DMSO system as the reaction field of the phosphorylase-catalyzed enzymatic polymerization. When the calcium alginate hydrogel beads including glucose-1-phosphate, maltoheptaose, and phosphorylase were suspended in DMSO and the system was slowly stirred at 40°C for 12 h, the reaction proceeded to produce amylose, which eluted to the DMSO solution. The obtained amylose was purified by the treatment with ion-exchange resins, and its structure was confirmed by the 1H NMR spectrum. The time-course experiment in the present system revealed that the phosphorylase-catalyzed enzymatic polymerization was carried out for 15 min on the inside of the calcium alginate hydrogel beads and the produced amylose was gradually eluted to the surrounding DMSO solution. The comparison of the present system with the general enzymatic polymerization in aqueous buffer solution suggested th...

Polyaniline (PANI) nanotubes were chemically synthesized from different aqueous buffer solutions: citric acid/phosphate (pH 3 and pH 4 as prepared) and HCl/phthalate (pH 3), beginning with 0.2 M an...

We report that solid-phase hydrothiolysis is an efficient method to convert resin-bound peptide thioesters to thioacids in aqueous buffer by using a total PEG-based resin. Also demonstrated is the use of the so-prepared peptide thioacids in chemoselective amide bond formation reactions.

Thiols, simple and complex, including polypeptide and protein thiols, react rapidly and selectively with esters of propiolic acid under very mild conditions (aqueous buffer, room temperature, pH 7) to give thioacrylates. These stable derivatives exhibit strong, characteristic ultraviolet spectra, maximal at 280–290 nm, molar absorbance ca 12,500. In a single, simple experiment the thiol is detected, its amount is estimated, it is stabilized against oxidation and disulfide scrambling, it is converted into a derivative amenable to isolation and structure elucidation procedures, and it is tagged with recognizable ultraviolet and NMR characteristics generated de novo. The reagents are stable and inexpensive and the procedures are quick, easy, sensitive and selective.

International audience; Persistent self-assembled polymersome capsules are shown to solubilise and stabilize a new hydrosulfide-containing (2), as well as hydroxylated (1), malachite green derivatives in their leuco-forms in aqueous buffer solution. Photoirradiation resulted in reversible hydroxide release/hydrogen sulfide generation. Notably, the efficient augmentation of H2S concentration to physiologically-relevant levels is shown.

Publisher Summary Solubility plays a crucial role in drug absorption in the gastrointestinal tract, accurate biological assays in vitro, and dosage form selection for in vivo administration. With this central role, measurement of solubility is a frequent activity of discovery scientists, and attention should be paid to accurate and relevant analyses. This chapter provides an overview of in silico and in vitro solubility methods. The total solubility of an ionizable compound at a particular pH can be estimated using its intrinsic solubility. Several software products for solubility are commercially available. One widely distributed software, QikProp, predicts several absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties based on regression models using molecular descriptors. Kinetic solubility is most appropriate for drug discovery for the following reasons. The purpose of kinetic solubility measurements is to identify compounds that do not have good kinetic solubility even in aqueous buffer containing DMSO, to guide modification of structures to improve solubility, and to guide formulation selection for animal dosing. Thermodynamic solubility data are most appropriate for crystalline material that is studied in detail during late drug discovery and development. Thermodynamic solubility studies are often performed to assist discovery and development formulation and in preparation for clinical development. Such studies provide perspectives on compound solubility in various physiological fluids to aid absorption studies, in various formulary solvents to aid formulation, and in solvents that are used to measure lipophilicity.

A new fluorescent turn-on probe (3) for the selective sensing and bioimaging of thiols is reported. In aqueous buffer solutions at physiological pH, thiols cleave the 2,4-dinitrobenzenesulfonyl group to release the red-emissive donor-acceptor fluorophore (4). The probe displays excellent immunity to interference from nitrogen and oxygen nucleophiles and the imaging of thiols in living cells is demonstrated.

Nitric oxide prodrugs derived from hindered secondary amines were prepared. The decomposition patterns of these prodrugs indicate that alpha-methyl groups around the nitrogen bearing the diazeniumdiolate group prolong their half-life in aqueous buffer.

Interaction of the dilacunary polyanion precursor [γ-GeW10O36]8- with Fe3+ ions in aqueous buffer medium (pH 4.8) results in the formation of two dimeric tungstogermanates depending on the reactant ratios. When using an Fe3+ to [γ-GeW10O36]8- ratio of 1:1, the asymmetric anion [K(H2O)(β-Fe2GeW10O37(OH))(γ-GeW10O36)]12- (1) is formed, whereas [{β-Fe2GeW10O37(OH)2}2]12- (2) is formed when using a ratio of 2:1. Single-crystal X-ray analyses were carried out on Cs3K9[K(H2O)(β-Fe2GeW10O37(OH))(γ-GeW10O36)]·19H2O (CsK-1), which crystallizes in the triclinic system, space group P1, a = 11.4547(2), b = 19.9181(5), c = 21.0781(6) A, α = 66.7977(12), β = 89.1061(12), γ = 84.4457(11)°, and Z = 2 and on Cs7K4Na[{β-Fe2GeW10O37(OH)2}2]·39H2O (CsKNa-2), which crystallizes in the monoclinic system, space group C2/m, a = 32.7569(13), b = 12.2631(5), c = 14.2895(5) A, β = 104.135(2)°, and Z = 2. Polyanion 1 consists of (β-Fe2GeW10O37) and (γ-GeW10O36) units linked via two Fe−O−W bridges and a central potassium ion. Two eq...

Hydrolysis of fenoxaprop-p-ethyl (FE), a widely used herbicide, was studied in aqueous buffer solutions at pH ranging from 4.0 to 10.0. The degradation kinetics, strongly dependent on pH values, followed first-order kinetics. FE was relatively stable in neutral media, whereas it degraded rapidly with decreasing or increasing pH. In acidic conditions (pH = 4, 5), the benzoxazolyl-oxy-phenyl ether linkage of FE was cleaved to form ethyl 2-(4-hydroxyphenoxy)propanoate (EHPP) and 6-chloro-2,3-dihydrobenzoxazol-2-one (CDHB). While in basic conditions (pH = 8, 9, 10), herbicidal activity fenoxaprop-p (FA) was formed via breakdown of the ester bond of the herbicide. Both the two pathways were concurrent in neutral conditions (pH = 6, 7). Toxicity studies on Daphnia magna showed that FE was most toxic to D. magna with 48 h EC(50) of 14.3 micromol/L, followed by FA (43.8 micromol/L), CDHB (49.8 micromol/L), and EHPP (333.1 micromol/L). Mode of toxic action analysis indicated that EHPP exhibited toxicity via polar narcosis, whereas CDHB belonged to reactive acing compound. The mixture toxicity of CDHB and EHPP was nonadditive and can be predicted by a response addition model. Therefore, the evaluation of overall FE toxicity to D. magna in the aquatic systems needs to consider the degradation of FE.

Luminescent tricarbonylrhenium(I) dipyridoquinoxaline indole complexes [Re(N−N)(CO)3(L)](CF3SO3) (N−N = dipyrido[3,2-f:2‘,3‘-h]quinoxaline (dpq), L = N-(3-pyridoyl)tryptamine (py-3-CONHC2H4-indole) (1a), N-(N-(3-pyridoyl)-6-aminohexanoyl)tryptamine (py-3-CONHC5H10CONHC2H4-indole) (1b); N−N = 2-(n-butylamido)dipyrido[3,2-f:2‘,3‘-h]quinoxaline (dpqa), L = py-3-CONHC2H4-indole (2a), py-3-CONHC5H10CONHC2H4-indole (2b)) and their indole-free counterparts [Re(N−N)(CO)3(py-3-CONH-Et)](CF3SO3) (py-3-CONH-Et = N-ethyl-(3-pyridyl)formamide; N−N = dpq (1c), dpqa (2c)) have been synthesized and characterized. The crystal structure of a related complex, [Re(dpqa)(CO)3(pyridine)](PF6), has also been studied. All the complexes exhibited triplet metal-to-ligand charge-transfer (3MLCT) (dπ(Re) → π*(N−N)) emission in fluid solutions at 298 K and in alcohol glass at 77 K. The emission quantum yields of the complexes were reduced upon changing from CH2Cl2 to aqueous buffer. The reduction was much more significant for the dpq...

Two types of host molecules based on phenolphthalein and two crown ethers have been constructed. These hosts made it possible to recognize the length of α,ω-diamines and transformed the binding events into detectable changes in color in an aqueous buffer solution.

The electrochemical and chemical oxidation of a series of C8-arylamine adducts of 2'-deoxyguanosine has been examined. The oxidations were found to be reversible by cyclic and square-wave voltammetry in both aqueous buffer and aprotic organic solvent. The mechanism of the oxidation in protic media was either one- or two-electron, depending on the aryl group. The chemical oxidation resulted in guanidinohydantoin and spiroiminodihydantoin rearrangement products similar to those observed for 8-oxo-7,8-dihydro-2'-deoxyguanosine.

We describe an unprecedented reaction between peptide selenoesters and peptide dimers bearing N-terminal selenocystine that proceeds in aqueous buffer to afford native amide bonds without the use of additives. The selenocystine-selenoester ligations are complete in minutes, even at sterically hindered junctions, and can be used in concert with one-pot deselenization chemistry. Various pathways for the transformation are proposed and probed through a combination of experimental and computational studies. Our new reaction manifold is also showcased in the total synthesis of two proteins.

We report a new chemodosimetric probe () for specific recognition of cysteine (Cys) in aqueous buffer and in whey protein isolated from fresh cow's milk. Using this reagent we could develop a luminescence-based methodology for estimation of Cys released from a commercially available Cys-supplement drug by aminoacylase-1 in live cells.

Herein, we report a novel strategy to accelerate the rate of DNA strand replacement reaction (DSRR) by polar organic solvents. DSRR plays a vital role in DNA nanotechnology but prolonged reaction time limits its further advancement. That is why it is extremely important to speed up the rate of DSRR. In this work, we introduce different polar organic solvents in both simple and complicated DSRR systems and observe that the rate constant is much more than in aqueous buffer. The rate acceleration of DSRR by polar organic solvents is very obvious and we believe that this strategy will extend the application of DNA nanotechnology in future.

Although native chemical ligation (NCL) is emerging as a powerful method for the assembly of (glyco)peptide building blocks, its applicability is reduced when peptide segments are poorly soluble in aqueous buffer. We have found that incorporating reactants in liposomes allows NCL of lipophilic peptides and lipopeptides. Furthermore, the reaction rates of liposome-mediated NCL are higher than traditional reaction conditions resulting in improved yields. [reaction: see text]

Aladan {β-[6′-(N,N-dimethyl)amino-2′-naphthoyl]alanine} and aladan-containing cyclodecapeptide [Ald6]- loloatin C were synthesized. The properties of their fluorescent behaviours both in aqueous buffer and micelles created by sodium dodecylsulfate (SDS) and dodecylphosphorylcholine (DPC) were further investigated. It was found that in either of micelle systems, [Ald6]loloatin C possesses red-shift emission maximum wavelength and higher quantum yield. This property could be useful in the research of peptide-cell membrane interaction.

In this paper we have investigated the kinetic decomposition of 2-chloroethylnitrososulfamides (CENS) in fetal calf serum. The study was monitored by on-line solid phase extraction linked to RP-LC-MS. We demonstrated that CENS are less stable in fetal calf serum than in aqueous buffer at pH 7.4 and 37 degrees C. Moreover, we have shown that partition coefficient of CENS can be correlated to the kinetics decomposition, and we observe that the stability is better for lipophilic CENS. The different metabolites after decomposition have been tentatively identified by RP-HPLC-MS. This study indicates the formation of several metabolites resulting from a mechanism of decomposition more complex than in aqueous phosphate buffer, nevertheless, leading to the same main products.

The horseradish peroxidase-catalyzed oxidative polymerization of substituted phenols with the use of hydrogen peroxide as an oxidizer in an aqueous-organic medium has been studied. The presence of an aqueous buffer controlling activity of the catalyst is the necessary condition for this reaction. The rate of the process decreases with a reduction in the fraction of water in the reaction mixture. It has been shown that the oxidizer should be gradually added in portions to the reaction mixture. The resulting oligomers convert into high-molecular-mass crosslinked products under heating.

Crystal solvates of hemin with pyridine were for the first time studied thermogravimetrically. Molecular complexes of the 1:1 and 1:2 compositions were isolated and their physicochemical characteristics determined. Molecular complex formation between hemin and pyridine in borate buffer solutions were studied. The influence of pyridine admixtures on the state of hemin in aqueous buffer solutions was estimated.

In agreement with our previous studies and those of other authors, it is shown that much better fits of retention time as a function of pH are obtained for acid–base analytes when pH is measured in the mobile phase, than when pH is measured in the aqueous buffer when buffers of different nature are used. However, in some instances it may be more practical to measure the pH in the aqueous buffer before addition of the organic modifier. Thus, an open methodology is presented that allows prediction of chromatographic retention of acid–base analytes from the pH measured in the aqueous buffer. The model presented estimates the pH of the buffer and the pKa of the analyte in a particular acetonitrile/water mobile phase from the pH and pKa values in water. The retention of the analyte can be easily estimated, at a buffer pH close to the solute pKa, from these values and from the retentions of the pure acidic and basic forms of the analyte. Since in many instances, the analyte pKa values in water are not known, the methodology has been also tested by using Internet software, at reach of many chemists, which calculates analyte pKa values from chemical structure. The approach is successfully tested for some pharmaceutical drugs.

A novel compound, p -(dimethylamino)benzamido-thiosemicarbazide ( 1 ), based on intramolecular charge transfer fluorescence was synthesized. In pH 7.4 Tris–HCl buffer, the emission intensity of 1 increased upon the addition of HSA or BSA due to the energy transfer from the protein to 1 and also the hydrophobic microenvironment provided by HSA or BSA. The binding constants between 1 and proteins were 1.41 × 10 5 mol −1 L for HSA and 8.68 × 10 4 mol −1 L for BSA, respectively. There is only one binding site. The interaction between 1 and proteins was further investigated by synchronous fluorescence.

We report a systematic analysis of the relationship between salt bridge composition and 14-helix structure within a family of model beta-peptides in aqueous buffer. We find an inverse relationship between side-chain length and the extent of 14-helix structure as judged by CD. Introduction of a stabilizing salt bridge pair within a previously reported beta-peptide ligand for hDM2 led to changes in structure that were detectable by NMR.

The versatile biosynthetic intermediate isochorismate decomposes in aqueous buffer by two competitive pathways, one leading to isoprephenate by a facile Claisen rearrangement and the other to salicylate via elimination of the enolpyruvyl side chain. Computation suggests that both processes are concerted but asynchronous pericyclic reactions, with considerable C-O cleavage in the transition state but relatively little C-C bond formation (rearrangement) or hydrogen atom transfer to the enolpyruvyl side chain (elimination). Kinetic experiments show that rearrangement is roughly 8-times more favorable than elimination. Moreover, transfer of the C2 hydrogen atom to C9 was verified by monitoring the decomposition of [2-(2)H]isochorismate, which was prepared chemoenzymatically from labeled shikimate, by (2)H NMR spectroscopy and observing the appearance of [3-(2)H]pyruvate. Finally, the isotope effects obtained with the C2 deuterated substrate are in good agreement with calculations assuming pericyclic reaction mechanisms. These results provide a benchmark for mechanistic investigations of isochorismate mutase and isochorismate pyruvate lyase, the enzymes that respectively catalyze the rearrangement and elimination reactions in plants and bacteria.

The interaction of bilirubin with albumin in aqueous buffer solutions is studied and the stability constants of the formed complex are determined. The adsorption of bilirubin and albumin from their individual and mixed solutions on the surface of highly dispersed silica is studied as a function of pH and albumin concentration. It is shown that the character of the adsorption of bilirubin from solutions containing albumin changes significantly compared to the adsorption from an individual solution.

The key to obtaining optimum performance of an enzyme is often a question of devising an effective method for its immobilization. This review describes a novel, versatile and effective methodology for enzyme immobilization, namely, as cross-linked enzyme aggregates (CLEAs). The method is exquisitely simple – involving precipitation of the enzyme from aqueous buffer followed by cross-linking of the resulting physical aggregates of enzyme molecules – and amenable to rapid optimization. It is applicable to a wide variety of enzymes, including cofactor-dependent oxidoreductases and lyases, and affords stable, recyclable catalysts with high retention of activity, sometimes higher than that of the free enzyme it was derived from. The enzyme does not need to be of high purity. Indeed, the methodology is essentially a combination of purification and immobilization in one step. The technique is also applicable to the preparation of combi-CLEAs, containing two or more enzymes, for use in one-pot, multi-step synthes...

A FRET-based fluorescence probe was developed for selective detection of H2S in aqueous buffer and inside living cells. For this probe, the FRET probe could be cleaved by H2S, and the fluorescence of FRET donor is released. The probe is highly selective to H2S over other biologically relevant species to give color change for naked eye observation. Confocal imaging indicated that the probe could monitor intracellular H2S level changes.

A new fluorescent chelator Oxa , having two metal-binding sites, was designed and synthesized in six steps. Oxa exhibited two distinctive dissociation constants for Zn 2+ ( K d 1 = 1.50 μM and K d 2 = 140 μM), with considerable fluorescence increase in aqueous buffer at pH 7.2. Affinities of Oxa for the other biologically important ions such as Ca 2+ ( K d 1 = 250 μM and K d 2 = 275 mM) and Mg 2+ ( K d 1 = 975 μM and K d 2 could not be determined) in the same conditions were also obtained.

To determine the degradation rates and degradation products of the herbicide flumioxazin in aqueous buffer solutions (pH 5, 7 and 9), its hydrolysis and photolysis were investigated at 30 degrees C in the dark, and in a growth chamber fitted with fluorescent lamps simulating the UV output of sunlight. The rate of hydrolysis of flumioxazin was accelerated by increasing pH. The t(1/2) values at pH 5, 7 and 9 were 16.4, 9.1 and 0.25 h, respectively. Two degradation products were detected and their structural assignments were made on the basis of LC-MS data. Degradation product I was detected in all buffer solutions while degradation product II was detected in acidic buffer only. Both degradation products appeared to be stable to further hydrolysis. After correcting for the effects of hydrolysis, the photolytic degradation rate also increased as a function of pH and was approximately 10 times higher at pH 7 than that at pH 5, showing t(1/2) values of 4.9 and 41.5 h, respectively. Degradation products formed by photolysis were the same as those formed by hydrolysis. Flumioxazin was degraded more extensively at high pH and should degrade in surface water.

We report an investigation on the interaction of the positively charged surfactant DDAB (didodecyldimethylammonium bromide) with preformed vesicles from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Addition of 1.9 mM DDAB to preformed POPC vesicles of different concentrations results in mixed vesicles, owing to the avid uptake of DDAB by POPC. This leads to the so-called matrix effect: when preformed POPC liposomes with a narrow size distribution are present in the aqueous solution, there is a fast formation process of mixed vesicles and the final size distribution closely resembles that of the preformed POPC liposomes. The final mixed vesicle system is stable in size and size distribution. The effect is independent of the initial size of the POPC vesicles and requires low relative concentrations of POPC with respect to DDAB (up to 1:4). Aside this, we report about the time stability of DDAB vesicles as a function of the aqueous buffer ionic strength and we investigate the interaction between...

The conformation of the non-glycosylated recombinant form of the extracellar domain of rat MOG (rMOG(1-125)) dissolved in different solvent conditions was studied by CD spectroscopy. The results show that rMOG(1-125) exhibits a predominantly beta sheet conformation in aqueous buffer solution at pH 7.5 and that this 'beta-form' is stabilized by zwitterionic phospholipids, DPC and LPCP. The alpha helical content of the protein can increase from 9% to up to 20% when TFE or anionic detergent LPAP and SDS are added.

Near-infrared (NIR) fluorochromes have become important reporter molecules for many biomedical applications, including FACS sorting, confocal microscopy, and more recently in vivo imaging. While the structures of several stable 800 nm fluorochromes have been published, their synthesis is often complex and there are difficulties in rapidly purifying these compounds in large quantities. Here we report on the synthesis of NIR820, ex/em = 790/820, with excellent physicochemical properties. Importantly, NIR820 is conveniently synthesized in a three-step reaction and can be purified by flash column chromatography rather than by HPLC. NIR820 is chemically stable and can be directly coupled to peptides during the solid-phase synthesis. In addition, NIR820 is also suitable for conjugation to proteins and other affinity molecules in aqueous buffer.

This paper presents a hypothesis relating the exclusion of a molecule (solute) from the surface of a protein in aqueous solution with the ability of that molecule to render surfaces “protein-resistant”, that is, resistant to the adsorption of proteins from aqueous buffer. While few current data test this hypothesis, it does suggest that surfaces presenting groups derived from certain osmolytesmolecules synthesized by cells to relieve osmotic stresswill be protein-resistant. These predictions were tested by constructing protein-resistant, self-assembled monolayers (SAMs) based on the osmolytes betaine and taurine. Examination of data from the literature also revealed that most of the known protein-resistant surfaces are based on displays of kosmotropesmolecules that stabilize the native structure of proteins. The connection between protein resistance, kosmotropicity, and biological function as an osmolyte may illuminate all three properties.

An engineered variant of lumazine synthase, a nonviral capsid protein with a negatively charged luminal surface, is shown to encapsulate up to 100 positively supercharged green fluorescent protein (GFP) molecules in vitro. Packaging can be achieved starting either from intact, empty capsids or from capsid fragments by incubation with cargo in aqueous buffer. The yield of encapsulated GFP correlates directly with the host/guest mixing ratio, providing excellent control over packing density. Facile in vitro loading highlights the unusual structural dynamics of this novel nanocontainer and should facilitate diverse biotechnological and materials science applications.

To permit rapid screening and characterization of novel cocaine hydrolases, as well as accurate measurement of cocaine levels in small samples of tissue, a radiometric assay was developed. The assay is based on selective, organic solvent partition of [3H]benzene-labeled cocaine or of [3H]benzoic acid liberated during enzymatic hydrolysis. With dilute samples the assay can be conducted entirely in scintillation vials and quantitated by addition of appropriate aqueous buffer and toluene-based fluor, making phase separation unnecessary. In this way, several hundred samples can be assayed in an afternoon, nanogram quantities of enzyme can be characterized without prior purification, and cocaine concentrations can be accurately measured in milligram samples of tissue after administration of [3H]cocaine in vivo.

Many different assays for measuring peptide-MHC interactions have been suggested over the years. Yet, there is no generally accepted standard method available. We have recently generated preoxidized recombinant MHC class I molecules (MHC-I) which can be purified to homogeneity under denaturing conditions (i.e., in the absence of any contaminating peptides). Such denatured MHC-I molecules are functional equivalents of "empty molecules". When diluted into aqueous buffer containing beta-2 microglobulin (beta2m) and the appropriate peptide, they fold rapidly and efficiently in an entirely peptide dependent manner. Here, we exploit the availability of these molecules to generate a quantitative ELISA-based assay capable of measuring the affinity of the interaction between peptide and MHC-I. This assay is simple and sensitive, and one can easily envisage that the necessary reagents, standards and protocols could be made generally available to the scientific community.

The dependence of the quantum yield of photogeneration of singlet oxygen with aluminum sulfophthalocyanines on the degree of their sulfonation was studied. Experiments showed that Fotosens preparation containing, on the average, about three sulfo groups in the macroring in aqueous buffer solution (pH 7.4) exists in the monomeric form and sensitizes singlet oxygen with a quantum yield ϕΔ 0.42±0.06. Aluminum tetra-4-sulfophthalocyanine forms dimers, and correspondingly ϕΔ is lower: 0.22±0.03. Sulfonated aluminum phthalocyanine containing, on the average, about two sulfo groups in the macroring is aggregated to a significant extent. Addition of Triton X-100 detergent results in partial deaggregation of the dye, and ϕΔ increases from ∼0.01 to 0.15±0.02. In the series including previously unknown aluminum phthalocyanines containing residues of phosphonic acids or their esters, and also aluminum and zinc octacarboxyphthalocyanines, the influence of the dye structure on the quantum yield of generation of singlet oxygen suggests participation of axial hydroxy groups in associate formation. These associates are not manifested spectroscopically and generate singlet oxygen inefficiently. The quantum yields ϕΔ for the monomers and associates are 0.3 and 0.1, respectively.

Monolithic silica columns prepared from tetramethoxysilane in a fused-silica capillary was octadecylsilylated, and evaluated by capillary electrochromatography. A plate height of 3 - 4 µm was obtained in an 80% acetonitrile-20% aqueous buffer (pH 8) under optimized conditions. An ODS phase having a high surface coverage, prepared by using octadecyldimethylchlorosilane, resulted in slow separation and a low column efficiency due to a slow electroosmotic flow. The results show that faster separation is achieved (i) with an ODS phase with a low surface coverage, (ii) with an ODS phase prepared by using octadecyltrichlorosilane, or (iii) by pressure-assisted CEC utilizing the high permeability of monolithic silica columns.

Assays traceable to high-level a reference materials are critical factors in ensuring accuracy of GFR estimates1. Serum cystatin C (Scys) is being proposed as a filtration marker that can be used with or as an alternative to serum creatinine (Scr) in equations to estimate GFR. The Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) published equations for estimating GFR from Scys in 2008 using serums assayed by the Cleveland Clinic Research Laboratory (CCRL) in 2003 using the Siemens-Dade-Behring (SDB) particle-enhanced immunonephelometric assay (PENIA) with the SDB BN II nephelometer2. At that time, Scys assays traceable to an internationally accepted reference material were not available. To foster consistent Scys results, the International Federation for Clinical Chemists (IFCC) Working Group for the Standardization of serum cystatin C and the Institute for Reference Materials and Measurements (IRMM) have collaborated for the production and characterization of a certified reference material (CRM)3, 4. This material, ERM-DA471/IFCC, was made available to laboratories by IRMM in fall 2010. Here we report on the re-expression of previously reported equations to estimate GFR from Scys for use with Scys values traceable to the new IRMM CRM The CKD-EPI Central Laboratory moved to the University of Minnesota (UMN) in 2009. At the time, comparisons between Scys measurement results at CCRL and UMN were made using serum calibration panels, which had been created in 2003 at CCRL and stored frozen at −70°5. The calibration panel included 40 reference sera pooled from at least 10 mixed-sex donors known to have serum creatinine values covering the range of 0.5 to 5.0 mg/dL. The panel was assayed for Scys in triplicate at CCRL in 2003 using the SDB measurement procedure as described above, and at the UMN in 2009 on a panel with one previous freeze thaw cycle using the same SDB PENIA reagent on a SDB ProSpec nephelometer. Conversion factors between the laboratories were determined using Deming linear regression6,7. To ensure stability over time, the calibration panel was retested on a previously unthawed panel at UMN in January 2011. ERM-DA471/IFCC CRM was reconstituted at UMN as instructed in its Certificate of Analysis yielding an aqueous buffer solution with a cystatin C concentration 5.48 mg/L (uncertainty 0.15 mg/L)3. Lower concentration solutions of the reference material were prepared by dilution of the neat reconstituted reference material with volumetric additions of PENIA reagent buffer to create target values of 1.37 mg/L, 2.74 mg/L, and 4.11 mg/L. The instrument PENIA reagent buffer was considered to have zero concentration. Accuracy of volumetric additions was checked by weight. The materials were assayed in duplicate on two separate days. Regression equations were used to compare the mean concentrations assigned by the SDB PENIA reagents on the ProSpec at UMN compared to the calculated values based on the certified value of the reference material. Intercepts that were very small and non-significant (p >0.05) were dropped from the regression. The calibration factor converts Scys results from UMN in 2009 (Scys-UMN’09) reported values to ERM-DA471/IFCC traceable values, which was then incorporated into the GFR estimating equation. Mean (SD, range) level of Scys measured in the calibration panel was 2.01 (0.74, 0.78-3.64) mg/L in CCRL in 2003, 1.67 (0.58, 0.68-2.97) mg/L in UMN in 2009, and 1.67 (0.59, 0.65-2.98) mg/L in UMN 2011. The mean (SD) difference between Scys values from CCRL in 2003 (Scys-CCRL’03) and Scys-UMN’09 was -0.34 (0.16) mg/L (p-value

Biomolecules evolve and function in densely crowded and highly heterogeneous cellular environments. Such conditions are often mimicked in the test tube by the addition of artificial macromolecular crowding agents. Still, it is unclear if such cosolutes indeed reflect the physicochemical properties of the cellular environment as the in-cell crowding effect has not yet been quantified. We have developed a macromolecular crowding sensor based on a FRET-labeled polymer to probe the macromolecular crowding effect inside single living cells. Surprisingly, we find that excluded-volume effects, although observed in the presence of artificial crowding agents, do not lead to a compression of the sensor in the cell. The average conformation of the sensor is similar to that in aqueous buffer solution and cell lysate. However, the in-cell crowding effect is distributed heterogeneously and changes significantly upon cell stress. We present a tool to systematically study the in-cell crowding effect as a modulator of biomolecular reactions.