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Tyrosine–tryptophan (YW) dyads are ubiquitous structural motifs in enzymes and play roles in proton-coupled electron transfer (PCET) and, possibly, protection from oxidative stress. Here, we describe the function of YW dyads in de novo designed 18-mer, β hairpins. In Peptide M, a YW dyad is formed between W14 and Y5. A UV hypochromic effect and an excitonic Cotton signal are observed, in addition to singlet, excited state (W*) and fluorescence emission spectral shifts. In a second Peptide, Peptide MW, a Y5–W13 dyad is formed diagonally across the strand and distorts the backbone. On a picosecond timescale, the W* excited-state decay kinetics are similar in all peptides but are accelerated relative to amino acids in solution. In Peptide MW, the W* spectrum is consistent with increased conformational flexibility. In Peptide M and MW, the electron paramagnetic resonance spectra obtained after UV photolysis are characteristic of tyrosine and tryptophan radicals at 160 K. Notably, at pH 9, the radical photolysis yield is decreased in Peptide M and MW, compared to that in a tyrosine and tryptophan mixture. This protective effect is not observed at pH 11 and is not observed in peptides containing a tryptophan–histidine dyad or tryptophan alone. The YW dyad protective effect is attributed to an increase in the radical recombination rate. This increase in rate can be facilitated by hydrogen-bonding interactions, which lower the barrier for the PCET reaction at pH 9. These results suggest that the YW dyad structural motif promotes radical quenching under conditions of reactive oxygen stress.

An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera’s allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation., Olivieri, Walker, Karamafrooz et al. show that the fusion of the dynamic J-domain to PKA-C (PKA-CDNAJB1) disrupts the internal allosteric network, attenuating the nucleotide/PKI binding cooperativity. This study suggests that the reduced allosteric cooperativity may contribute to the pathology that PKA-CDNAJB1 drives.

In the nucleus, the spatiotemporal regulation of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is orchestrated by an intrinsically disordered protein kinase inhibitor, PKI, which recruits the CRM1/RanGTP nuclear exporting complex. How the PKA-C/PKI complex assembles and recognizes CRM1/RanGTP is not well understood. Using NMR, SAXS, fluorescence, metadynamics, and Markov model analysis, we determined the multi-state recognition pathway for PKI. After a fast binding step in which PKA-C selects PKI’s most competent conformations, PKI folds upon binding through a slow conformational rearrangement within the enzyme’s binding pocket. The high-affinity and pseudo-substrate regions of PKI become more structured and the transient interactions with the kinase augment the helical content of the nuclear export sequence, which is then poised to recruit the CRM1/RanGTP complex for nuclear translocation. The multistate binding mechanism featured by PKA-C/PKI complex represents a paradigm on how disordered, ancillary proteins (or protein domains) are able to operate multiple functions such as inhibiting the kinase while recruiting other regulatory proteins for nuclear export.

Antimicrobial peptides (AMPs) are short peptides active against a wide range of pathogens and, therefore, they are considered a useful alternative to conventional antibiotics. We have identified a new AMP in a transcriptome derived from the Antarctic fish Trematomus bernacchii. This peptide, named Trematocine, has been investigated for its expression both at the basal level and after in vivo immunization with an endemic Antarctic bacterium (Psychrobacter sp. TAD1). Results agree with the expected behavior of a fish innate immune component, therefore we decided to synthesize the putative mature sequence of Trematocine to determine the structure, the interaction with biological membranes, and the biological activity. We showed that Trematocine folds into a &alpha, helical structure in the presence of both zwitterionic and anionic charged vesicles. We demonstrated that Trematocine has a highly specific interaction with anionic charged vesicles and that it can kill Gram-negative bacteria, possibly via a carpet like mechanism. Moreover, Trematocine showed minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against selected Gram-positive and Gram-negative bacteria similar to other AMPs isolated from Antarctic fishes. The peptide is a possible candidate for a new drug as it does not show any haemolytic or cytotoxic activity against mammalian cells at the concentration needed to kill the tested bacteria.

Paramagnetic relaxation enhancement (PRE) measurements constitute a powerful approach for detecting both permanent and transient protein-protein interactions. Typical PRE experiments require an intrinsic or engineered paramagnetic site on one of the two interacting partners; while a second, diamagnetic binding partner is labeled with stable isotopes ((15)N or (13)C). Multiple paramagnetic labeled centers or reversed labeling schemes are often necessary to obtain sufficient distance restraints to model protein-protein complexes, making this approach time consuming and expensive. Here, we show a new strategy that combines a modified pulse sequence ((1)H(N)-Γ(2)-CCLS) with an asymmetric labeling scheme to enable the detection of both intra- and inter-molecular PREs simultaneously using only one sample preparation. We applied this strategy to the non-covalent dimer of ubiquitin. Our method confirmed the previously identified binding interface for the transient di-ubiquitin complex, and at the same time, unveiled the internal structural dynamics rearrangements of ubiquitin upon interaction. In addition to reducing the cost of sample preparation and speed up PRE measurements, by detecting the intra-molecular PRE this new strategy will make it possible to measure and calibrate inter-molecular distances more accurately for both symmetric and asymmetric protein-protein complexes.

The increasing resistance to conventional antibiotics is an urgent problem that can be addressed by the discovery of new antimicrobial drugs such as antimicrobial peptides (AMPs). AMPs are components of innate immune system of eukaryotes and are not prone to the conventional mechanisms that are responsible of drug resistance. Fish are an important source of AMPs and, recently, we have isolated and characterized a new 22 amino acid residues peptide, the chionodracine (Cnd), from the Antarctic icefish Chionodraco hamatus. In this paper we focused on a new Cnd-derived mutant peptide, namely Cnd-m3a, designed to improve the selectivity against prokaryotic cells and the antimicrobial activity against human pathogens of the initial Cnd template. Cnd-m3a was used for immunization of rabbits, which gave rise to a polyclonal antibody able to detect the peptide. The interaction kinetic of Cnd-m3a with the Antarctic bacterium Psychrobacter sp. (TAD1) was imaged using a transmission electron microscopy (TEM) immunogold method. Initially the peptide was associated with the plasma membrane, but after 180 min of incubation, it was found in the cytoplasm interacting with a DNA target inside the bacterial cells. Using fluorescent probes we showed that the newly designed mutant can create pores in the outer membrane of the bacteria E. coli and Psychrobacter sp. (TAD1), confirming the results of TEM analysis. Moreover, in vitro assays demonstrated that Cnd-m3a is able to bind lipid vesicles of different compositions with a preference toward negatively charged ones, which mimics the prokaryotic cell. The Cnd-m3a peptide showed quite low hemolytic activity and weak cytotoxic effect against human primary and tumor cell lines, but high antimicrobial activity against selected Gram – human pathogens. These results highlighted the high potential of the Cnd-m3a peptide as a starting point for developing a new human therapeutic agent.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-021-02006-3

This is a review of the status of the universe as described by the standard cosmological model combined with the inflationary paradigm. Their key features and predictions, consistent with the WMAP (Wilkinson Microwave Anisotropies Probe) and Planck Probe 2013 results, provide a significant mechanism to generate the primordial gravitational waves and the density perturbations which grow over time, and later become the large-scale structure of the universe—from the quantum fluctuations in the early era to the structure observed 13.7 billion later, our epoch. In the single field slow-roll paradigm, the primordial quantum fluctuations in the inflaton field itself translate into the curvature and density perturbations which grow over time via gravitational instability. High density regions continuously attract more matter from the surrounding space, the high density regions become more and more dense in time while depleting the low density regions. At late times the highest density regions peaks collapse into the large structure of the universe, whose gravitational instability effects are observed in the clustering features of galaxies in the sky. Thus, the origin of all structure in the universe probably comes from an early era where the universe was filled with a scalar field and nothing else.

Distinctin, a 47-residue heterodimeric peptide with potent antimicrobial activity, comprises two monomeric units linked covalently by a disulfide bond between Cys19 from the 22-residue A chain and Cys23 from the 25-residue B chain. Previous synthetic strategies involved assemblies of the two individual chains, followed by their co-oxidation to form the connecting disulfide bridge, and resulted in a mixture of three species: two homodimers and one heterodimer. Here, we report synthesis of exclusively heterodimeric distinctin, using recently developed tactics for directed disulfide bridge formation. Material prepared this way was characterized and found to be suitable for more detailed structural studies. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 479-484, 2012.

SBIC 2009 Abstract We investigated the time dependence of the degradation of three alkyltin derivatives by a nine amino acid linear peptide (I1LGCWCYLR9) containing a CXC motif derived from the primary sequence of stannin, a membrane protein involved in alkyltin toxicity. We mon- itored the reaction kinetics using the intrinsic fluorescence of the tryptophan residue in position 5 of the peptide and found that all of the alkyltins analyzed are progressively degraded to dialkyl derivatives, following a pseudoenzy- matic reaction mechanism. The end point of the reactions is the formation of a covalent complex between the disub- stituted alkyltin and the peptide cysteines. These data agree with the speciation profiles proposed for polysubstituted alkyltins in the environment and reveal a possible biotic degradation pathway for these toxic compounds.

In class 1a ribonucleotide reductase (RNR), a substrate-based radical is generated in the α2 subunit by long-distance electron transfer involving an essential tyrosyl radical (Y122O·) in the β2 subunit. The conserved W48 β2 is ∼10 Å from Y122OH; mutations at W48 inactivate RNR. Here, we design a beta hairpin peptide, which contains such an interacting tyrosine–tryptophan dyad. The NMR structure of the peptide establishes that there is no direct hydrogen bond between the phenol and the indole rings. However, electronic coupling between the tyrosine and tryptophan occurs in the peptide. In addition, downshifted ultraviolet resonance Raman (UVRR) frequencies are observed for the radical state, reproducing spectral downshifts observed for β2. The frequency downshifts of the ring and CO bands are consistent with charge transfer from YO· to W or another residue. Such a charge transfer mechanism implies a role for the β2 Y-W dyad in electron transfer., Tyrosine-tryptophan dyads are known to mediate electron transfer reactions in a range of different proteins. Here, the authors study a beta hairpin peptide, probing the tyrosine-tryptophan interaction and showing no hydrogen bonding but rather charge transfer between the tyrosyl radical and tryptophan'.

In this paper, we review our past and current efforts toward the elucidation of the biological chemistry of organotin compounds. In particular, we cover two prominent aspects of organotin compounds: their reactivity toward biological dithiols, and their degradation (or metabolization) mechanism using a combination of experimental and computational techniques.

Chionodracine (Cnd) is a 22-residue peptide of the piscidin family expressed in the gills of the Chionodraco hamatus as protection from bacterial infections. Here, we report the effects of synthetic Cnd on both Psychrobacter sp. TAD1 and Escherichia coli bacteria, as well as membrane models. We found that Cnd perforates the inner and outer membranes of Psychrobacter sp. TAD1, making discrete pores that cause the cellular content to leak out. Membrane disruption studies using intrinsic and extrinsic fluorescence spectroscopy revealed that Cnd behaves similarly to other piscidins, with comparable membrane partition coefficients. Membrane accessibility assays and structural studies using NMR in detergent micelles show that Cnd adopts a canonical topology of antimicrobial helical peptides, with the hydrophobic face toward the lipid environment and the hydrophilic face toward the bulk solvent. The analysis of Cnd free energy of binding to vesicles with different lipid contents indicates a preference for charged phospholipids and a more marked binding to native E. coli extracts. Taken with previous studies on piscidin-like peptides, we conclude that Cnd first adsorbs to the membrane, and then forms pores together with membrane fragmentation. Since Cnd has only marginal hemolytic activity, it constitutes a good template for developing new antimicrobial agents.

The complete assignment of the 1H and 13C NMR spectra of bendaline (BNDL) was performed by mono-dimensional and homo- and hetero-correlated two-dimensional NMR experiments. The interaction between bendaline and albumin was also studied by the analysis of the motional parameters spin-lattice relaxation times, allowing the motional state of the BNDL free and bound with albumin to be defined. In absence of albumin the indazolacetic and benzylic moieties are characterized by roughly the same mobility and by positive sigma (cross-relaxation rates) values. In the presence of the macromolecule, the indazolacetic and benzylic moieties and the lysine change their motional behaviour to different extents, as indicated by correlation times. Data obtained in absence and in presence of the protein show that the molecular moiety of the bendaline most involved in the binding with albumin is the fragment H-4 H-5. The binding constant was evaluated at 2.4x10(3)M(-1).

The kinetics of the reaction catalyzed by human erythrocyte Acetylcholinesterase (AChE) is studied in the presence of its inhibitor Tacrine (1,2,3,4-tetrahydro-9-acridinamine), and of two newly synthesized compounds, 6-metoxy-Tacrine and N -eptyl-Tacrine. The proposed kinetic model describes the rate of the enzymatic reaction in terms of competitive and uncompetitive mixed inhibition of the three different inhibitors. The kinetic parameters describing the rate of the reaction are obtained. The competitive and uncompetitive inhibition constants of the different inhibitors are reported and the mixed competitive–uncompetitive inhibition is discussed.

Antimicrobial peptides (AMPs) provide a primordial source of immunity, conferring upon eukaryotic cells resistance against bacteria, protozoa, and viruses. Despite a few examples of anionic peptides, AMPs are usually relatively short positively charged polypeptides, consisting of a dozen to about a hundred amino acids, and exhibiting amphipathic character. Despite significant differences in their primary and secondary structures, all AMPs discovered to date share the ability to interact with cellular membranes, thereby affecting bilayer stability, disrupting membrane organization, and/or forming well-defined pores. AMPs selectively target infectious agents without being susceptible to any of the common pathways by which these acquire resistance, thereby making AMPs prime candidates to provide therapeutic alternatives to conventional drugs. However, the mechanisms of AMP actions are still a matter of intense debate. The structure-function paradigm suggests that a better understanding of how AMPs elicit their biological functions could result from atomic resolution studies of peptide-lipid interactions. In contrast, more strict thermodynamic views preclude any roles for three-dimensional structures. Indeed, the design of selective AMPs based solely on structural parameters has been challenging. In this chapter, we will focus on selected AMPs for which studies on the corresponding AMP-lipid interactions have helped reach an understanding of how AMP effects are mediated. We will emphasize the roles of both liquid- and solid-state NMR spectroscopy for elucidating the mechanisms of action of AMPs.

This work presents a new method for the simple and inexpensive determination of atrazine. The method is based on the use of a novel, partially disposable, plant tissue bioelectrode, which is sensitive to a variety of mono- and polyphenols. The biosensor is obtained by coupling a thin slice of potato ( Solanum tuberosum ) tissue, which contains high levels of the enzyme polyphenoloxidase (PPO), to a commercial O 2 -selective Clark electrode. The concentration of atrazine in aqueous samples can be determined thanks to its inhibitory power toward the catalytic activity of PPO. The low cost of this device and its good analytical performance suggest its application in the field of environmental analysis, especially in the continuous monitoring of atrazine in risk areas.

The effects of gravity on the Schwarzschild’s black-hole behavior is described on using the thermodynamic equations of state for contractile materials. Its entropy and temperature, obtained by using classical principles, reproduce the results derived from quantum field theories and statistical mechanics. The given results show that, by using the gravitational dynamics to reproduce the thermodynamic equation TdS/dx = F gravity , there is no way to establish the entropic origin of gravity, because the results can be seen the other way around.

A novel analytical system for the in-flow quantitative determination of acetylcholine (ACh) and acetylcholinesterase (AChE) inhibitors is presented. Reliable and reproducible values in the monitoring of ACh, of carbamic acid derivatives and of organophosphorous compounds have been obtained thanks to a device realized by coupling an AChE-based whole plant tissue column bioreactor with a traditional choline oxidase (ChO) biosensor. AChE was extracted from the inner part of a grapefuit shell (Albedum pomi citreum), coupled to chitin by glutaraldehyde and immobilized into a glass column bioreactor, whose outlet was connected to a thermostated flow-through cell incorporating the ChO biosensor. A sharp improvement in the sensitivity limits was obtained by recycling the sample solution into the bioreactor. The efficacy of the analytical system here presented was checked on standard solutions of ACh and on three of the most commonly used inhibitors of AChE (aldicarb, malathion and paraoxon), widely employed mainly as the active components of pesticides and insecticides.

Distinctin is a 47-residue antimicrobial peptide, which interacts with negatively charged membranes and is active against Gram-positive and Gram-negative bacteria. Its primary sequence comprises two linear chains of 22 (chain 1) and 25 (chain 2) residues, linked by a disulfide bridge between Cys19 of chain 1 and Cys23 of chain 2. Unlike other antimicrobial peptides, distinctin in the absence of the lipid membrane has a well-defined three-dimensional structure, which protects it from protease degradation. Here, we used static solid-state NMR spectroscopy in mechanically aligned lipid bilayers (charged or zwitterionic) to study the topology of distinctin in lipid bilayers. We found that this heterodimeric peptide adopts an ordered conformation absorbed on the surface of the membrane, with the long helix (chain 2), approximately parallel to the lipid bilayer (~5° from the membrane plane) and the short helix (chain 1) forming a ~24° angle with respect to the bilayer plane. Since the peptide does not disrupt the macroscopic alignment of charged or zwitterionic lipid bilayers at lipid-to-protein molar ratio of 50:1, it is possible that higher peptide concentrations might be needed for pore formation, or alternatively, distinctin elicits its cell disruption action by another mechanism.

An amperometric biosensor for the direct determination of L-glutamate was developed by chemical bonding of L-glutamate oxidase (GAO) on a carboxylic Nylon membrane with polyazetidine prepolymer (PAP), and using a hydrogen peroxide electrode as indicating sensor. The biosensor is specific for L-glutamate and the peculiar analytical properties (linearity range, reproducibility, accuracy) were experimentally determined. Furthermore, the same basic biosensor was also modified to be used and characterized for the direct determination of L-glutamine. This L-glutamine biosensor was obtained by coimmobilizing, on two separate membranes, glutamic acid oxidase and glutaminase (GMN) on the same biosensor. The two sensors were then used for the determination of glutamate and L-glutamine contained in pharmaceutical formulations and the results were compared with those obtained by other analytical methods.

The present work deals with the quantitative determination of catechol and other biologically active polyphenols (catecholamines) by means of plant tissue electrodes built up by a whole tissue containing an oxidizing enzyme (polyphenol oxidase, PPO) coupled with an O2 gas-selective electrode. Several tissues have been employed to prepare the biocatalytic layer of the biosensors. The analytical characteristics of the plant tissue electrodes here proposed, tested in catechol standard solutions, are comparable to those of traditional PPO-based enzyme electrodes. Moreover, the whole-tissue biocatalytic membranes are more stable than those prepared by using purified enzymes, thus ensuring longer life times of application. The biosensors presented here are also suitable for the quantitative determination of other catecholamines (epinephrine, norepinephrine, dopamine, l-DOPA).

LL-37 is the only cathelicidin-derived polypeptide found in humans. Its eclectic function makes this peptide one of the most intriguing chemical defense agents, with crucial roles in moderating inflammation, promoting wound healing, and boosting the human immune system. LL-37 kills both prokaryotic and eukaryotic cells through physical interaction with cell membranes. In order to study its active conformation in membranes, we have reconstituted LL-37 into dodecylphosphocholine (DPC) micelles and determined its three-dimensional structure. We found that, under our experimental conditions, this peptide adopts a helix-break-helix conformation. Both the N- and C-termini are unstructured and solvent exposed. The N-terminal helical domain is more dynamic, while the C-terminal helix is more solvent protected and structured (high density of NOEs, slow H/D exchange). When it interacts with DPC, LL-37 is adsorbed on the surface of the micelle with the hydrophilic face exposed to the water phase and the hydrophobic face buried in the micelle hydrocarbon region. The break between the helices is positioned at K12 and is probably stabilized by a hydrophobic cluster formed by I13, F17, and I20 in addition to a salt bridge between K12 and E16. These results support the proposed nonpore carpet-like mechanism of action, in agreement with the solid-state NMR studies, and pave the way for understanding the function of the mature LL-37 at the atomic level.

The structure and aggregation state of amyloid beta-peptide (Abeta) in membrane-like environments are important determinants of pathological events in Alzheimer's disease. In fact, the neurotoxic nature of amyloid-forming peptides and proteins is associated with specific conformational transitions proximal to the membrane. Under certain conditions, the Abeta peptide undergoes a conformational change that brings the peptide in solution to a "competent state" for aggregation. Conversion can be obtained at medium pH (5.0-6.0), and in vivo this appears to take place in the endocytic pathway. The combined use of (1)H NMR spectroscopy and molecular dynamics-simulated annealing calculations in aqueous hexafluoroisopropanol simulating the membrane environment, at different pH conditions, enabled us to get some insights into the aggregation process of Abeta, confirming our previous hypotheses of a relationship between conformational flexibility and aggregation propensity. The conformational space of the peptide was explored by means of an innovative use of principal component analysis as applied to residue-by-residue root-mean-square deviations values from a reference structure. This procedure allowed us to identify the aggregation-prone regions of the peptide.

Two acetylcholinesterase (AChE) inhibitors structurally related to Tacrine, 6-methoxytacrine ( 1a ) and 9-heptylamino-6-methoxytacrine ( 1b ), and their interaction with Electrophorus Electricus AChE were investigated. The complete assignment of the 1 H and 13 C NMR spectra of 1a and 1b was performed by mono-dimensional and homo- and hetero-correlated two-dimensional NMR experiments. This study was undertaken to elucidate the interaction modes between AChE and 1a and 1b in solution, using NMR. The interaction between the two inhibitors and AChE was studied by the analysis of the motional parameters non-selective and selective spin–lattice relaxation times, thereby allowing the motional state of 1a and 1b , both free and bound with AChE, to be defined. The relaxation data pointed out the ligands molecular moiety most involved in the binding with AChE. The relevant ligand/enzyme interaction constants were also evaluated for both compounds and resulted to be 859 and 5412 M −1 for 1a and 1b , respectively.

Pardaxins are a class of ichthyotoxic peptides isolated from fish mucous glands. Pardaxins physically interact with cell membranes by forming pores or voltage-gated ion channels that disrupt cellular functions. Here we report the high-resolution structure of synthetic pardaxin Pa4 in sodium dodecylphosphocholine micelles, as determined by (1)H solution NMR spectroscopy. The peptide adopts a bend-helix-bend-helix motif with an angle between the two structure helices of 122 +/- 9 degrees , making this structure substantially different from the one previously determined in organic solvents. In addition, paramagnetic solution NMR experiments on Pa4 in micelles reveal that except for the C terminus, the peptide is not solvent-exposed. These results are complemented by solid-state NMR experiments on Pa4 in lipid bilayers. In particular, (13)C-(15)N rotational echo double-resonance experiments in multilamellar vesicles support the helical conformation of the C-terminal segment, whereas (2)H NMR experiments show that the peptide induces considerable disorder in both the head-groups and the hydrophobic core of the bilayers. These solid-state NMR studies indicate that the C-terminal helix has a transmembrane orientation in DMPC bilayers, whereas in POPC bilayers, this domain is heterogeneously oriented on the lipid surface and undergoes slow motion on the NMR time scale. These new data help explain how the non-covalent interactions of Pa4 with lipid membranes induce a stable secondary structure and provide an atomic view of the membrane insertion process of Pa4.

A new class of plant tissue biosensors has been set up and applied to the determination of biogenic diamines. All the biosensors presented in this work are based on the amperometric determination of H2O2 produced by the enzymatic oxidation of diamines (putrescine and cadaverine) by the diamine oxidase (DAO) contained in the cotyledon of legumes (pea and lentil). Putrescine and cadaverine were quantitatively determined by means of these plant tissue electrodes in a range of concentrations between 0.5 and 320 μM and between 0.5 and 200 μM, respectively. The same lentil tissue-based sensor was then used, in combination with a lysine decarboxylase (LDC) membrane, for the realization of a hybrid enzyme electrode for the determination of lysine. The performances of the sensor here proposed, tested in lysine standard solutions, were comparable to those of traditional lysine bienzyme electrodes. A similar enzyme electrode was assembled for the determination of ornithine: in this case the lentil tissue diamine oxidase sensor was used in combination with an adequate amount of ornithine decarboxylase (ODC), added directly into the measuring cell. The effect of carbonic anhydrase (CA) on the speed of the LDC- and ODC-catalysed reactions was also evaluated. The synergistic interaction observed between the decarboxylation reaction, operated by either LDC or ODC, and the faciliated transport of CO2, operated by CA, is discussed both in terms of its analytical relevance and of its broader physiopathologic implications.

A new type of biosensor based on the coupling of an enzyme to an ion-selective membrane containing a conducting polymer is evaluated. The results obtained with the enzyme field- effect transistor (ENFET) and the ion-selective electrode (ISE) for the determination of creatinine and urea are compared. The presence of the conducting polymer significantly lowers the detection limit for creatinine by one decade to 10 −7 and 10 −4 M for the ENFET and ISE, respectively. The determination of urea in urine and serum with the ENFET was carried out, and the results correlated well with those obtained by spectrophotometry.

The kinetics of the reaction catalyzed by y-aminobutyric acid transaminase (GABA-T) is studied both in the absence and in the presence two inhibitors : n-dipropylacetate (n-DPA) and 4-amino-2-azide-butyric acid (GABA azide). A novel kinetic model is proposed, that is based on an experimentally derived rate equation, which describes the kinetics of the transamination process accounting for the competitive inhibition of the ketoacid and of the added inhibitor (n-DPA, GABA azide). The consequence of the ketoacid inhibition on the primary and secondary double reciprocal plots of the initial velocity against the substrates concentration is examined. The kinetic parameters which describe the rate of the transamination reaction are derived and compared with data reported in the literature. The higher inhibition power of GABA azide with respect to n-DPA is also assessed and discussed.