Combinatorial Peptide Library for Probing the Selectivity of the s-1 Subsite of Proteases

A combinatorial tetrapeptide library, Suc-Ala-Phe-Arg-AA₁-OR, in which R = p-formamidobenzyl ester and AA₁ = 17 of the 20 natural occurring amino acids, has been synthesized chemically and separated by a reverse phase HPLC. The library was used to study the s-1 subsite specificity of various proteases. The preferred substrate at the s-1 subsite of chymotrypsin is in the order of Trp > Tyr > Phe > Met > Leu. This agreed with the reported data that the favored substrate at the s-1 subsite for chymotrypsin-catalyzed hydrolysis is an aromatic amino acid residue. The hydrophobic amino acid residues at this subsite can be hydrolysized after a longer incubating time. This procedure of selective hydrolysis of a peptide library was used to probe the selectivity of s-1 subsites of four proteases isolated from Bacillus stearothermophilus, subtilisin Carlsberg, subtilisin BPN' and an engineered protease subtilisin 8397. The protease from Bacillus stearothermophilus favored the substrate with residue Lys, and Arg at the s-1 subsite as a trypsin-like protease. The relative reactivities of amino acid residues in the protease-catalyzed hydrolysis of the library can be used as a fingerprint to identify the protease in a protease family.     

The formation of iron carbonyl radical anions in the reactions of iron carbonyls with trimethylamine N-oxide

The interaction of iron carbonyls, Fe(CO)₅, Fe₂(CO)₉ and Fe₃(CO)₁₂ with Me₃NO occurs according to a one-electron redox-disproportionation scheme giving rise to iron carbonyl radical anions: Fe₂(CO)₈^·− (1), Fe₃(CO)₁₂^·− (2), Fe₃(CO)₁₁^·− (3) and Fe₄(CO)₁₃^·− (4). The role of Me₃NO, inducing CO-substitution, consists of the generation of reactive 17-electron species with a labile coordination sphere in which the substitution for other ligands occurs, resulting from fast ligand and electron exchange in the confines of the ETC-reaction.     

A combinatorial approach for determining protease specificities: application to interleukin-1β converting enzyme (ICE)

Background: Interleukin-1β converting enzyme (ICE/caspase-1) is the protease responsible for interleukin-1β (IL-1β) production in monocytes. It was the first member of a new cysteine protease family to be identified. Members of this family have functions in both inflammation and apoptosis.Results: A novel method for identifying protease specificity, employing a positional-scanning substrate library, was used to determine the amino-acid preferences of ICE. Using this method, the complete specificity of a protease can be mapped in the time required to perform one assay. The results indicate that the optimal tetrapeptide recognition sequence for ICE is WEND, not YVAD, as previously believed, and this led to the synthesis of an unusually potent aldehyde inhibitor, Ac-WEND-CHO (K_i=56 pM). The structural basis far this potent inhibition was determined by X-ray crystallography.Conclusions: The results presented in this study establish a positional-scanning library as a powerful tool for rapidly and accurately assessing protease specificity. The preferred sequence for ICE (WEND) differs significantly from that found in human pro-interleukin-1β (YVHD), which suggests that this protease may have additional endogenous substrates, consistent with evidence linking it to apoptosis and IL-1α production.     

Interconversion of acetyl- and terminal-carbonyl groups on labeled (η5-indenyl)(CO)2Fe13C(O)CH3

The ¹³C-labeled (95–99%) acetyl complex (η⁵-In)(CO)₃Fe¹³C(O)CH₃ (8) (In = indenyl) has been prepared by acylating In(CO)₂Fe⁻ Na⁺ (1) with CH₃ ¹³C(O)Cl. All of the starting 1 must be consumed in this reaction (at −78°C), or 45% of the product results as In(CO)(¹³CO)FeC(O)CH₃ (9). Once isolated, neither 8 nor mixtures of 8 and 9 further redistribute or lose this label after pressurizing under 800 atm CO, or after heating in heptane, THF, or acetonitrile solution. Treating 8 with even trace amounts of 1 or of Cp(CO)₂Fe⁻ Na⁺ (5) rapidly interconverts the acetyl and terminal carbonyls, thus transforming 8 into mixtures of 8 and 9. A mechanism is proposed that involves a labile metalla-β-diketonate In(CO)Fe(Fp-CO)(CH₃¹³CO)⁻ Na⁺.     

Substrate turnover and inhibitor binding as selection parameters in directed evolution of blood coagulation factor Xa

A library of blood coagulation factor Xa (FXa)-trypsin hybrid proteases was generated and displayed on phage for selection of derivatives with the domain "architecture" of trypsin and the specificity of FXa. Selection based on binding to soybean trypsin inhibitor only provided enzymatically inactive derivatives, due to a specific mutation of serine 195 of the catalytic triad to a glycine, revealing a significant selection pressure for proteolytic inactive derivatives. By including a FXa peptide substrate in the selection mixture, the majority of the clones had retained serine at position 195 and were enzymatically active after selection. Further, with the inclusion of bovine pancreatic trypsin inhibitor, in addition to the peptide substrate, the selected clones also retained FXa specificity after selection. This demonstrates that affinity selection combined with appropriate deselection provides a simple strategy for selection of enzyme derivatives that catalyse a specific reaction.     

Substrate phage as a tool to identify novel substrate sequences of proteases

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A substrate phage library has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.     

Mapping of the active site of proteases in the 1960s and rational design of inhibitors/drugs in the 1990s

For several decades the specificity of proteases has been presented as an active site divided into subsites, using the nomenclature of Schechter & Berger from 1967 (S1, S2... for subsites of the active site; P1, P2... for residues of the substrate occupying the corresponding subsites). At early stages of the research (1960s) it was realized that the size of the active site was larger than expected and important interactions occur in regions remote from the catalytic site. Since the active site was found to be large it was divided into subsites, and a procedure to map it up was developed. The map provides information on the size of the active site (number of subsites), the properties of each subsite (free energy of ligand binding, nature of binding forces, etc.), and it enables rational design of new substrates and inhibitors. Already in 1968 inhibitors with binding constants ten thousand fold higher than available inhibitors, were prepared. The model of a large active site was initially met with strong opposition. Before long, however, predictions of the model (size of the active site, interactions in subsites remote from the catalytic site) were confirmed by X-ray crystallography (1970). During the 1990s proteolytic enzymes received renewed attention in biology and medicine, they became therapeutic targets, and protease inhibitors were successfully applied in the treatment of AIDS and hypertension. The model of large active site divided into subsites, proposed 38 years ago, stood the test of time. This model is still in use in basic research to evaluate enzyme activity, and in pharmaceutical research for the development of inhibitors/drugs.     

Fluorescence quenching in molecular recognition by hydrogen bonding

Steady-state fluorescence and single photon timing have been used to study the effect of the presence of hydrogen bonding on the intermolecular quenching of pyrene covalently linked to a guanine-like receptor I by an aliphatic amine (N,N-dimethylpropylamine) covalently linked to cytosine derivative II. By comparing the fluorescence quenching of I by II with that of 10methylpyrene (1-MP) by triethylamine (TEA), as a model system in which no hydrogen bonding can occur, one could possibly analyze the effect of the hydrogen bonding between receptor and substrate as a quenching as it leads to a higher local concentration of donor and acceptor. While the quenching of I by II was observed with an apparent rate constant k_q of (1.78 ± 0.10) × 10⁹ M⁻¹ s⁻¹ and (8.72 ± 0.42) × 10⁸ M⁻¹ s⁻¹ in toluene and acetonitrile, respectively, no quenching could be observed in methanol. Upon excitation of 1-MP, no quenching by II could be detected in the same concentration range as used in the quenching of I. Quenching of I and of 1-MP by TEA (⩾ 10⁻² M) in toluene leads to exciplex formation with maxima centred at 540 and 514 nm, respectively. The rate constants of exciplex formation and dissociation of I with TEA were analyzed using a global compartmental analysis. The following values were obtained for the rate constants: k₀₁ = (9.70 ± 0.01) × 10⁶ s⁻¹, k₂₁ = (1.12 ± 0.003) × 10⁹ M⁻¹ s⁻¹, k₀₂ = (5.24 ± 0.01) × 10⁷ s⁻¹ and k₁₂ = (7.74 ± 0.08) × 10⁶ s⁻¹. Quenching of I by TEA in the presence of III, a hydrogen-bonding system without an alkyl amine substituent, leads to exciplex formation centred at 538 nm. The rate constant values for the exciplex formation and dissociation of I with TEA in the presence of III were: k₀₁ = (9.32 ± 0.08) × 10⁶ s⁻¹, k₂₁ = (9.32 ± 0.003) × 10⁸ M⁻¹ s⁻¹, k₀₂ = (6.16 ± 0.03) × 10⁷ s⁻¹ and k₁₂ = (21.90 ± 0.3) × 10⁶ s⁻¹. The apparent rate constants k_q for this system was (7.26 ± 0.56) × 10⁶ M⁻¹ s⁻¹. The observed decrease in the rate of exciplex formation of I with TEA in the presence of III could suggest that the guanine-like moiety in I forms hydrogen bonds with the cytosine-like moiety and this could decrease the electron affinity of I. The rate constant of exciplex dissociation increased, indicating that the exciplex is less stable in the presence of III. Because of the single exponential decay of I in the presence and absence of II and of the agreement between steady-state and transient fluorescence measurements, the information available for quantitative analysis of the association between I and II is limited.     

Amphiphilic peroxynitrite decomposition catalysts in liposomal assemblies

Background: Peroxynitrite (ONOO⁻), a toxic biological oxidant, has been implicated in many pathophysiological conditions. The water-soluble porphyrins 5,10,15,20-tetrakis(N-methyl-4′-pyridyl)porphinato iron(III) (FeTMPyP) and manganese(III) (MnTMPyP) have recently emerged as potential drugs for ONOO⁻ detoxification, and FeTMPyP has demonstrated activity in models of ONOO⁻ related disease states. We set out to develop amphiphilic analogs of FeTMPyP and MnTMPyP suitable for liposomal delivery in sterically stabilized liposomes (SLs).Results: Three amphiphilic iron porphyrins (termed 1a-c) and three manganese porphyrins (termed 2a-c) bound to liposomes and catalyzed the decomposition of ONOO⁻. The polyethylene-glycol-linked metalloporphyrins 1b and 2b proved the most effective of these catalysts, rapidly decomposing ONOO⁻ with second-order rate constants (k_cat) of 2.9 × 10⁵ M⁻¹s⁻¹ and 5.0 × 10⁵ M⁻¹ s⁻¹, respectively, in dimyristoylphosphatidylcholine liposomes. Catalysts 1b and 2b also bound to SLs, and these metal loporphyrin-SL constructs efficiently catalyzed ONOO⁻ decomposition (k_cat ≈ 2 × 10⁵ M⁻¹ s⁻¹). The analogous metalloporphyrins 1a and 2a, which are not separated from the vesicle membrane surface by polyethylene glycol linkers, were significantly less effective (k_cat ≈ 3.5 × 10⁴ M⁻¹ s⁻¹).Conclusions: For these amphiphilic analogs of FeTMPyP and MnTMPyP, the polarity of the environment of the metalloporphyrin headgroup is intimately related to the efficiency of the catalyst; a polar aqueous environment is essential for effective catalysis of ONOO⁻ decomposition. Thus, catalysts 1b and 2b react rapidly with ONOO⁻ and are potential therapeutic agents that, unlike their water-soluble TMPyP analogs, could be administered as liposomal formulations in SLs. These SL-bound amphiphilic metalloporphyrins may prove to be highly effective in the exploration and treatment of ONOO⁻ related disease states.     

Metal complexes of anionic 3-borane-1-alkylimidazol-2-ylidene derivatives

Addition of BH₃·thf to 1-alkylimidazoles (alkyl=methyl, butyl) and 1-methylbenzimidazole leads to BH₃ adducts, which are deprotonated by BuLi to yield the organolithium compounds (L)Li⁺(1b–d)⁻. In the solid state (thf)Li⁺1b⁻ is dimeric. The acyl–iron complexes (thf)₃Li⁺(3b,d)⁻ are formed from (thf)Li⁺(1b,d)⁻ and Fe(CO)₅. (L)Li⁺(1a–c)⁻ react with [CpFe(CO)₂X], however, the only complex obtained is [CpFe(CO)₂1a] (5a). The analogous reaction of (L)Li⁺1a⁻ with the pentadienyl complex [(C₇H₁₁)Fe(CO)₂Br] yields the corresponding iron compound 6a. Their compositions follow from spectroscopic data. Treatment of Cp₂TiCl with (L)Li⁺1a⁻ leads to [Cp₂Ti1a] (7a), which could not be oxidized with PbCl₂ to give the corresponding Ti(IV) complex. The compounds [Li(py)₄]⁺9a⁻ and [Li(L)₄]⁺(10b–d)⁻ are obtained when (L)Li⁺1⁻ are reacted with VCl₃ and ScCl₃. The X-ray structure analysis of the vanadium complex reveals a distorted tetrahedron of the anion [V(1a)₄]⁻ with two smaller and four larger CVC angles. The scandium compound [Li(dme)₂⁺10c⁻] has a different structure: the distorted tetrahedron of the anion [Sc(1c)₄]⁻ contains two larger (140.2 and 142.9°) and four smaller CScC angles (93.9–98.7°). This arrangement allows the formation of four bridging BHSc 3c,2e bonds to give an eight-fold coordination. The anion 10c⁻ is formally a 16e complex.     

Vertebrate collagenase inhibitor. II. Tetrapeptidyl hydroxamic acids.

To develop a potent and specific collagenase inhibitor, a series of tetrapeptidyl hydroxamic acids were synthesized, based on the previous findings with tripeptidyl derivatives (Chem. Pharm. Bull., 38, 1007-1011, 1990). Among the series of tetrapeptidyl derivatives synthesized, R-Gly-Pro-Leu-Ala-NHOH and R-Gly-Pro-D-Leu-D-Ala-NHOH were found to be highly specific and potent inhibitors against vertebrate collagenase with an IC50 of 10(-6) M order, where R stands for Boc or acyl group. Analysis of their structure-activity relationships showed a characteristic feature of the substrate-binding site of collagenase as follows: 1) the S1 subsite forms a shallow hydrophobic pocket, although glycine residue corresponds to the subsite of the natural collagen substrate: 2) the S2 subsite constitutes a bulky pocket with less requirement for hydrophobicity: 3) the S3 subsite preferentially accommodates Pro residue: and 4) the accommodation of the P4-P1 subsites of peptidyl collagenase inhibitor to the S4-S1 subsites is required to form a tight binding of its hydroxamic acid moiety to the zinc ion at the catalytic site of the enzyme. The introduction of an enantiometric dipeptide unit, D-Leu-D-Ala, to the P2-P1 subsites demonstrated an increased binding capacity to the extended S4-S1 subsites of collagenase, thus providing proteinase-resistant inhibitor.     

The circular dichroism spectra of the cyclodextrin complex with 1,4-benzocyclooctenedione

The assignment of the absorption spectra of 1,4-benzocyclooctenedione (1) is reported by measuring the circular dichroism spectra of the β-cyclodextrin complex with 1. It is concluded from the signs of the circular dichroism bands that the first (16.6 × 10³−27.4 × 10³ cm⁻¹) absorption band is composed of two electronic transitions having perpendicular polarizations with respect to the long axis of 1, the second (27.4 × 10³−35.8 × 10³ cm⁻¹) absorption band has the transition dipole moment parallel to the long axis of 1 and the third (35.8 × 10³−44.3 × 10³ cm⁻¹) absorption band is composed of two electronic transitions having perpendicular and parallel polarizations with respect to the long axis of 1.     

A N,N′-diacetate benzodioxotetraazamacrocycle and its transition metal complexes

Reaction of 2,9-dioxo-1,4,7,10-tetraazabicyclo[1.10.1]hexadeca-1(11),13,15-triene-4,7-diacetic acid (H₂L1) with CuCl₂ · 2H₂O in ethanol at pH 6 led to the monomeric benzodioxochlorocomplex [Cu(L′1)Cl] (1) (HL′1 = monoethylesther of H₂L1). X-ray structural analysis has shown that in complex 1 the Cu is five-coordinated by two nitrogen and two oxygen atoms of the macrocycle and by a chloride, displaying a square pyramidal coordination geometry. One of the acetate arms does not coordinate to the Cu and has suffered an in situ ethanolic esterification reaction. The protonation constants of H₂L1 and the stability constants of its complexes with Cu²⁺, Ni²⁺, Zn²⁺, Cd²⁺ and Pb²⁺ were determined by potentiometric methods and in some cases by ¹H NMR spectroscopy. The stability constants of the complexes follow the trend [Ni(H₋₁L1)]⁻ > [Cu(H₋₁L1)]⁻ ≫ [Pb(H₋₁L1)]⁻ > [Zn(H₋₁L1)]⁻ > [Cd(H₋₁L1)]⁻, probably due to steric requirements. Spectroscopic measurements (absorption and EPR) at different pH values have shown the effect of the pH on the coordination sphere of the Cu complexes.     

Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl₂, KCl, and BaCl, and partially inhibited by CuCl₂, CoCl₂ and totally inhibited by AlCl₃ and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k _cat/K _m of 10,666 mM^?1?s^?1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k _cat/K _m of 7,500 mM^?1?s^?1. Basic and acidic side chain-containing amino acids performed best at subsite S₁. Subsites S₂, S₃, S^′ ₂, and S^′ ₁, S^′ ₃ showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k _cat/K _m were observed for the subsites S₂, S₃, and S^′ _2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.     

The loading of polyoxometalates based compound on reduced graphene oxide,a composite material for electrical energy storage and tetracycline removal

A polyoxometalate based composite material (NiPW₁₂NP/FrGO) was synthesized successfully, in which the nanoparticle of a polyoxometalate compound (NiPW₁₂NP) distributes on carboxylate group functionalized reduced graphene oxide (FrGO) homogenously. There exist intensive chemical bonds between NiPW₁₂NP and FrGO, which guarantees the stability of this composite material. When employed as a cathode material, NiPW₁₂NP/FrGO exhibits high specific capacitance, remarkable rate capability and long-term stability. When the current density is 4 A g⁻¹, a specific capacitance as high as 437.6 F g⁻¹ is achieved by NiPW₁₂NP/FrGO. With NiPW₁₂NP/FrGO serving as cathode and MnO₂ acting as anode, a high performance asymmetric supercapacitor (ASC) is assembled, which possesses a high energy density of 12.96 W h·kg⁻¹ at 0.67 kW kg⁻¹. It also shows a good rate capability, when the current density increases from 4 to 12 A g⁻¹, its specific capacitances decreases from 115.2 to 90.9 F g⁻¹, with 78.9% capacitance retention. After 5000 cycles charge-discharge experiments, 94.3% of its capacitance can be maintained, which exhibits good stability. Furthermore, NiPW₁₂NP/FrGO composite material also shows excellent tetracycline adsorption ability with capacity 288.28 mg g⁻¹, the adsorption can be well described with Temkin model, which suggests electrostatic attraction dominates the adsorption process.     

Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes

Background: The proteasome is a multicatalytic protease complex responsible for most cytosolic protein breakdown. The complex has several distinct proteolytic activities that are defined by the preference of each for the carboxyterminal (P1) amino acid residue. Although mutational studies in yeast have begun to define substrate specificities of individual catalytically active β subunits, little is known about the principles that govern substrate hydrolysis by the proteasome.Results: A series of tripeptide and tetrapeptide vinyl sulfones were used to study substrate binding and specificity of the proteasome. Removal of the aromatic amino-terminal cap of the potent tripeptide vinyl sulfone proteasome inhibitor 4-hydroxy-3-iodo-2-nitrophenyl-leucinyl-leucinyl-leucine vinyl sulfone resulted in the complete loss of binding and inhibition. Addition of a fourth amino acid (P4) to the tri-leucine core sequence fully restored inhibitory potency. 1251-labeled peptide vinyl sulfones were also used to examine inhibitor binding and to determine the correlation of subunit modification with inhibition of peptidase activity. Changing the amino acid in the P4 position resulted in dramatically different profiles of β-subunit modification.Conclusions: The P4 position, distal to the site of hydrolysis, is important in defining substrate processing by the proteasome. We observed direct correlations between subunit modification and inhibition of distinct proteolytic activities, allowing the assignment of activities to individual β subunits. The ability of tetrapeptides, but not tripeptide vinyl sulfones, to act as substrates for the proteasome suggests there could be a minimal length requirement for hydrolysis by the proteasome. These studies indicate that it is possible to generate inhibitors that are largely specific for individual β subunits of the proteasome by modulation of the P4 and carboxy-terminal vinyl sulfone moieties.     

Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34

A protease from newly isolated Bacillus circulans M34 was purified by Q‐Sepharose anion exchange chromatography and Sepharose–bacitracin affinity chromatography followed by (NH₄)₂SO₄ precipitation. The molecular mass of the purified enzyme was determined using SDS–PAGE. The optimum pH and temperature for protease activity were 11 and 50°C, respectively. The effect of various metal ions on protease activity was investigated. Alkaline protease from Bacillus circulans M34 wase activated by Zn²⁺, Cu²⁺ and Co²⁺ up to 31%. The purified protease was found to be stable in the organic solvents, surfactants and oxidizing agent. The substrate specificity of purified protease was investigated towards different substrates. The protease was almost completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The kinetic parameters of the purified protease, maximum rate (V_max) and Michaelis constant (K_m), were determined using a Lineweaver–Burk plot. Copyright © 2015 John Wiley & Sons, Ltd.     

Dynamic Bio-Barcode Assay Enables Electrochemical Detection of a Cancer Biomarker in Undiluted Human Plasma: A Sample-In-Answer-Out Approach

There is a need for biosensing systems that can be operated at the point-of-care (POC) for disease screening and diagnostics and health monitoring. In spite of this, simple to operate systems with the required analytical sensitivity and specificity in clinical samples, using a sample-in-answer-out approach, remain elusive. Reported here is an electrochemical bio-barcode assay (e-biobarcode assay) that integrates biorecognition with signal transduction using molecular (DNA/protein) machines and signal readout using nanostructured electrodes. The e-biobarcode assay eliminates multistep processing and uses a single step for analysis following sample collection into the reagent tube. A clinically relevant performance for the analysis of prostate specific antigen (PSA) in undiluted and unprocessed human plasma: a log-linear range of 1 ng mL⁻¹–200 ng mL⁻¹ and a LOD of 0.4 ng mL⁻¹, was achieved. The e-biobarcode assay offers a realistic solution for biomarker analysis at the POC.     

Accurate measurement of conformational equilibria using NMR chemical shifts at the fast exchange limit: Dual anancomeric models for 1-methyl-1-chlorocyclohexane

3-Methyl- and cis-3,5-dimethyl- derivatives of 1-methyl-1-chlorocyclohexane (1) provide anancomeric model compounds from which to derive reliable estimates of the ¹³C NMR chemical shifts of the 1-methyl substituents in the two chair conformers of 1 at the fast exchange limit and thence ΔH° = 4.74 kJ mol⁻¹, ΔS° = 1.9 J mol⁻¹ K⁻¹ for 1E→1A in toluene.