Methylation Of Carboxyl Groups Research Articles

Enhancement of protein flocculant properties through carboxyl group methylation and the relationship with protein structural changes

Some proteins will function as flocculants to clear water of suspended particles. Prior work has shown that covalent methylation of a protein’s carboxyl groups can enhance that protein’s flocculant activity; this effect is usually attributed to the reduction in negative charges on the protein. We present results generated using four proteins which show that much of the flocculant activity enhancement occurs in a very short time at the beginning of the methylation reaction, before most of the carboxyl groups have been methylated. A variety of techniques are used to show that the methylation reaction has unintended effects on protein structure and aggregation, and that these effects may play an important role in enhancing protein flocculant activity. The observed enhancement in flocculant activity results from both charge and structural modification.

Transcriptomic response of Arabidopsis thaliana roots to naproxen and praziquantel

Exposition to pharmaceutical compounds released to the environment is considered as a potential risk for various organisms. We exposed Arabidopsis thaliana plants to naproxen (NAP) and praziquantel (PZQ) in 5 µM concentration for 2 days and recorded transcriptomic response in their roots with the aim to estimate ecotoxicity and to identify gene candidates potentially involved in metabolism of both compounds. Nonsteroidal anti-inflammatory drug NAP up-regulated 105 and down-regulated 29 genes (p-value ≤ 0.1, fold change ≥ 2), while anthelmintic PZQ up-regulated 389 and down-regulated 353 genes with more rigorous p-value ≤ 0.001 (fold change ≥ 2). High number of up-regulated genes coding for heat shock proteins and other genes involved in response to biotic and abiotic stresses as well as down-regulation of genes involved in processes such as cell proliferation, transcription and water transport indicates serious negative effect of PZQ. NAP up-regulated mostly genes involved in various biological processes and signal transduction and down-regulated mainly genes involved in signal transduction and electron transport or energy pathways. Further, two cytochrome P450s (demethylation) and one methyltransferase (methylation of carboxyl group) were identified as candidates for phase I and several glutathione- and glycosyltransferases (conjugation) for phase II of NAP metabolism. Cytochrome P450s, glutathione and glycosyltransferases seem to play role also in metabolism of PZQ. Up-regulation of several ABC and MATE transporters by NAP and PZQ indicated their role in transport of both compounds.

SEC-MALLS analysis of TEMPO-oxidized celluloses using methylation of carboxyl groups

Two cellouronic acids [sodium (1 → 4)-β-polyglucuronates, CUAs] and one 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized wood cellulose (TOC) became soluble in 8 % lithium chloride/N,N-dimethylacetamide (LiCl/DMAc) after the methylation of C6 carboxyl groups in these samples using trimethylsilyldiazomethane (TMSD). The obtained solutions were diluted to 1 % LiCl/DMAc and subjected to size-exclusion chromatography combined with multi-angle laser-light scattering (SEC-MALLS). Neither depolymerization nor side reactions took place during methylation; this was confirmed by SEC-MALLS and nuclear magnetic resonance analyses, using CUAs as models. The SEC-MALLS analysis of the original wood cellulose and the carboxyl-methylated TOC prepared from it, using 1 % LiCl/N,N-dimethyl-2-imidazolidinone and 1 % LiCl/DMAc, respectively, as eluents, showed that the weight-average degree of polymerization of the original wood cellulose decreased from 3,100 to 2,210 through TEMPO-mediated oxidation. The molecular-mass distributions of the original wood cellulose and the TOC both consisted of one large peak with a small shoulder, indicating that some of the oxidized hemicelluloses remained in the TOC. The combination of methylation of carboxyl groups in polysaccharides using TMSD and subsequent SEC-MALLS analysis using 1 % LiCl/DMAc as an eluent may be applicable not only to TOCs, but also to other polysaccharides with carboxyl groups, for evaluation of their molecular-mass parameters.

A novel, mild and selective methylation of carboxyl groups in cellulosic pulps 10th EWLP, Stockholm, Sweden, August 25–28, 2008

Abstract A method for selective, fast, and near-quantitative methylation of carboxyl groups in the presence of aliphatic hydroxyl groups of cellulose was developed. The main advantage of the method is the complete maintenance of cellulose integrity; no degradation of cellulose whatsoever during the methylation procedure was observed, making the protocol extremely useful in advanced cellulose analytics. Trimethylsilyl diazomethane (TMS-DAM) was used as the methylation agent. The methylation conditions were pre-optimized by means of carbohydrate model compounds with low-molecular weight, such as derivatives of glucuronic acid and hexenuronic acid, and then re-optimized for methylation of cellulose. The influence of reaction parameters, such as time, temperature, solvents, and concentration, was also determined. The reaction kinetics was monitored by fluorescence labeling of the remaining carboxyl groups according to the common FDAM protocol. A general protocol for the fast methylation of carboxyl groups in pulp samples is presented. The methylation with TMS-DAM is a very fast process, both in comparison to general heterogeneous reactions on cellulosic pulps, and especially in comparison to other methylation approaches. In dissolving and paper grade pulps, methylation of xylan carboxyls proceeded faster than methylation of cellulosic carboxyls. The methylation rate and kinetics are dependent on the pulp type. As application examples, electrokinetic measurements were used to demonstrate the “blocked” reactivity of carboxyl groups in spin-coated films of methylated pulp samples, and ICP-MS was used to demonstrate the drastically decreased metal ion binding capacity of pulp after such methylation.

Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis of hyaluronan oligosaccharides

A new method is presented for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronan (HA) with bacterial hyaluronidase (E.C. 4.2.2.1, from Streptomyces hyalurolyticus) using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS). Mixtures containing HA oligosaccharides of tetrasaccharide (4-mer)–34-mer were analyzed using this method. The carboxyl groups of the glucuronate residues in the prepared HA oligomers, were modified as the acidic form ( COOH), sodium salts ( COONa), organic ammonium salts, or methylesters before MALDI-TOFMS measurement. Among these samples, the methylester form of glucuronate residues in HA oligosaccharides, prepared by methylation using trimethylsilyl diazomethane, afforded high sensitivity for spectra. This simple modification method for carboxyl group methylation of acidic polysaccharides [Hirano et al., Carbohydr. Res., 340, (2005) 2297–2304] provides samples suitable for MALDI-TOF mass spectrometric analysis throughout a significantly enhanced range of masses.

Functional group analysis by H NMR/chemical derivatization for the characterization of organic aerosol from the SMOCC field campaign

Abstract. Water soluble organic compounds (WSOC) in aerosol samples collected in the Amazon Basin in a period encompassing the middle/late dry season and the beginning of the wet season, were investigated by H NMR spectroscopy. HiVol filter samples (PM2.5 and PM>2.5) and size-segregated samples from multistage impactor were subjected to H NMR characterization. The H NMR methodology, recently developed for the analysis of organic aerosol samples, has been improved by exploiting chemical methylation of carboxylic groups with diazomethane, which allows the direct determination of the carboxylic acid content of WSOC. The content of carboxylic carbons for the different periods and sizes ranged from 12% to 20% of total measured carbon depending on the season and aerosol size, with higher contents for the fine particles in the transition and wet periods with respect to the dry period. A comprehensive picture is presented of WSOC functional groups in aerosol samples representative of the biomass burning period, as well as of transition and semi-clean atmospheric conditions. A difference in composition between fine (PM2.5) and coarse (PM>2.5) size fractions emerged from the NMR data, the former showing higher alkylic content, the latter being largely dominated by R-O-H (or R-O-R') functional groups. Very small particles (<0.14 μm), however, present higher alkyl-chain content and less oxygenated carbons than larger fine particles (0.42–1.2 μm). More limited variations were found between the average compositions in the different periods of the campaign.

Methylating peptides to prevent adduct ion formation also directs cleavage in collision-induced dissociation mass spectrometry.

We investigated the effect of N-terminal amino group and carboxyl group methylation on peptide analysis by electrospray mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Permethylation of the N-terminal amino group and the carboxyl groups can reduce metal ion adducts but does not enhance sensitivity in electrospray as previously observed for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. N-terminal trimethylated peptides exhibit collision-induced dissociation (CID) tandem mass spectra that differ from their unmodified analogs; the results support the mobile proton hypothesis of peptide fragmentation. A permanent positive charge at the N-terminus leads to competition between permanent-charge directed processes and loss of the N-terminal trimethyl amino group. Carboxyl methylation has no effect on fragmentation behavior other than to shift the mass of fragments containing methylated carboxyl groups. Comparison of regular and tandem mass spectra of different methylated peptides allowed probing the location of incomplete methylation, the proton displaced by alkali metal ions and the purity of a mass-selected methylated peptide ion.

On the Methylation of Carboxyl Groups with Methanesulfonyl Chloride in Pyridine.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Graphical Abstracts

Synth. Commun. 2003, 33, 2575 On the Methylation of Carboxyl Groups with Methanesulfonyl Chloride in Pyridine Peter A. Jacobi* and E. Hampton Sessions Jr. Burke Chemical Laboratory, Dartmouth College, Hanover, New Hampshire 03755, USA In our hands methylation of carboxylic acids does not occur employing MsCl/pyridine.

On the Methylation of Carboxyl Groups with Methanesulfonyl Chloride in Pyridine

In a recent article Siddiqui et al. described a novel conversion of carboxylic acids to methyl esters,[1a] employing the reagent system methanesulfonyl chloride (MsCl) in pyridine at 0°C (Fig. 1).[...

ChemInform Abstract: A New Reagent for the Methylation of Carboxyl Groups.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Myelin proteolipid protein-induced aggregation of lipid vesicles: efficacy of the various molecular species.

The different molecular species that form the myelin proteolipid protein family were isolated by size-exclusion and ion-exchange chromatography in organic solvents and their adhesive properties were tested using a vesicle aggregation assay. Addition of the major proteolipid (PLP) to phosphatidylcholine-cholesterol vesicles caused their clustering as determined by increase in O.D.(450 nm) and by transmission electron microscopy. A small fraction of the aggregated vesicles underwent fusion as determined by resonance energy transfer experiments. Vesicle aggregation by PLP, but not the dissociation of the aggregates, was influenced by pH suggesting that electrostatic interactions are important only during cluster formation. Cleavage of disulfide bonds and methylation of carboxyl groups in PLP greatly reduced the aggregating activity, indicating that the process is dependent on the protein's conformation. Unexpectedly, the proteolipid DM-20 was also effective at inducing the clustering of neutral lipid vesicles. In contrast, three protein fractions comprising the naturally-occurring PLP fragments 1-107/112, 113/125-276 and 129/131-276, bearing different net charges, displayed a much lower activity. In addition, trypsin digestion of PLP resulted in a progressive decrease in the protein's ability to induce vesicle aggregation which coincided with the disappearance of the full-length molecule. Together, these results suggest that even large PLP fragments cannot fulfill the adhesive function of the intact protein.

A new reagent for the methylation of carboxyl groups

A new reagent for the preparation of methyl esters of carboxylic acids is described. The reaction involves treatment of acids with methanesulfonyl chloride in pyridine at 0°C.

Characterization of Prenylcysteines That Interact with P-glycoprotein and Inhibit Drug Transport in Tumor Cells

Prenylcysteine methyl esters that represent the C-terminal structures of prenylated proteins demonstrate specific substrate-like interactions with P-glycoprotein (Zhang, L., Sachs, C. W., Fine, R. L., and Casey, P. J. (1994) J. Biol. Chem. 269, 15973-15976). The simplicity of these compounds provides a unique system for probing the structural specificity of P-glycoprotein substrates. We have further assessed the structural elements of prenylcysteines involved in the interaction with P-glycoprotein. Carboxyl group methylation, a modification in many prenylated proteins, plays an essential role of blocking the negative charge at the free carboxylate. Substitution of the methyl ester with a methyl amide or simple amide does not change the ability of the molecule to stimulate P-glycoprotein ATPase activity, but substitution with a glycine is not tolerated unless the carboxyl group of glycine is methylated. The presence of a nitrogen atom, which is found in many P-glycoprotein substrates and modifiers, is also essential for prenylcysteines to interact with P-glycoprotein. The structure at the nitrogen atom can, however, influence the type of interaction. Acetylation of the free amino group of prenylcysteine/results in a significant loss in the ability of prenylcysteines to stimulate P-glycoprotein ATPase activity. Instead, certain acetylated prenylcysteines behave as inhibitors of this activity. In studies using MDR1-transfected human breast cancer cells, the acetylated prenylcysteine analogs inhibit P-glycoprotein-mediated drug transport and enhance the steady-state accumulation of [3H]vinblastine, [3H]colchicine, and [3H]taxol. These inhibitors do not, however, affect drug accumulation in parental cells. These studies provide a novel approach for designing P-glycoprotein inhibitors that could prove effective in reversing the phenotype of multidrug resistance in tumor cells.

Macromolecular sieving by glomerular basement membrane in vitro: Effect of polycation or biochemical modifications

To evaluate the effect of biochemical modifications not possible in vivo, filters of dog glomerular basement membrane (GBM) were constructed in ultrafiltration cells in vitro. The sieving coefficients (SCs) of three protein markers of differing size and charge (native, anionic bovine albumin—BSA; cationized BSA—cBSA; and immunoglobulin G—IgG) were determined using filters of differing amounts of control GBM, and under varying transmembrane pressures ( ΔP). Flow rates did not increase proportionately with increasing ΔP, indicating filter compressibility. Protein SCs did not change with changing ΔP, but did decrease with increasing filter thickness. Control filters showed a small but definite charge selectivity (SC cBSA − SC BSA >0); a much greater degree of size selectivity (SC cBSA − SC IgG) was observed. Hexadimethrine (HDM), a polycation which causes proteinuria in vivo, led to marked increases in protein SCs. In contrast, removal of the major population of intrinsic GBM negative charges by carboxyl group methylation only produced a small increase in the filtration of BSA, with no change in filtration of cBSA or IgG. Other biochemical modifications (heparinase or neuraminidase treatment) had no effect on filter permselectivity. Carboxyl group methylation essentially abolished filter binding of cationized ferritin, which showed substantial binding to control filters. These in vitro studies provide confirmatory evidence for a direct effect of HDM on the permselective properties of GBM. In addition, biochemical modification studies suggest a fundamental difference between the binding of an exogenous polycation to GBM anionic sites and the removal of intrinsic charges.

Water-soluble carrier proteins having carboxyl spacer groups.

Three water-soluble bovine serum albumin derivatives with carboxyl spacer groups have been synthesized as carrier proteins for direct preparation of conjugates between proteins and low molecular haptens with amino groups. N-Glycylaminomethanesulfonic acid, a newly developed solubilizing reagent for proteins, was first introduced into bovine serum albumin through its carboxyl groups. After the methylation of carboxyl groups remaining unreacted in the modified albumin, N-succinylglycine and N-bromoacetyl-β-alanine, both carboxyl spacer reagents, were reacted with the amino or imidazole groups of the modified albumin, respectively, to give two water-soluble albumin derivatives. Another water-soluble albumin derivatives. Another water-soluble albumin derivative was obtained by treating the modified albumin with N-bromoacetyl-β-alanine followed by amidination of the remaining amino groups of the albumin with methylacetimidate. Glycine ethyl ester, selected as a model hapten that has an amino group, was reacted with these albumin derivatives to form conjugates, and 4-15 ester moieties can be incorporated into one molecule of the albumin derivatives without loss of their water-solubility.

Depression of human sperm motility by inhibition of enzymatic methylation

Alteration of membrane fluidity during enzymatic methylation of membrane phosphatidylethanolamine (PE) and neutralization of negative charges of membrane proteins due to methylation of carboxyl groups may contribute to sperm motility. Therefore, enzymatic phospholipid methylation and carboxymethylation, and the consequences of their inhibition on motility, were studied using human sperm. These studies gave the following results. Human sperm homoganates contained two phospholipid N-methyltransferases (PMT) which converted PE to phosphatidylcholine (PC) in the presence of S-adenosylmethionine (SAM). The first PMT converted PE to phosphatidyl- N-methylethanolamine (PME). In had a K m of 4.0 μM and a pH optimum of 8.0. The second PMT converted PME to phosphatidyl- N, N-dimethylethanolamine and PC. It had a K m of 71μM and a pH optimum of 10.0. Spermatozoa also contained protein carboxymethylase (PCM) and methyl aceptor protein (MAP). The intracellular levels of S-adenosylhomocysteine (SAH), an inhibitor of SAM-mediated methylations, were increased by adding adenosine (100 μM), l-homocysteine thiolactone (L-HCT, 10 μM), and erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA, 10 μM), an inhibitor of adenosine deaminase, to human sperm ejaculates that had been diluted with sodium phosphate buffer at pH 7.4 and 25°. The motility index of each sperm suspension was determined every hour for 4 hr. In the presence of the mixture of adenosine, L-HCT and EHNA, the motility index was depressed by 57%. Under similar conditions, phospholipid methylation was depressed by 48%. Similar experiments were also conducted in the presence of 3-deazaadenosine (Deaza, 80 μM), a selective inhibitor of SAH hydrolase. In the presence of adenosine and L-HCT, Deaza depressed the motility index by 60% and phospholipid methylation by 86%. The potencies of SAH in the inhibition of phospholipid methylation and protein carboxymethylation in sperm homogenates had the following order: PMT I > PCM > PMT II. These observations indicate that the PMT system and/or the PCM-MAP system play a significant role in the regulation of human sperm motility.

Enzymatic methylation of carboxyl groups of chromaffin granule membrane proteins.

Carboxyl groups of membrane and soluble proteins from bovine adrenal medulla chromaffin granules were enzymatically methylated. The methylated peptides were resolved using gel electrophoresis under acidic conditions in the presence of N-cetylpyridinium chloride. There was a selective methylation of two groups of membrane peptides which did not correspond to any of the chromaffin granule soluble proteins. Dopamine beta-hydroxylase, an acidic protein accounting for up to 25% of the membrane proteins, was a poor substrate for protein carboxylmethylase. The methyl esters of membrane proteins were more labile than those of the chromaffin granule soluble proteins. At all pH values tested, membrane protein-methyl esters were hydrolyzed three times more rapidly than the soluble protein-methyl esters.

Carboxyl group methylation of loganic acid by a cell-free enzyme preparation from Vinca rosea.

Carboxyl group methylation of loganic acid by a cell-free enzyme preparation from Vinca rosea.

Cation exchange in plant roots

Abstract Methylation of carboxyl groups reduced the cation exchange capacity of plant roots to almost zero. Deamination of the roots did not affect their cation exchange capacity.