Structural characterization of a rhamnose-binding glycoprotein (lectin) from Spanish mackerel (Scomberomorous niphonius) eggs

A rhamnose-binding glycoprotein (lectin), named SML, was isolated from the eggs of Spanish mackerel (Scomberomorous niphonius) by affinity and ion-exchange chromatographies. SML was composed of a non-covalently linked homodimer. The SML subunit was composed of 201 amino acid residues with two tandemly repeated domains, and contained 8 half-Cys residues in each domain, which is highly homologous to the N-terminal lectin domain of calcium-independent α-latrotoxin receptor in mammalian brains. Each domain has the same disulfide bonding pattern; Cys10–Cys40, Cys20–Cys99, Cys54–Cys86 and Cys67–Cys73 were located in the N-terminal domain, and Cys108–Cys138, Cys117–Cys195, Cys152–Cys182 and Cys163–Cys169 were in the C-terminal domain. SML was N-glycosylated at Asn168 in the C-terminal domain. The structure of the sugar chain was determined to be NeuAc-Galβ1-4GlcNAcβ1-2Manα1-6-(NeuAc-Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-Asn.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Single-cell proteomic analysis of glucosinolate-rich S-cells in Arabidopsis thaliana

Single-cell analysis is essential for understanding the processes of cell differentiation and metabolic specialisation in rare cell types. The amount of single proteins in single cells can be as low as one copy per cell and is for most proteins in the attomole range or below; usually considered as insufficient for proteomic analysis. The development of modern mass spectrometers possessing increased sensitivity and mass accuracy in combination with nano-LC–MS/MS now enables the analysis of single-cell contents. In Arabidopsis thaliana, we have successfully identified nine unique proteins in a single-cell sample and 56 proteins from a pool of 15 single-cell samples from glucosinolate-rich S-cells by nanoLC–MS/MS proteomic analysis, thus establishing the proof-of-concept for true single-cell proteomic analysis. Dehydrin (ERD14_ARATH), two myrosinases (BGL37_ARATH and BGL38_ARATH), annexin (ANXD1_ARATH), vegetative storage proteins (VSP1_ARATH and VSP2_ARATH) and four proteins belonging to the S-adenosyl-l-methionine cycle (METE_ARATH, SAHH1_ARATH, METK4_ARATH and METK1/3_ARATH) with associated adenosine kinase (ADK1_ARATH), were amongst the proteins identified in these single-S-cell samples. Comparison of the functional groups of proteins identified in S-cells with epidermal/cortical cells and whole tissue provided a unique insight into the metabolism of S-cells. We conclude that S-cells are metabolically active and contain the machinery for de novo biosynthesis of methionine, a precursor for the most abundant glucosinolate glucoraphanine in these cells. Moreover, since abundant TGG2 and TGG1 peptides were consistently found in single-S-cell samples, previously shown to have high amounts of glucosinolates, we suggest that both myrosinases and glucosinolates can be localised in the same cells, but in separate subcellular compartments. The complex membrane structure of S-cells was reflected by the presence of a number of proteins involved in membrane maintenance and cellular organisation.     

Caenorhabditis elegans proteins captured by immobilized Galβ1-4Fuc disaccharide units: assignment of 3 annexins

Galβ1-4Fuc is a key structural motif in Caenorhabditis elegans glycans and is responsible for interaction with C. elegans galectins. In animals of the clade Protostomia, this unit seems to have important roles in glycan–protein interactions and corresponds to the Galβ1-4GlcNAc unit in vertebrates. Therefore, we prepared an affinity adsorbent having immobilized Galβ1-4Fuc in order to capture carbohydrate-binding proteins of C. elegans, which interact with this disaccharide unit. Adsorbed C. elegans proteins were eluted with ethylenediaminetetraacetic acid (EDTA) and followed by lactose (Galβ1-4Glc), digested with trypsin, and were then subjected to proteomic analysis using LC–MS/MS. Three annexins, namely NEX-1, -2, and -3, were assigned in the EDTA-eluted fraction. Whereas, galectins, namely LEC-1, -2, -4, -6, -9, -10, and DC2.3a, were assigned in the lactose-eluted fraction. The affinity of annexins for Galβ1-4Fuc was further confirmed by adsorption of recombinant NEX-1, -2, and -3 proteins to the Galβ1-4Fuc column in the presence of Ca²⁺. Furthermore, frontal affinity chromatography analysis using an immobilized NEX-1 column showed that NEX-1 has an affinity for Galβ1-4Fuc, but no affinity toward Galβ1-3Fuc and Galβ1-4GlcNAc. We would hypothesize that the recognition of the Galβ1-4Fuc disaccharide unit is involved in some biological processes in C. elegans and other species of the Protostomia clade.     

Optimizing the connectivity in disulfide-rich peptides: alpha-conotoxin SII as a case study

We describe a strategy for the efficient, unambiguous assignment of disulfide connectivities in alpha-conotoxin SII, of which approximately 30% of its mass is cysteine, as an example of a generalizable technique for investigation of cysteine-rich peptides. alpha-Conotoxin SII was shown to possess 3-8, 2-18, and 4-14 disulfide bond connectivity. Sequential disulfide bond connectivity analysis was performed by partial reduction with Tris(2-carboxyethyl)phosphine and real-time mass monitoring by direct-infusion electrospray mass spectrometry (ESMS). This method achieved high yields of the differentially reduced disulfide bonded intermediates and economic use of reduced peptide. Intermediates were alkylated with either N-phenylmaleimide or 4-vinylpyridine. The resulting alkyl products were assigned by ESMS and their alkyl positions sequentially identified via conventional Edman degradation. The methodology described allows a more efficient, rapid, and reliable assignment of disulfide bond connectivity in synthetic and native cysteine-rich peptides.     

Structures of the novel α-glucosyl linked diterpene glycosides from Stevia rebaudiana

From the commercial extract of the leaves of Stevia rebaudiana, two new minor diterpene glycosides having α-glucosyl linkage were isolated besides the known steviol glycosides including stevioside, steviolbioside, rebaudiosides A–F, rubusoside and dulcoside A. The structures of the two compounds were identified as 13-[(2-O-(3-α-O-d-glucopyranosyl)-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (1), and 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (2), on the basis of extensive NMR and MS spectral data as well as chemical studies.     

Assignment of all four disulfide Bridges in echistatin

Echistatin is a 49-amino-acid protein fromEchis carinatus venom. It contains four disulfide bonds. Since the disulfide bonding is critical for biological activity, it is very important to assign the disulfide linkage in this protein. Echistatin was incubated in 250 mM oxalic acid at 100°C for 4 hr under nitrogen. Under these conditions, many overlapping disulfide-containing peptides were identified by ionspray mass spectrometry. Ionspray MS/MS data indicate that the four disulfide bonds are Cys 2–Cys 11, Cys 7–Cys 32, Cys 8–Cys 37, and Cys 20–Cys 39. To our knowledge, this is the first time all four disulfide bonds in echistatin have been assigned in one experiment without disulfide bond exchange. This approach, which combines oxalic acid hydrolysis and ionspray MS/MS, may be very useful for assigning disulfide bridges in other proteins from the disintegrin family.     

Disulfide bond structure of the atrial natriuretic peptide receptor extracellular domain: conserved disulfide bonds among guanylate cyclase-coupled receptors

The disulfide bond structure of the extracellular domain of rat atrial natriuretic peptide (ANP) receptor (NPR-ECD) has been determined by mass spectrometry (MS) and Edman sequencing. Recombinant NPR-ECD expressed in COS-1 cells and purified from the culture medium binds ANP with as high affinity as the natural ANP receptor. Reaction with iodoacetic acid yielded no S-carboxymethylcysteine, indicating that all six Cys residues in NPR-ECD are involved in disulfide bonds. Electrospray ionization MS of NPR-ECD deglycosylated by peptide-N-glycosidase F gave a molecular mass of 48377.5+/-1.6 Da, which was consistent with the presence of three disulfide bonds. Liquid chromatography MS analysis of a lysylendopeptidase digest yielded three cystine-containing fragments with disulfide bonds Cys(60)-Cys(86), Cys(164)-Cys(213) and Cys(423)-Cys(432) based on their observed masses. These bonds were confirmed by Edman sequencing of each of the three fragments. No evidence for an inter-molecular disulfide bond was found. The six Cys residues in NPR-ECD, forming a 1-2, 3-4, 5-6 disulfide pairing pattern, are strictly conserved among A-type natriuretic peptide receptors and are similar in B-type receptors. We found that in other families of guanylate cyclase-coupled receptors, the Cys residues involved in 1-2 and 5-6 disulfide pairs are conserved in nearly all, suggesting an important contribution of these disulfide bonds to the receptor's structure and function.     

Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

Protein S-glutathionylation is a posttranslational modification that links oxidative stimuli to reversible changes in cellular function. Protein–glutathione mixed disulfide (PSSG) is commonly quantified by reduction of the disulfide and detection of the resultant glutathione species. This methodology is susceptible to contamination by free unreacted cellular glutathione (GSH) species, which are present in 1000-fold greater concentration. A liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based method was developed for quantification of glutathione and glutathione disulfide (GSSG), which was used for the determination of PSSG in biological samples. Analysis of rat liver samples demonstrated that GSH and GSSG coprecipitated with proteins similar to the range for PSSG in the sample. The use of [¹³C₂,⁵N]GSH and [¹³C₄,⁵N₂]GSSG validated these results and demonstrated that the release of GSH from PSSG did not occur during sample preparation and analysis. These data demonstrate that GSH and GSSG contamination must be accounted for when determining PSSG content in cellular/tissue preparations. A protocol for rinsing samples to remove the adventitious glutathione species is demonstrated. The fragmentation patterns for glutathione were determined by high-resolution mass spectrometry, and candidate ions for detection of PSSG on protein and protein fragments were identified.     

Newly Detected Specific Hydrogenation of the Conjugated Double Bond of Unsaturated Alkaloid Lactones by Aspergillus sp.

A new isolate of Aspergillus sp. hydrogenated the γ,δ-double bond of securinine (143 mg l⁻¹) to give 14,15-dihydrosecurinine at over 98% (w/w) yield after 8 h. It also hydrogenated the C11(13) double bond of 3-hydroxy-1(10),3,11(13)-guaiatriene-12,6-olide-2-one (HGT) (200 mg l⁻¹) to give 3-hydroxy-1(10),3-guaiadiene-12,6-olide-2-one with over 98% (w/w) conversion after 24 h.     

Identification of the major glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca sativa L. (salad rocket)

The major and structurally unique glucosinolate (GLS) in leaves of Eruca sativa L. (salad rocket) was identified as 4-mercaptobutyl GLS. Both 4-methylthiobutyl GLS and 4-methylsulfinylbutyl GLS were also present, but at lower concentrations. The 4-mercaptobutyl GLS was observed to oxidise under common GLS extraction conditions, generating a disulfide GLS that may be reduced efficiently by tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to reform the parent molecule. The identities of 4-mercaptobutyl GLS and of the corresponding dimeric GLS were confirmed by LC/MS, MS/MS and NMR. Myrosinase treatment of an enriched GLS fraction or of the purified dimer GLS generated a mixture of unique bi-functional disulfides, including bis-(4-isothiocyanatobutyl) disulfide (previously identified elsewhere). TCEP reduction of the purified dimer, followed by myrosinase treatment, yielded only 4-mercaptobutyl ITC. GLS-derived volatiles generated by autolysis of fresh seedlings and true leaves were 4-mercaptobutyl ITC (from the newly identified GLS), 4-methylthiobutyl ITC (from 4-methylthiobutyl GLS) and 4-methylsulfinylbutyl ITC (from 4-methylsulfinyl-butyl GLS); no unusual bi-functional disulfides were found in fresh leaf autolysate. These results led to the conclusion that, in planta, the new GLS must be present as 4-mercaptobutyl GLS and not as the disulfide found after extraction and sample concentration. This new GLS and its isothiocyanate are likely to contribute to the unique odour and flavour of E. sativa.     

Acidolysis-based component mapping of glycosaminoglycans by reversed-phase high-performance liquid chromatography with off-line electrospray ionization–tandem mass spectrometry: Evidence and tags to distinguish different glycosaminoglycans

Diverse monosaccharide analysis methods have been established for a long time, but few methods are available for a complete monosaccharide analysis of glycosaminoglycans (GAGs) and certain acidolysis-resistant components derived from GAGs. In this report, a reversed-phase high-performance liquid chromatography (RP–HPLC) method with pre-column 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization was established for a complete monosaccharide analysis of GAGs. Good separation of glucosamine/mannosamine (GlcN/ManN) and glucuronic acid/iduronic acid (GlcA/IdoA) was achieved. This method can also be applied to analyze the acidolysis-resistant disaccharides derived from GAGs, and the sequences of these disaccharides were confirmed by electrospray ionization–collision-induced dissociation–tandem mass spectrometry (ESI–CID–MS/MS). These unique disaccharides could be used as markers to distinguish heparin/heparan sulfate (HP/HS), chondroitin sulfate/dermatan sulfate (CS/DS), and hyaluronic acid (HA).     

Chemical Synthesis of Human β-Defensin (hBD)-1, -2, -3 and -4: Optimization of the Oxidative Folding Reaction

Reduced human β-defensin (hBD)-1, -2, -3 and -4 synthesized by Boc chemistry were subjected to oxidative folding reaction under optimal conditions. Among the factors affecting the oxidative folding in the presence of reduced and oxidized glutathione (GSH/GSSG), the buffer concentration and reaction temperature were essential for the predominant formation of the native disulfide structure. The homogeneity of the four synthetic hBDs was confirmed by analytical procedures using RP-HPLC, IEX-HPLC, capillary zone electrophoresis (CZE) and MALDI-TOF MS as well as sequencing, although high temperature (70 °C) was used for the RP-HPLC analysis of hBD-3 and hBD-4 to exclude the influence of equilibrium with the respective conformers having native disulfide pairing. All synthetic hBDs were shown to possess the native disulfide structure by sequential analyses and mass measurements with cystine segments obtained by enzymatic digestion. Upon digestion of hBD-1 and hBD-4 with proline specific endopeptidase, the Cys-X bond was found to be reproducibly cleaved together with the Pro-X bond although the cleavage of Cys-X afforded the appropriate cystine segments for determining the disulfide structure of hBD-1 and hBD-4. With respect to antimicrobial activity against E. coli, the four synthetic hBDs of high homogeneity possessed the same potencies as those reported previously.Australian Peptide Conference Issue     

Assignment of the Disulfide Bridges in Bothropstoxin-I, a Myonecrotic Lys49 PLA2 Homolog from Bothrops jararacussu Snake Venom

Bothropstoxin-I (BthTX-I), a Lys49 phospholipase A₂ homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV₈) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV₈ digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-I disulfide bonds follow the normal pattern of group II PLA₂s.     

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from <Emphasis Type="Italic">Asclepias fruticosa</Emphasis> latex

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZα vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.     

Identification of a disulfide bridge essential for structure and function of the voltage-gated Ca(2+) channel α(2)δ-1 auxiliary subunit

Voltage-gated calcium (Ca(V)) channels are transmembrane proteins that form Ca(2+)-selective pores gated by depolarization and are essential regulators of the intracellular Ca(2+) concentration. By providing a pathway for rapid Ca(2+) influx, Ca(V) channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. Ca(V) channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α(1) subunit, which typically contains associated β and α(2)δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α(2)δ-1 is post-translationally cleaved into an extracellular α(2) polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α(2)δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α(2)) and Cys1047 (in the extracellular domain of δ) prevented the association of the α(2) and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α(2)δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (Ca(V)2.2α(1)/β(3)) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α(2)δ-1 function.     

Capturing of the free cysteine residue in the ligand-binding site by affinity labeling of the ORL1 nociceptin receptor

All of the δ, μ, and κ opioid receptors have a free thiol group of the Cys residue in the ligand-binding site, although its functional role is not yet known. In order to examine whether or not a similar Cys is also present in the ORL1 nociceptin receptor, we attempted to identify it by affinity labeling using a specific antagonist peptide. We first treated ORL1-expressing COS-7 cell membrane preparations with the thiol-alkylation reagent N-ethylmaleimide (NEM) to perform a binding assay using [³H]nociceptin as a tracer and nociceptin, an ORL1 agonist, or Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH₂, a nociceptin/ORL1 antagonist, as a competitor. It was suggested that ORL1 has a free Cys in its ligand-binding site, since the NEM treatment reduced the population of ligand-binding sites. This was further confirmed by affinity labeling using Cys(Npys)-Arg-Tyr-Tyr-Arg-Ile-Lys-NH₂ with the SNpys group that can react with a free thiol group, resulting in the formation of a disulfide bond. This affinity labeling was approximately 23 times more specific than NEM alkylation. The results revealed that the ORL1 nociceptin receptor does contain a free Cys residue in the ligand-binding site.     

Isolation and identification of oligomeric procyanidins from Crataegus leaves and flowers

Oligomeric procyanidins were isolated from the leaves and flowers of hawthorn (Crataegus laevigata). A trimer, epicatechin-(4β→8)-epicatechin-(4β→6)-epicatechin, and a pentamer consisting of (−)-epicatechin units linked through C-4β/C-8 bonds have been isolated from hawthorn for the first time, in addition to known procyanidins including dimers B-2, B-4 and B-5, trimers C-1 and epicatechin-(4β→6)-epicatechin-(4β→8)-epicatechin, and tetramer D-1. A fraction containing a hexamer was also found.