In situ alkylation with acrylamide for identification of cysteinyl residues in proteins during one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Cysteinyl residues in proteins were alkylated with acrylamide during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to yield a thioether derivative, cys-S-beta-propionamide (PAM cys). The process was termed in situ alkylation with acrylamide. Using this method, the recovery of PAM-cys peptides from bovine serum albumin (BSA) was 88.6% at 10 picomol in one-dimensional (1-D) SDS-PAGE and 97.1% at 50 picomol in two-dimensional (2-D) SDS-PAGE. The coverage of tryptic peptide of BSA in 1-D and 2-D SDS-PAGE was 83.7% and 81.1%, respectively. The advantages of in situ alkylation with acrylamide were the following: (i) cysteinyl peptides were effectively derived in a single PAM cys and then proteins were precisely identified using databases; (ii) marked reduction of salts compared with post alkylation, e.g., using carboxymethylamide (CAM), resulting in higher signal intensity and wider coverage of cysteinyl peptides from PAM cys, compared with those of CAM derivatives, in mass spectrometry peptide mapping; and (iii) shorter duration by excluding the processes of post alkylation and desalting before peptide mapping.     

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH₄) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH₄ and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure–function relationships in the CuMMOs are inadequately understood. Here, we describe genetic and physiological characterization of a novel member of the CuMMO family that has an unusual physiological substrate range (C₂–C₄ alkanes) and a distinctive bacterial host (Mycobacterium). The Mycobacterial CuMMO genes (designated hmoCAB) were amenable to heterologous expression in M. smegmatis—this is the first example of recombinant expression of a complete and highly active CuMMO enzyme. The apparent specific activity of recombinant cells containing hmoCAB ranged from 2 to 3 nmol min^–1 per mg protein on ethane, propane and butane as substrates, and the recombinants could also attack ethene, cis-dichloroethene and 1,2-dichloroethane. No detectable activity of recombinants or wild-type strains was seen with methane. The specific inhibitor allylthiourea strongly inhibited growth of wild-type cells on C₂–C₄ alkanes, and omission of copper from the medium had a similar effect, confirming the physiological role of the CuMMO for growth on alkanes. The hydrocarbon monooxygenase provides a new model for studying this important enzyme family, and the recombinant expression system will enable biochemical and molecular biological experiments (for example, site-directed mutagenesis) that were previously not possible.     

An in-gel digestion procedure that facilitates the identification of highly hydrophobic proteins by electrospray ionization-mass spectrometry analysis

A procedure is described for in-gel tryptic digestion of proteins that allows the direct analysis of eluted peptides in electrospray ionization (ESI) mass spectrometers without the need of a postdigestion desalting step. It is based on the following principles: (a) a thorough desalting of the protein in-gel before digestion that takes advantage of the excellent properties of acrylamide polymers for size exclusion separations, (b) exploiting the activity of trypsin in water, in the absence of inorganic buffers, and (c) a procedure for peptide extraction using solvents of proven efficacy with highly hydrophobic peptides. Quality of spectra and sequence coverage are equivalent to those obtained after digestion in ammonium bicarbonate for hydrophilic proteins detected with Coomassie blue, mass spectrometry-compatible silver or imidazole-zinc but are significantly superior for highly hydrophobic proteins, such as membrane proteins with several transmembrane domains. ATPase subunit 9 (GRAVY 1.446) is a membrane protein channel, lipid-binding protein for which both the conventional in-gel digestion protocol and in solution digestion failed. It was identified with very high sequence coverage. Sample handling after digestion is notably simplified as peptides are directly loaded into the ESI source without postdigestion processing, increasing the chances for the identification of hydrophobic peptides.     

A novel insight into the cost–benefit model for the evolution of botanical carnivory

Background The cost–benefit model for the evolution of botanical carnivory provides a conceptual framework for interpreting a wide range of comparative and experimental studies on carnivorous plants. This model assumes that the modified leaves called traps represent a significant cost for the plant, and this cost is outweighed by the benefits from increased nutrient uptake from prey, in terms of enhancing the rate of photosynthesis per unit leaf mass or area (A_N) in the microsites inhabited by carnivorous plants.Scope This review summarizes results from the classical interpretation of the cost–benefit model for evolution of botanical carnivory and highlights the costs and benefits of active trapping mechanisms, including water pumping, electrical signalling and accumulation of jasmonates. Novel alternative sequestration strategies (utilization of leaf litter and faeces) in carnivorous plants are also discussed in the context of the cost–benefit model.Conclusions Traps of carnivorous plants have lower A_N than leaves, and the leaves have higher A_N after feeding. Prey digestion, water pumping and electrical signalling represent a significant carbon cost (as an increased rate of respiration, R_D) for carnivorous plants. On the other hand, jasmonate accumulation during the digestive period and reprogramming of gene expression from growth and photosynthesis to prey digestion optimizes enzyme production in comparison with constitutive secretion. This inducibility may have evolved as a cost-saving strategy beneficial for carnivorous plants. The similarities between plant defence mechanisms and botanical carnivory are highlighted.     

Protocol for Increasing the Sensitivity of MS-Based Protein Detection in Human Chorionic Villi

An important step in the proteomic analysis of missing proteins is the use of a wide range of tissues, optimal extraction, and the processing of protein material in order to ensure the highest sensitivity in downstream protein detection. This work describes a purification protocol for identifying low-abundance proteins in human chorionic villi using the proposed “1DE-gel concentration” method. This involves the removal of SDS in a short electrophoresis run in a stacking gel without protein separation. Following the in-gel digestion of the obtained holistic single protein band, we used the peptide mixture for further LC–MS/MS analysis. Statistically significant results were derived from six datasets, containing three treatments, each from two tissue sources (elective or missed abortions). The 1DE-gel concentration increased the coverage of the chorionic villus proteome. Our approach allowed the identification of 15 low-abundance proteins, of which some had not been previously detected via the mass spectrometry of trophoblasts. In the post hoc data analysis, we found a dubious or uncertain protein (PSG7) encoded on human chromosome 19 according to neXtProt. A proteomic sample preparation workflow with the 1DE-gel concentration can be used as a prospective tool for uncovering the low-abundance part of the human proteome.     

Diversity of active aerobic methanotrophs along depth profiles of arctic and subarctic lake water column and sediments

Methane (CH₄) emitted from high-latitude lakes accounts for 2–6% of the global atmospheric CH₄ budget. Methanotrophs in lake sediments and water columns mitigate the amount of CH₄ that enters the atmosphere, yet their identity and activity in arctic and subarctic lakes are poorly understood. We used stable isotope probing (SIP), quantitative PCR (Q-PCR), pyrosequencing and enrichment cultures to determine the identity and diversity of active aerobic methanotrophs in the water columns and sediments (0–25 cm) from an arctic tundra lake (Lake Qalluuraq) on the north slope of Alaska and a subarctic taiga lake (Lake Killarney) in Alaska''s interior. The water column CH₄ oxidation potential for these shallow (∼2 m deep) lakes was greatest in hypoxic bottom water from the subarctic lake. The type II methanotroph, Methylocystis, was prevalent in enrichment cultures of planktonic methanotrophs from the water columns. In the sediments, type I methanotrophs (Methylobacter, Methylosoma and Methylomonas) at the sediment-water interface (0–1 cm) were most active in assimilating CH₄, whereas the type I methanotroph Methylobacter and/or type II methanotroph Methylocystis contributed substantially to carbon acquisition in the deeper (15–20 cm) sediments. In addition to methanotrophs, an unexpectedly high abundance of methylotrophs also actively utilized CH₄-derived carbon. This study provides new insight into the identity and activity of methanotrophs in the sediments and water from high-latitude lakes.     

Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.     

Matrix layer sample preparation: an improved MALDI-MS peptide analysis method for proteomic studies

The analytical performance of MALDI-MS is highly influenced by sample preparation and the choice of matrix. Here we present an improved MALDI-MS sample preparation method for peptide mass mapping and peptide analysis, based on the use of the 2,5-dihydroxybenzoic acid matrix and prestructured sample supports, termed: matrix layer (ML). This sample preparation is easy to use and results in a rapid automated MALDI-MS and MS/MS with high quality spectra acquisition. The between-spot variation was investigated using standard peptides and statistical treatment of data confirmed the improvement gained with the ML method. Furthermore, the sample preparation method proved to be highly sensitive, in the lower-attomole range for peptides, and we improved the performance of MALDI-MS/MS for characterization of phosphopeptides as well. The method is versatile for the routine analysis of in-gel tryptic digests thereby allowing for an improved protein sequence coverage. Furthermore, reliable protein identification can be achieved without the need of desalting sample preparation. We demonstrate the performance and the robustness of our method using commercially available reference proteins and automated MS and MS/MS analyses of in-gel digests from lung tissue lysate proteins separated by 2-DE.     

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2_hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97–125 + Tyr), interacted specifically with isolated K2_hPg. However, the binding strength of VEK30 (K_D = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (K_D = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH₂ and COOH termini of VEK30. VEK64 (PAM residues 83–145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2_hPg with nearly the same affinity as PAM (K_D = 1–2 nm). However, addition of two PAM residues (Arg¹²⁶-His¹²⁷) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2_hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg¹¹³, His¹¹⁴, Glu¹¹⁶, Arg¹²⁶, His¹²⁷), mutation of which reduced PAM binding affinity for K2_hPg by ∼1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence.     

Standardisation of rapid in-gel digestion by mass spectrometry

In-gel digestion has been standardised using a poly(propylene) disposable. We designed a four-step rapid and simple in-gel digestion protocol which is carried out in one self-contained reaction tube avoiding keratin contamination. In order to quantify the efficiency of in-gel digestion, we developed a rapid on-column peptide acetylation protocol. Results show that trypsin in-gel uptake is increased and in-gel digestion is 90% complete within 15 min. We further show that spectrum quality, peptide yield and sequence coverage for mass spectrometric analysis are enhanced. We utilise 2-D PAGE separation of photosystem II from barley to demonstrate that the protocol facilitates identification of highly hydrophobic membrane proteins.     

Spatial patterns of photosynthesis in thin- and thick-leaved epiphytic orchids: unravelling C3–CAM plasticity in an organ-compartmented way

Background and Aims

A positive correlation between tissue thickness and crassulacean acid metabolism (CAM) expression has been frequently suggested. Therefore, this study addressed the question of whether water availability modulates photosynthetic plasticity in different organs of two epiphytic orchids with distinct leaf thickness.

Methods

Tissue morphology and photosynthetic mode (C₃ and/or CAM) were examined in leaves, pseudobulbs and roots of a thick-leaved (Cattleya walkeriana) and a thin-leaved (Oncidium ‘Aloha’) epiphytic orchid. Morphological features were studied comparing the drought-induced physiological responses observed in each organ after 30 d of either drought or well-watered treatments.

Key Results

Cattleya walkeriana, which is considered a constitutive CAM orchid, displayed a clear drought-induced up-regulation of CAM in its thick leaves but not in its non-leaf organs (pseudobulbs and roots). The set of morphological traits of Cattleya leaves suggested the drought-inducible CAM up-regulation as a possible mechanism of increasing water-use efficiency and carbon economy. Conversely, although belonging to an orchid genus classically considered as performing C₃ photosynthesis, Oncidium ‘Aloha’ under drought seemed to express facultative CAM in its roots and pseudobulbs but not in its leaves, indicating that such photosynthetic responses might compensate for the lack of capacity to perform CAM in its thin leaves. Morphological features of Oncidium leaves also indicated lower efficiency in preventing water and CO₂ losses, while aerenchyma ducts connecting pseudobulbs and leaves suggested a compartmentalized mechanism of nighttime carboxylation via phosphoenolpyruvate carboxylase (PEPC) (pseudobulbs) and daytime carboxylation via Rubisco (leaves) in drought-exposed Oncidium plants.

Conclusions

Water availability modulated CAM expression in an organ-compartmented manner in both orchids studied. As distinct regions of the same orchid could perform different photosynthetic pathways and variable degrees of CAM expression depending on the water availability, more attention should be addressed to this in future studies concerning the abundance of CAM plants.     

Synthesis of two configurational isomers of a 14-membered tetraaza macrocycle bearing N-CH2CH2CONH2 pendent arms and their copper(II) complexes: Crystal structures of the complexes

Two isomers of 1,8-bis(N-carbamoylethyl)-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane (L²) bearing two N-CH₂CH₂CONH₂ groups, C-meso-L² and C-racemic-L², have been prepared and characterized. Each isomer reacts with Cu(II) ion to form a five-coordinate complex, [Cu(C-meso-L²)](ClO₄)₂ (1) or [Cu(C-racemic-L²)](ClO₄)₂ (2), in which only one pendent amide group is coordinated to the metal ion. The crystal structure of 1 · CH₃CN shows that the complex possesses trans-III-type N-configuration and has a slightly distorted square-pyramidal coordination geometry with a relatively long axial Cu-O (N-CH₂CH₂CONH₂) bond (2.207(3) Å). On the other hand, 2 exhibits trans-V configuration and has a slightly distorted trigonal bipyramidal coordination geometry with a very short equatorial Cu-O (N-CH₂CH₂CONH₂) bond (2.007(3) Å); the Cu-O distance is distinctly shorter than the Cu-N distances (2.062(4)-2.090(4) Å). The complex 1 exhibits a d-d transition band at approximately 565 nm, whereas the band for 2 is observed at approximately 770 nm.     

Identification of Proteins by Matrix-Assisted Laser Desorption Ionization–Mass Spectrometry Following In-Gel Digestion in Low-Salt, Nonvolatile Buffer and Simplified Peptide Recovery

Matrix-assisted laser desorption ionization–mass spectrometry is an efficient analytical method for large-scale identification of proteins separated by two-dimensional polyacrylamide gel electrophoresis. Following in-gel digestion, the salt present in the peptide extracts is usually removed by chromatography prior to analysis. Desalting is a labor-intensive and time-consuming step, limiting the total number of samples that can be processed daily. We improved the daily sample output by performing the in-gel protein digestion in low-salt, nonvolatile buffer and simplifying the recovery of the generated peptides, collecting them in a small volume by sonication. This technique is routinely used for identification of proteins ofHaemophilus influenzaeand human brain. The methodology described facilitates the analytical process and allows the analysis of hundreds of proteins per day. Furthermore, it represents an essential step toward process automation.     

Computational and Experimental Studies on β-Sheet Breakers Targeting Aβ1–40 Fibrils

In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aβ_1–40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17–21 region of the Aβ_1–40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-sheet breakers, namely Ac-LPFFD-NH₂ and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH₂) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH₂). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that β-sheet breakers are able to inhibit in vitro fibril formation and prevent the β sheet folding of portions of the Aβ_1–40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH₂ β-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aβ_1–40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aβ_1–40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17–21 Aβ_1–40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH₂ β-sheet breaker.     

Matrix-assisted laser desorption–ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures

In theory, peptide mass fingerprinting by matrix assisted laser desorption–ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower μl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.     

Heterologous Expression and Efficient Secretion of Chitosanase from Microbacterium sp. in Escherichia coli

A recombinant expression vector, pCT7-CHISP6H, was constructed for the secretory expression of mature peptide of chitosanase (mMschito) from Microbacterium sp. OU01. The vector contains several elements, including T7 promoter, signal peptide sequence of mschito, 6 × His-tag sequence and PmaCI restriction enzyme cloning site. In pCT7-CHISP6H, mMschito was fused into signal peptide sequence of mschito gene to construct recombinant plasmid pCT7-CHISP6H-mMschito. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and then expressed. The recombinant protein was secreted into the Luria–Bertani broth and the chitosanase activity in supernatant of the culture could reach up to 67.56 U/mL. The rmMschito in the broth supernatant was purified using HisTrap™ FF Crude column and the purified rmMschito was shown to be apparent homogeneity by 12 % SDS–PAGE analysis. Detected by 4700 MALDI-TOF–TOF-MS, the molecular weight of the purified rmMschito was 26,758.1875 and it was consistent with the predicted molecular weight. Chitosan (degree of deacetylation of 99 %) was mostly hydrolyzed into chitopentaose, chitotriose, and chitobiose by the purified rmMschito.     

Shortened derivatives from native antimicrobial peptide LyeTx I: In vitro and in vivo biological activity assessment

In the continuing search for novel antibiotics, antimicrobial peptides are promising molecules, due to different mechanisms of action compared to classic antibiotics and to their selectivity for interaction with microorganism cells rather than with mammalian cells. Previously, our research group has isolated the antimicrobial peptide LyeTx I from the venom of the spider Lycosa erythrognatha. Here, we proposed to synthesize three novel shortened derivatives from LyeTx I (LyeTx I mn; LyeTx I mnΔK; LyeTx I mnΔKAc) and to evaluate their toxicity and biological activity as potential antimicrobial agents. Peptides were synthetized by Fmoc strategy and circular dichroism analysis was performed, showing that the three novel shortened derivatives may present membranolytic activity, like the original LyeTx I, once they folded as an alpha helix in 2.2.2-trifluorethanol and sodium dodecyl sulfate. In vitro assays revealed that the shortened derivative LyeTx I mnΔK presents the best score between antimicrobial (↓ MIC) and hemolytic (↑ EC₅₀) activities among the synthetized shortened derivatives, and LUHMES cell-based NeuriTox test showed that it is less neurotoxic than the original LyeTx I (EC₅₀ [LyeTx I mnΔK] ⋙ EC₅₀ [LyeTx I]). In vivo data, obtained in a mouse model of septic arthritis induced by Staphylococcus aureus, showed that LyeTx I mnΔK is able to reduce infection, as demonstrated by bacterial recovery assay (∼10-fold reduction) and scintigraphic imaging (less technetium-99m labeled-Ceftizoxime uptake by infectious site). Infection reduction led to inflammatory process and pain decreases, as shown by immune cells recruitment reduction and threshold nociception increment, when compared to positive control group. Therefore, among the three shortened peptide derivatives, LyeTx I mnΔK is the best candidate as antimicrobial agent, due to its smaller amino acid sequence and toxicity, and its greater biological activity.     

Indentification of venom proteins of spider S. huwena on two-dimensional electrophoresis gel by N-terminal microsequencing and mass spectrometric peptide mapping

Venom proteins of the spider Selenocosmia huwena were separated by two-dimensional gel electrophoresis, with the separation in the first dimension on a wide range of immobilized pH (3–10) gradients. Over 300 protein spots were presented on a silver-stained 2D gel. The protein spots with molecular weight > 10 kDa were analyzed, after electrotransferring to polyvinyldene difluoride (PVDF) membrane, by N-terminal microseqencing. Some of the silver-stained protein spots with molecular weight over 10 kDa were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including (1) in-gel reduction, alkylation, and enzymatic digestion; (2) extraction and desalting by using the pipette tip containing a small C18 microcolumn (Ziptip); and (3) direct MAIDI-TOF mass analysis and protein database searching. Several known toxins such as HWTX-I, HWTX-II, HWTX-IV, and SHL-I were identified and some new components were found among these protein spots.     

Design,synthesis and biological evaluation of novel N-phosphorylated and O-phosphorylated tacrine derivatives as potential drugs against Alzheimer’s disease

In this work, we designed, synthesised and biologically investigated a novel series of 14 N- and O-phosphorylated tacrine derivatives as potential anti-Alzheimer’s disease agents. In the reaction of 9-chlorotacrine and corresponding diamines/aminoalkylalcohol we obtained diamino and aminoalkylhydroxy tacrine derivatives. Next, the compounds were acid to give final products 6–13 and 16–21 that were characterised by ¹H, ¹³C, ³¹P NMR and MS. The results of the docking studies revealed that the designed phosphorus hybrids, in theory can bind to AChE and BChE. All compounds exhibited significantly lower AutoDock Vina scores compared to tacrine. The inhibitory potency evaluation was performed using the Ellman’s method. The most inhibitory activity against AChE exhibited compound 8 with an IC₅₀ value of 6.11 nM and against BChE 13 with an IC₅₀ value of 1.97 nM and they were 6- and 12-fold potent than tacrine. Compound 19 showed the lack of hepatocytotoxicity in MTT assay.     

The reactivity of affinity labels: A kinetic study of the reaction of alkyl halides with thiolate anions—a model reaction for protein alkylation

Factors have been investigated which govern the electrophilic reactivity of alkyl halides with thiolate anions in aqueous solution. In the series of alkyl halides studied, some are potential metal-directed affinity labels, while others are frequently used in protein modification. Previous data on the kinetics of this type of alkylation are compared with the present results. The influence of electronic, polar, and steric factors on alkyl halide reactivity is seen. The following order of reactivity for alkyl halides bearing different α substituents was observed: RCH₂CH(X)COOCH₃ > RCH₂CH(X)CONH₂ > RCH₂CH(X)COOH > RCH₂CH₂X > RCH₂CH(X)CH₂OH. The metal-directed affinity labels are imidazole derivatives, some of which have substituents in their imidazole ring. The effect of the imidazole ring and of ring substitution on reactivity is seen. The nucleophilic reactivity of thiols is highly pH dependent since the thiolate anion (RS^?) is the reactive species, but only minor differences emerged between different free thiolates.