SH Reagents Research Articles

Theoretical study of S + SH reaction on its ground state HS2(X2 A ″) potential energy surface

ABSTRACT In this study, we explore the initial state selected dynamics of the S + SH ( X 2 Π ) → S 2 ( X 3 ∑ ) + H reaction through the lens of time-dependent quantum dynamics (QD), quasi-classical trajectory (QCT), and statistical quantum mechanical (SQM) approaches, utilising the electronic ground state HS 2 (X 2 A ′′ ) potential energy surface (PES). We present total reaction probabilities and integral cross sections (ICSs) as part of our findings. Our results indicate that at low and moderate collision energies, the reaction predominantly follows an indirect pathway, forming meta-stable quasi-bound complexes. Additionally, we observe that the rotational excitation of the SH reagent initially leads to a decrease and then an increase in reactivity. We conduct a comprehensive analysis and comparison of the outcomes from QD, QCT, and SQM methodologies.

О СУБСТРАТНОЙ СПЕЦИФИЧНОСТИ И НЕКОТОРЫХ СВОЙСТВАХ ВНЕКЛЕТОЧНОЙ ОКСИДАЗЫ ИЗ БАЗИДИОМИЦЕТА NEONOTHOPANUS NAMBI

An extracellular enzyme with oxidase activity was isolated from the mycelium of the higher fungus Neonothopanus nambi by mild treatment of the biomass with β-glucosidase. A substrate specificity and some properties of the isolated extracellular oxidase were studied in the present work. Experiments revealed that the extracellular oxidase exhibited activity with most phenolic compounds chosen as model substrates. It is important to note that the enzyme exhibited a catalytic function in the reactions without the addition of exogenous hydrogen peroxide and other mediators. The highest catalytic activity of the enzyme was observed with veratryl alcohol and dihydric phenols, hydroquinone and guaiacol. The enzyme showed lower activity with aromatic azo compounds (ABTS, DAB, o-dianisidine). In reactions with dihydric phenol resorcinol and monophenol, the enzyme efficiency was extremely low. The kinetic parameters of the enzymatic reactions with actively oxidized substrates were determined. The addition of a divalent metal ion chelator (EDTA) did not affect the activity of the enzyme, while the addition of the SH reagent (DTT) increased the catalytic efficiency of the studied oxidase. The totality of the data obtained indicates that the extracellular oxidase of the N. nambi fungus catalyzes the oxidation of a wide range of aromatic compounds under slightly acidic and neutral conditions without the addition of additional mediators (in particular, hydrogen peroxide). This creates the prerequisites for studying the applicability of the enzyme in biomedical analytics.

Copper-Catalyzed Fluoroalkylphosphorothiolation of Alkynes for the Synthesis of (E)-β-Fluoroalkyl Vinyl Phosphorothioates.

A convenient copper-catalyzed three-component radical-based fluoroalkylphosphorothiolation of terminal alkynes with (iPrO)2P(O)SH and fluoroalkylation reagent for the synthesis of a variety of (E)-β-fluoroalkyl vinyl phosphorothioates with excellent regioselectivity and stereoselectivity has been developed. All the starting materials used in this reaction are highly stable and readily available. Thus, this process features with mild reaction conditions, simple operation and good functional group tolerance (>40 examples). Furthermore, this modular reaction system allows the late-stage functionalization of bioactive molecules.

ГИДРОФОБНЫЕ АРОМАТИЧЕСКИЕ ТИО(ДИТИО) СОЕДИНЕНИЯ В КАЧЕСТВЕ ХРОМОГЕННЫХ АНАЛИТИЧЕСКИХ РЕАГЕНТОВ ДЛЯ ФОТОМЕТРИЧЕСКОГО ОПРЕДЕЛЕНИЯ ТИОЛОВ ПО МЕРКАПТО-ГРУППЕ. II. ЕДИНАЯ МОЛЕКУЛЯРНАЯ МОДЕЛЬ АНАЛИТИЧЕСКОГО РЕАГЕНТА

The concept of a unified molecular model is proposed for the directed synthesis of chromogenic analytical reagents for the mercapto group in ("thiol-thiol") substitution reactions (SH reagents). The unified molecular model of the SH reagent includes an aromatic chromophore-containing thio fragment covalently bound by an ordinary bond on a sulfide sulfur atom to an electrophilic, relatively soft one [in terms of the principle of hard and soft acids and bases (the HSAB principle)] reaction center. The characteristics of the analytical effect of the SH reagent are determined by the structure of the thio fragment. The required change in the reactivity and selectivity of the SH reagent are achieved mainly by varying the softness (stiffness) of the reaction center. Spectral characteristics of analytical effects, reactivity and selectivity of SH reagents (obtained earlier and synthesized for the first time in this work) – hydrophobic aromatic thio (dithio) compounds: thiocyanates, symmetric disulfides, and thiomercuric compounds – were studied in an aprotic dipolar DMF medium. In support of the concept of the unified molecular model for compounds containing identical thiofragments it was established: - spectral characteristics of the analytical effect of all three types of compounds (thiocyanates, disulfides and thiomercuric compounds) are the same in the DMF medium. - in accordance with the principle of HSAB, the reactivity increases in the sequence thiocyanate < disulfide <<thiomercuric compound, which corresponds to an increase in the softness of the reaction center in the series: -С << - S-<< -Hg-. The universality of the concept of the unified molecular model follows from consideration of the spectral and kinetic characteristics not only of Ellman reagent, but as well of thio(dithio)azo compounds, thiocyanate and thiomercuric compound containing the same 3-carboxy, 4-nitrophenylthio fragment.

Structure/function relationships of the human mitochondrial ornithine/citrulline carrier by Cys site-directed mutagenesis. Relevance to mercury toxicity

The effect of SH reagents on the human mitochondrial ornithine/citrulline carrier (hORC) was studied. Site-directed Cys mutants were employed to gain information on structure/function relationships. The substitutions of each Cys by Ala did not alter the hORC activity measured as [3H]ornithine/ornithine antiport in proteoliposomes. N‑ethylmaleimide inhibited the transport of WT with IC50 of 149 μM. C51A, C50A and C132A showed a much higher IC50. MTSEA and MTSET also inhibited the WT with IC50 of 0.40 μM and 1.60 μM, respectively. C51A and C132A showed much higher IC50 values for both reagents. The triple mutant C50/51/132A showed an IC50 for the three reagents that was higher than that of the single mutants. The data strongly suggests that C132, C50 and C51 are involved in inhibition of hORC. Inhibition of WT and mutants by CuPhenanthroline, an S-S forming reagent, suggested that C132 may form disulfides with C50 or C51, impairing the transporter function. The structure/function relationships information deriving from the inhibition studies, were corroborated by the homology structural model of the transporter. The effect of HgCl2 and methyl mercury was also tested on hORC in the light of their capacity to bind thiol residues. Both reagents potently inhibit the transporter.

Epoxycyclohexenedione-Type Compounds Make Up a New Class of Inhibitors of the Bovine Mitochondrial ADP/ATP Carrier.

Through the extensive screening of our chemical library, we found epoxycyclohexenedione (ECHD)-type compounds (AMM-59 and -120) as unique inhibitors of the bovine heart mitochondrial ADP/ATP carrier (AAC). This study investigated the mechanism of inhibition of AAC by ECHDs using submitochondrial particles (SMPs). Proteomic analyses of ECHD-bound AAC as well as biochemical characterization using different SH reagents showed that ECHDs inhibit the function of AAC by covalently binding primarily to Cys57 and secondarily to Cys160. Interestingly, AAC remarkably aggregated in SMPs upon being incubated with high concentrations of ECHDs for a long period of time. This aggregation was observed under both oxidative and reductive conditions of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of SMP proteins, indicating that aggregation is not caused by intermolecular S-S linkages. ECHDs are the first chemicals, to the best of our knowledge, to induce prominent structural alteration in AAC without forming intermolecular S-S linkages. When all solvent-accessible cysteines (Cys57, Cys160, and Cys257) were previously modified by N-ethylmaleimide, the aggregation of AAC was completely suppressed. In contrast, when Cys57 or Cys160 is selectively modified by a SH reagent, the covalent binding of ECHDs to a residual free residue of the two cysteines is sufficient to induce aggregation. The aggregation-inducing ability of another ECHD analogue (AMM-124), which has an alkyl chain that is shorter than those of AMM-59 and -120, was significantly less efficient than that of the two compounds. On the basis of these results, the mechanism underlying the aggregation of AAC induced by ECHDs is discussed.

Novel insights into the transport mechanism of the human amino acid transporter LAT1 (SLC7A5). Probing critical residues for substrate translocation

BackgroundLAT1 (SLC7A5) is the transport competent unit of the heterodimer formed with the glycoprotein CD98 (SLC3A2). It catalyzes antiport of His and some neutral amino acids such as Ile, Leu, Val, Cys, Met, Gln and Phe thus being involved in amino acid metabolism. Interestingly, LAT1 is over-expressed in many human cancers that are characterized by increased demand of amino acids. Therefore LAT1 was recently acknowledged as a novel target for cancer therapy. However, knowledge on molecular mechanism of LAT1 transport is still scarce. MethodsCombined approaches of bioinformatics, site-directed mutagenesis, chemical modification, and transport assay in proteoliposomes, have been adopted to unravel dark sides of human LAT1 structure/function relationships. ResultsIt has been demonstrated that residues F252, S342, C335 are crucial for substrate recognition and C407 plays a minor role. C335 and C407 cannot be targeted by SH reagents. The transporter has a preferential dimeric structure and catalyzes an antiport reaction which follows a simultaneous random mechanism. ConclusionsCritical residues of the substrate binding site of LAT1 have been probed. This site is not freely accessible by molecules other than substrate. Similarly to LeuT, K+ has some regulatory properties on LAT1. General significanceThe collected data represent a solid basis for deciphering molecular mechanism underlying LAT1 function.

Stimulation by pro-apoptotic valinomycin of cytosolic NADH/cytochrome c electron transport pathway—Effect of SH reagents

Intrinsic and extrinsic apoptosis are both characterised by the presence of cytochrome c (cyto-c) in the cytosol. We present data on the extra-mitochondrial NADH oxidation catalysed by exogenous (cytosolic) cyto-c, as a possible answer to the paradox of apoptosis being an energy-dependent program but characterized by the impairment of the respiratory chain. The reduction of molecular oxygen induced by the cytosolic NADH/cyto-c pathway is coupled to the generation of an electrochemical proton gradient available for ATP synthesis. Original findings show that SH reagents inhibit the NADH/cyto-c system with a conformational change mechanism. The mitochondrial integrity-test of sulfite oxidase unequivocally demonstrates that this enzyme (120kDa) can be released outside but exogenous cyto-c (12.5kDa) does not permeate into mitochondria. Valinomycin at 2nM stimulates both the energy-dependent reversible mitochondrial swelling and the NADH/cyto-c oxidation pathway. The pro-apoptotic activity of valinomycin, as well as to the dissipation of membrane potential, can be also ascribed to the increased activity of the NADH/cyto-c oxidation pathway useful as an additional source of energy for apoptosis. It can be speculated that the activation of the NADH/cyto-c system coupled to valinomycin-induced mitochondrial osmotic swelling may represent a strategy to activate apoptosis in confined solid tumours.

Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris — functional and kinetic asymmetry revealed in proteoliposomes

The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni2+-chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [3H]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na+. Internal Na+ slightly stimulated the antiport. Optimal activity was found at pH7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (≥70%) and by mercurials and methanethiosulfonates (≥80%). Heterologous antiport of [3H]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [3H]glutamine/glutamine antiport Km values of 0.097 and 1.8mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na+ on the external side was 32mM. The homology structural model of the hASCT2 protein was built using the GltPh of Pyrococcus horikoshii as template. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism.

Novel chiral tool, (R)-2-octanol dehydrogenase, from Pichia finlandica: purification, gene cloning, and application for optically active α-haloalcohols

A novel enantioselective alcohol dehydrogenase, (R)-2-octanol dehydrogenase (PfODH), was discovered among methylotrophic microorganisms. The enzyme was purified from Pichia finlandica and characterized. The molecular mass of the enzyme was estimated to be 83,000 and 30,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzyme was an NAD(+)-dependent secondary alcohol dehydrogenase and showed a strict enantioselectivity, very broad substrate specificity, and high tolerance to SH reagents. A gene-encoding PfODH was cloned and sequenced. The gene consisted of 765 nucleotides, coding polypeptides of 254 amino acids. The gene was singly expressed and coexpressed together with a formate dehydrogenase as an NADH regenerator in an Escherichia coli. Ethyl (S)-4-chloro-3-hydroxybutanoate and (S)-2-chloro-1-phenylethanol were synthesized using a whole-cell biocatalyst in more than 99% optical purity.

The Unwound Portion Dividing Helix IV of NhaA Undergoes a Conformational Change at Physiological pH and Lines the Cation Passage

pH and Na(+) homeostasis in all cells requires Na(+)/H(+) antiporters. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and their pH regulation. Functional studies of NhaA in the membrane have yielded valuable information regarding its functionality in situ at physiological pH. Here, we Cys-scanned the discontinuous transmembrane segment (TM) IV (helices IVp and IVc connected by an extended chain) of NhaA to explore its functionality at physiological pH. We then tested the accessibility of the Cys replacements to the positively charged SH reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) and the negatively charged 2-sulfonatoethyl methanethiosulfonate (MTSES) in intact cells at pH 8.5 and 6.5 and in parallel tested their accessibility to MTSET in high-pressure membranes at both pH values. We found that the outer membrane of E. coli TA16 acts as a partially permeable barrier to MTSET. Overcoming this technical problem, we revealed that (a) Cys replacement of the most conserved residues of TM IV strongly increases the apparent K(m) of NhaA to both Na(+) and Li(+), (b) the cationic passage of NhaA at physiological pH is lined by the most conserved and functionally important residues of TM IV, and (c) a pH shift from 6.5 to 8.5 induces conformational changes in helix IVp and in the extended chain at physiological pH.

Endogenous Molecules Stimulating N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA)

Fatty acid amide hydrolase (FAAH) plays the central role in the degradation of bioactive N-acylethanolamines such as the endocannabinoid arachidonoylethanolamide (anandamide) in brain and peripheral tissues. A lysosomal enzyme referred to as N-acylethanolamine-hydrolyzing acid amidase (NAAA) catalyzes the same reaction with preference to palmitoylethanolamide, an endogenous analgesic and neuroprotective substance, and is therefore expected as a potential target of therapeutic drugs. In the in vitro assays thus far performed, the maximal activity of NAAA was achieved in the presence of both nonionic detergent (Triton X-100 or Nonidet P-40) and the SH reagent dithiothreitol. However, endogenous molecules that might substitute for these synthetic compounds remain poorly understood. Here, we examined stimulatory effects of endogenous phospholipids and thiol compounds on recombinant NAAA. Among different phospholipids tested, choline- or ethanolamine-containing phospholipids showed potent effects, and 1 mM phosphatidylcholine increased NAAA activity by 6.6-fold. Concerning endogenous thiol compounds, dihydrolipoic acid at 0.1-1 mM was the most active, causing 8.5-9.0-fold stimulation. These results suggest that endogenous phospholipids and dihydrolipoic acid may contribute in keeping NAAA active in lysosomes. Even in the presence of phosphatidylcholine and dihydrolipoic acid, however, the preferential hydrolysis of palmitoylethanolamide was unaltered. We also investigated a possible compensatory induction of NAAA mRNA in brain and other tissues of FAAH-deficient mice. However, NAAA expression levels in all the tissues examined were not significantly altered from those in wild-type mice.

Kinetics of Inactivation of NADP+-Linked Isocitrate Dehydrogenase from Germinating Mung Bean (Vigna radiata)

Metal chelating agent EDTA inhibits the activity of mung-bean NADP+-linked isocitrate dehydrogenase (ICDH) in a competitive manner. The activity of the Apo-enzyme was restored by divalent metal ions with the order of effectiveness found to be Mn 2+> Mg2+ > Zn2+ > Co2+ > Cu2+. here appeared to be a single type of metal binding site that was saturated either with 0.5 mM of Mn2+ or with 2.5 mM of Mg2+. ADP, ATP and NADPH inhibit the enzyme in competitive manner. On titration with 5, 5’-dithiobis (2-nitrobenzoate), i.e. DTNB, the mung bean isocitrate dehydrogenase showed 4.0 reactive -SH groups per molecule. The denatured ICDH enzyme of mung bean possess 8.1-SH groups per molecule. The blocking of this group with -SH reagents, lead to the inactivation of mung bean ICDH enzyme. Time-dependent inactivation of ICDH with iodoacetamide and Nethylmaleimide (NEM) revealed decay in the activity in a single exponential manner.

D-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637

Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. D-Amino acid dehydrogenase is a flavoenzyme that digests free neutral D-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 D-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial D-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to D-proline. The enzyme mediated electron transport from D-proline to coenzyme Q(1), thus distinguishing it from D-amino acid oxidase. The apparent K(m) and V(max) values were 40.2 mM and 25.0 micromol min(-1) mg(-1), respectively, for dehydrogenation of D-proline, and were 8.2 microM and 12.3 micromol min(-1) mg(-1), respectively, for reduction of Q(1). The respective pH and temperature optima were 8.0 and 37 degrees C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian D-amino acid oxidase than other bacterial D-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from D-proline to a c-type cytochrome was suggested spectrophotometrically.

Thiol-disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum).

Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5'-phosphate-dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S--S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored --SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two --SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free --SH groups. This allowed the oriented conjugation of alliinase, via the --SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.

Structure-based Functional Study Reveals Multiple Roles of Transmembrane Segment IX and Loop VIII–IX in NhaA Na+/H+ Antiporter of Escherichia coli at Physiological pH

The three-dimensional crystal structure of Escherichia coli NhaA determined at pH 4 provided the first structural insights into the mechanism of antiport and pH regulation of a Na(+)/H(+) antiporter. However, because NhaA is activated at physiological pH (pH 6.5-8.5), many questions pertaining to the active state of NhaA have remained open including the structural and physiological roles of helix IX and its loop VIII-IX. Here we studied this NhaA segment (Glu(241)-Phe(267)) by structure-based biochemical approaches at physiological pH. Cysteine-scanning mutagenesis identified new mutations affecting the pH dependence of NhaA, suggesting their contribution to the "pH sensor." Furthermore mutation F267C reduced the H(+)/Na(+) stoichiometry of the antiporter, and F267C/F344C inactivated the antiporter activity. Tests of accessibility to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide, a membrane-impermeant positively charged SH reagent with a width similar to the diameter of hydrated Na(+), suggested that at physiological pH the cytoplasmic cation funnel is more accessible than at acidic pH. Assaying intermolecular cross-linking in situ between single Cys replacement mutants uncovered the NhaA dimer interface at the cytoplasmic side of the membrane; between Leu(255) and the cytoplasm, many Cys replacements cross-link with various cross-linkers spanning different distances (10-18 A) implying a flexible interface. L255C formed intermolecular S-S bonds, cross-linked only with a 5-A cross-linker, and when chemically modified caused an alkaline shift of 1 pH unit in the pH dependence of NhaA and a 6-fold increase in the apparent K(m) for Na(+) of the exchange activity suggesting a rigid point in the dimer interface critical for NhaA activity and pH regulation.

The high affinity choline transporter: topology and choline binding site as studied by site‐directed mutagenesis

The high affinity choline transporter (CHT1) is expressed in cholinergic neuron. CHT1 takes up choline and provides it for acetylcholine synthesis. CHT1 has two putative glycosylation sites (N69, N301). We found that N69Q mutant was glycosylated as well as wild type CHT1, but N301Q mutant was not glycosylated and not expressed on the cell surface. We have constructed a number of mutants substituted with cysteine and examined the accessibility of impermeable SH reagent to each cysteine residue in these mutants expressed in HEK293 cells. Obtained results indicated that CHT1 has the extracellular amino terminus and 13 transmembrane segments. Several acidic or basic amino acid residues in the putative third and 12th transmembrane segments were indicated to be involved in the choline uptake activity. In particular, the E451D mutant had approximately five times higher Km for choline uptake and five times lower Kd for HC3 binding as compared with wild type hCHT1, indicating that the E451 residue is involved in recognition of choline and HC3.

4-N-Trimethylaminobutyraldehyde Dehydrogenase: Purification and Characterization of an Enzyme fromPseudomonassp. 13CM

4-N-trimethylaminobutyraldehyde dehydrogenase from Pseudomonas sp. 13CM was purified 14-fold to apparent homogeneity by hydrophobic chromatography on a Phenyl-Toyopearl, and affinity chromatography was done on a 5'-AMP Sepharose4B in the presence of dithiothreitol. The enzyme was found to be a trimer with identical 55 kDa subunits. The isoeletric point was found to be 5.5. The optimum temperature and pH were 40 degrees C and pH 10.0. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters, and analog inhibition. The K(m) values for 4-N-trimethylaminobutyraldehyde, 4-dimethylaminobutyraldehyde, and NAD(+) were 7.4, 51, and 125 microM respectively. The enzyme was inhibited by SH reagents, and by heavy metal ions.

Chiral high-performance liquid chromatography analysis of α-amino acid mixtures using a novel SH reagent— N- R-mandelyl- l-cysteine and traditional enantiomeric thiols for precolumn derivatization

Several chiral thiols, i.e. traditionally used enantiomerically pure SH reagents and novel N- R-mandelyl- l-cysteine ( R-NMC) containing additional chiral center, have been applied as co-reagents in precolumn derivatization with o-phthalaldehyde for enantiomeric HPLC analysis of individual α-amino acids and their mixtures. The R-NMC-derived isoindoles as well as adducts with other thiols have a characteristic absorption maximum at 340 nm, and are highly fluorescent allowing detection of 10 μg/l of an amino acid. Investigated 19 amino acids were analyzed separately and in a mixture by a gradient HPLC after precolumn derivatization. The chromatographic behavior of formed isoindoles substantially differs for each of the thiols used for modification. In contrast to traditional enantiomeric thiols application of diastereomeric R-NMC provides higher resolution for α-amino acid enantiomers, with l, d-elution order (except for Arg). Combined use of R-NMC and other thiol enlarges the possibilities of this method, allowing accurate chiral analysis of complex amino acid mixtures.

Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents

During substrate translocation mitochondrial carriers cycle between the cytoplasmic-state (c-state) with substrate-binding site open to the intermembrane space and matrix-state (m-state) with the binding site open to the mitochondrial matrix. Here, the accessibility of Cys-58, Cys-136 and Cys-155 of the rat mitochondrial carnitine/acylcarnitine carrier (CAC) to membrane-impermeable SH reagents was examined as a function of the conformational state. Reconstituted mutant CACs containing the combinations Cys-58/Cys-136, Cys-58/Cys-155, and Cys-136/Cys-155 transport carnitine with a ping-pong mechanism like the wild-type, since increasing substrate concentrations on one side of the membrane decreased the apparent affinity for the substrate on the other side. In view of this mechanism, the effect of SH reagents on the transport activity of mutant CACs was tested by varying the substrate concentration inside or outside the proteoliposomes, keeping the substrate concentration on the opposite side constant. The reagents MTSES, MTSEA and fluorescein-5-maleimide did not affect the carnitine/carnitine exchange activity of the mutant carrier with only Cys-58 in contrast to mutant carriers with Cys-58/Cys-136, Cys-58/Cys-155 or Cys-136/Cys-155. In the latter, the inhibitory effect of the reagents was more pronounced when the intraliposomal carnitine concentration was increased, favouring the m-state of the carrier, whereas the effect was less when the concentration of carnitine was increased in the external compartment of the proteoliposomes, favouring the c-state. Moreover, the mutant carrier proteins with Cys-136/Cys-155, Cys-58/Cys-136 or Cys-58/Cys-155 were more fluorescent when extracted from fluorescein-5-maleimide-treated proteoliposomes containing 15 mM internal carnitine as compared to 2.5 mM. These results are discussed in terms of conformational changes of the carrier occurring during substrate translocation.