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PubMed1979

Chemical modification of lactose repressor protein using N-substituted maleimides.

Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore. The reaction with repressor cysteine residues has been characterized. Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents. Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281. The reaction is sulfhydryl-specific. Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments. Effects of the maleimide reagents on biological activity have been determined. Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA. This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding.

Cysteine↗
PubMed1978

New protein reagents. Synthesis and properties of halogenated maleimides and isomaleimides.

The title compounds were investigated as examples of potential hetero-bifunctional protein reagents having an electrophilic moiety in addition to a maleimide or isomaleimide ring. The synthesis of such products by cyclization of the corresponding maleamic acids gives lower yields in the maleimide than in the isomaleimide series, especially when the second function is labile, owing to the more drastic reaction conditions necessary to form the symmetric isomer. On the other hand, a comparison of the isomeric imides showed that despite some differences in reactivity, isomaleimides can be used instead of maleimides for chemical modification of proteins. Therefore, if the synthesis of new maleimide reagents containing a second fragile functional group is difficult, they can be designed in the isoimide form.

Cyclization↗
PubMedDate not supplied

More useful maleimide compounds for the conjugation of Fab' to horseradish peroxidase through thiol groups in the hinge.

Nine different maleimide compounds were evaluated for the conjugation of Fab' to horseradish peroxidase through thiol groups in the hinge. The compounds evaluated were succinimidyl maleimidoacetate (I), succinimidyl 4-maleimidobutyrate (II), succinimidyl 6-maleimidohexanoate (III), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (IV), succinimidyl m-maleimidobenzoate (V), succinimidyl 4-(p-maleimidophenyl)butyrate (VI), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (VII), sulfosuccinimidyl m-maleimidobenzoate (VIII), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (IX). Maleimide groups of I-IV and VII were fairly stable at pH 7.0 at 30 degrees C, while those of the other compounds were significantly decomposed. I-III and VII-IX were sufficiently soluble in the reaction mixture for the introduction of maleimide groups, while the others were more or less precipitated during the reaction. II and III were the most effective in the introduction of maleimide groups and gave the highest recovery of peroxidase in the conjugate, which reached 80%. From these results, II and III were judged to be the most useful, and IV and VII were judged to be fairly useful.

Horseradish Peroxidase↗
PubMed1976

Impermeant maleimides. Identification of an exofacial component of the human erythrocyte hexose transport mechanism.

The facilitated diffusion of D-glucose across human erythrocyte membranes requires an exofacial (outer surface) sulfhydryl group which can be alkylated by the impermeant reagents glutathione-maleimide and dextran-maleimide. The irreversible inhibition produced by these reagents is asymmetric; inhibition of glucose efflux considerably exceeds that of influx when transport is assayed in the absence of glucose on the opposite side of the membrane. Both D-glucose and cytochalasin B protect the exofacial transport site from alkylation by the impermeant maleimides. This masking effect provides the basis for a two-step procedure for differential labeling of the outer transport site with radioactive glutathione-maleimide. The method labels clearly and consistently a component of the membrane proteins which migrates in sodium dodecyl sulfate-polyacrylamide gels between Coomassie brilliant blue-stained Bands 4.2 and 5, corresponding to an apparent molecular weight of 65,000 to 70,000. Transport studies after inhibition with N-ethylmaleimide suggest that the hexose mechanism also requires a second sulfhydryl group which is not accessible at the cell surface.

Binding Sites↗
PubMed2003

Synthesis of 6H-pyrrolo[3',4':2,3][1,4]diazepino[6,7,1-hi]indole-8,10(7H,9H)-diones using 3-bromo-4-(indol-1-yl)maleimide scaffold.

Series of 3-arylalkyl- or 3-alkylamino-4-(indol-1-yl)maleimides and bis(indol-1-yl)maleimides were synthesised. The cyclization of the 3-substituted 4-(indol-1-yl)maleimides under the action of acids resulted in the formation of diazepine[1,4] derivatives with indoline and maleimide nuclei annelated. These compounds readily produced the corresponding indolomaleimidodiazepines[1,4] after dehydrogenation.

Journal Article↗
PubMed1978

Effects on mitochondrial K flux of pH, K concentration, and N-ethyl maleimide.

Based on published evidence that cation transport in mitochondria is not significantly dependent on a membrane potential, it is suggested that the process of mitochondrial cation transport may be nonelectrogenic. These experiments focused on the possibility that K+ flux into rat liver mitochondria may be directly coupled, via an energy-linked carrier mechanism, to OH- influx or H+ efflux. The dependence of the unidirectional K+ influx on the external K+ concentration indicates involvement of a saturable mechanism. Increasing the external pH from 7.0 to 8.0 increases the apparent V max of the K+ influx without significantly altering the apparent Km for K+. The pH dependence is greater in the presence of N-ethyl maleimide, a known inhibitor of the mitochondrial Pi/OH- exchange mechanism. N-Ethyl maleimide decreases the apparent V max at pH 7.0 and increases it at pH 8.0. Evidence indicates that both N-ethyl maleimide and a high external Pi concentration may stimulate the K+ influx at alkaline external pH (8.0) by preventing net exchanges between endogenous Pi and external OH-. An apparent first-order dependence of the K+ influx on the external OH- concentration is observed in the presence of N-ethyl maleimide. These results are consistent with a possible role of external OH- as a cosubstrate of the K+ transport mechanism.

Animals↗
PubMed1979

Reversible uncoupling of photophosphorylation by a new bifunctional maleimide.

A new bifunctional maleimide that contains a disulfide bond has been synthesized. This maleimide, dithiobis-N-ethylmaleimide (DTEM), like another bifunctional maleimide, o-phenylenebismaleimide, is about 500-fold more effective as an inhibitor of photophosphorylation than N-ethylmaleimide. Thylakoids must be illuminated in the presence of DTEM before the assay of phosphorylation for the inhibition to occur. Phosphoryalation in thylakoids treated with DTEM in the light is uncoupled and proton permeability of the treated thylakoids is enhanced. This uncoupling of photophosphorylation in thylakoids treated with DTEM can be reversed by thiol compounds. The addition of 50 mM dithiothreitol restores H+ uptake in thylakoids treated with DTEM in the light to control levels and partially reverses the inhibition of phosphorylation. Evidence is provided to show that DTEM cross-links groups within the gamma subunit of the coupling factor 1, and that the cross-link is broken by high concentrations of thiols. These results suggest that cross-linking is the cause for the increased proton permeability in thylakoids treated with bifunctional maleimides in the light.

Chloroplasts↗
PubMed1987

The use of N-[beta-(4-diazophenyl)ethyl]maleimide as a heterobifunctional agent in developing enzyme immunoassay for neurotensin.

A heterobifunctional crosslinking agent N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM) was newly synthesized and characterized to possess the maleimide group with a stability greater than that previously reported for N-(4-diazophenyl)maleimide. Using the peptide hormone neurotensin (NT) as a model hapten, DPEM was used in the conjugation reaction with bovine serum albumin (BSA) and with beta-D-galactosidase (beta-Gal) in developing an enzyme immunoassay (EIA) for NT. The NT-DPEM-BSA conjugate elicited anti-NT antibodies in rabbits and the NT-beta-Gal conjugate behaved as an enzyme marker of NT in the EIA. The EIA developed double antibody was reproducible and sensitive in detecting NT at concentrations as low as 30 fmol per tube. The specificity of anti-NT serum seems to be primarily toward the carboxy-terminal region of NT, showing cross-reactions with such NT fragments as NT2-13, NT8-13, and NT1-8 for 120, 22, and less than 0.1%, respectively. The utility of this assay was also demonstrated by measuring the NT immunoreactivity in several rat organs. DPEM could be useful for developing EIAs for other peptide hormones (even those which contain neither a free amino group nor a free carboxyl group), using the imidazole, phenolic, or indole group(s) of amino acids as a binding site for carrier proteins.

Cross-Linking Reagents↗
PubMed2006

Labeling of low-density lipoproteins using the 18F-labeled thiol-reactive reagent N-[6-(4-[18F]fluorobenzylidene)aminooxyhexyl]maleimide.

The novel thiol-group-selective bifunctional 18F-labeling agent N-[6-(4-[18F]fluoro-benzylidene)aminooxyhexyl]maleimide ([18F]FBAM) has been developed. The bifunctional labeling precursor N-(6-aminoxyhexyl)maleimide containing a thiol-reactive maleimide group and a carbonyl-group-reactive aminooxy group was prepared in only three steps in a total chemical yield of 59%. Subsequent radiolabeling with 4-[18F]fluorobenzaldehyde gave the bifunctional 18F-labeling agent [18F]FBAM in a radiochemical yield of 29%. In a typical experiment, 3.88 GBq of [18F]fluoride could be converted into 723 MBq of [18F]FBAM within 69 min. Conjugation of [18F]FBAM with thiol groups was exemplified with the cysteine-containing tripeptide glutathione and with various apolipoproteins of human low-density lipoprotein (LDL) subfractions. The latter was evaluated with respect to the uptake of [18F]FBAM-LDL subfractions in human hepatoma cells (HepG2) in vitro. In vivo biodistribution studies in rats revealed high stability for [18F]FBAM-LDL subfractions. Moreover, the metabolic fate of [18F]FBAM-LDL subfractions in vivo was delineated by dynamic positron emission tomography studies using a dedicated small animal tomograph. Data were compared to former studies that used the NH2-reactive 18F-labeling agent N-succinimidyl-4-[18F]fluorobenzoate. The compound [18F]FBAM can be considered as an excellent prosthetic group for the selective and mild 18F labeling of thiol-group-containing biomolecules suitable for subsequent investigations in vitro and in vivo.

Animals↗
PubMed2004

Interactions of LY333531 and other bisindolyl maleimide inhibitors with PDK1.

LY333531, BIM-1, BIM-2, BIM-3, and BIM-8 are bisindolyl maleimide-based, nanomolar protein kinase C inhibitors. LY333531, a PKCbeta-specific inhibitor, is in clinical trials against diabetes and cardiac ventricular hypertrophy complications. Specificity analysis with a panel of 29 protein kinases reveals that these bisindolyl maleimide inhibitors also inhibit PDK1, a key kinase from the insulin signaling pathway, albeit in the lower microM range. To understand the molecular basis of inhibition, the PDK1 kinase domain was cocrystallized with these bisindolyl maleimide inhibitors. The inhibitor complexes represent the first structural description of this class of compounds, revealing their unusual nonplanar conformation within the ATP binding site and also explaining the higher inhibitory potential of LY33331 compared to the BIM compounds toward PDK1. A combination of site-directed mutagenesis and essential dynamics analysis gives further insight into PDK1 and also PKC inhibition by these compounds, and may aid inhibitor design.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMedDate not supplied

Differential reactivity of maleimide and bromoacetyl functions with thiols: application to the preparation of liposomal diepitope constructs.

The comparative reactivity of maleimide and bromoacetyl groups with thiols (2-mercaptoethanol, free cysteine, and cysteine residues present at the N-terminus of peptides) was investigated in aqueous media. These studies were performed (i) with water-soluble functionalized model molecules, i.e., polyoxyethylene-based spacer arms that could also be coupled to lipophilic anchors destined to be incorporated into liposomes, and (ii) with small unilamellar liposomes carrying at their surface these thiol-reactive functions. Our results indicate that an important kinetic discrimination (2-3 orders of magnitude in terms of rate constants) can be achieved between the maleimide and bromoacetyl functions when the reactions with thiols are performed at pH 6.5. The bromoacetyl function which reacts at higher pH values (e.g., pH 9.0) retained a high chemoselectivity; i.e., under conditions where it reacted appreciably with the thiols of, e.g., HS-peptides, it did react with other nucleophilic functions such as alpha- and epsilon-amino groups or imidazole, which could also be present in peptides. This differential reactivity was applied to design chemically defined and highly immunogenic liposomal diepitope constructs as synthetic vaccines, i.e., vesicles carrying at their surface two different peptides conjugated each to a specific amphiphilic anchor. This was realized by coupling sequentially at pH 6.5 and 9.0 two HS-peptides to preformed vesicles containing lipophilic anchors functionalized with maleimide and bromoacetyl groups [Boeckler, C., et al. (1999) Eur. J. Immunol. 29, 2297-2308].

Acetates↗
PubMed2002

Synthesis, characterization, photophysical properties, and biological labeling studies of a series of luminescent rhenium(I) polypyridine maleimide complexes.

We report the synthesis, characterization, and photophysical properties of a series of rhenium(I) polypyridine maleimide complexes [Re(N-N)(CO)(3)(py-3-mal)](CF(3)SO(3)) [N-N = 1,10-phenanthroline, phen (1), 2,9-dimethyl-1,10-phenanthroline, 2,9-Me(2)-phen (2), 3,4,7,8-tetramethyl-1,10-phenanthroline, 3,4,7,8-Me(4)-phen (3), 4,7-diphenyl-1,10-phenanthroline, 4,7-Ph(2)-phen (4), 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline, 2,9-Me(2)-4,7-Ph(2)-phen (5), 2,2'-biquinonine, biq (6); py-3-mal = N-(3-pyridyl)maleimide]. The X-ray crystal structure of complex 2 has been investigated. Upon excitation, the complexes exhibit intense and long-lived photoluminescence in fluid solutions at 298 K. The emission wavelengths range from 514 to 654 nm, and the emission lifetimes fall in the microsecond time scale. The luminescence is assigned to originate from a metal-to-ligand charge-transfer MLCT [dpi(Re) --> pi*(diimine)] triplet excited state. As the maleimide group can react with the sulfhydryl group to form a stable thioether moiety, these complexes have been used as thiol-specific luminescent labels for a thiolated oligonucleotide, glutathione, and bovine serum albumin and human serum albumin. The photoluminescence properties of the labeled biological species have also been investigated.

Animals↗
PubMed1992

Excimer fluorescence of pyrene-maleimide-labeled tubulin.

Excimer-forming cysteines in tubulin are detected by the presence of excimer fluorescence in N-(1-pyrenyl)maleimide-labeled tubulin. The ratio of excimer/monomer fluorescence of labeled protein remained unchanged upon its dilution. These results indicating that both partner of each pair(s) of cysteine are located in the same subunit. The excimer fluorescence is insensitive to prior treatment of tubulin with either colchicine or GTP, indicating that pairs of cysteines protected by those drugs are not involved in excimer formation. This excimer fluorescence of N-(1-pyrenyl)maleimide-labeled tubulin disappeared upon treatment with SDS, guanidinium chloride (GdmCl) and urea. Studies with GdmCl induced unfolding of N-(1-pyrenyl)maleimide-labeled tubulin showed that the loss of excimer fluorescence precedes subunit dissociation. The loss of both colchicine-binding activity and the excimer fluorescence with increasing temperature indicates a major conformational change of the tubulin molecule at elevated temperatures.

Guanidine↗
PubMedDate not supplied

[Inhibition of respiration and phosphorylation by some maleimide derivatives].

The inhibitory effect of maleimide and ten its N-substituted mono- and bifunctional derivatives was studied in isolated rat liver mitochondria. It was found that all these compounds blocked protein and non-protein SH-groups and were the potent inhibitors of the respiratory chain. In the inhibitory activity of unsubstituted maleimide its alkylating properties play the dominant role. The introduction of some substitutors in the maleimide molecule lends the properties of hydrophobic inhibitors to these derivatives. A high toxicity of all compound studied was shown in in vivo experiments.

Animals↗
PubMed1997

Cysteine-specific radioiodination of proteins with fluorescein maleimide.

A protocol is described for coupling of carrier-free iodine to protein sulfhydryl groups via fluorescein maleimide. 125I is first coupled to fluorescein maleimide in the presence of chloramine T. Iodination is stopped with sodium thiosulfate, and the iodine-substituted fluorescein maleimide is reacted with free cysteines of the protein. Excess label is then removed by gel-permeation chromatography. The procedure avoids exposition of the protein to oxidative conditions and does not require purification of the labeled carrier reagent. Suitability of the method for a given protein can be evaluated spectrophotometrically without employing radioactivity. It can be applied under denaturing conditions and may be particularly useful with mutant proteins carrying engineered single cysteine residues at sites that are not functionally critical.

Bacterial Proteins↗
PubMed1984

Cytochemistry of the chromatin replication band in hypotrichous ciliated protozoa staining with silver and thiol-specific coumarin maleimide.

A modification of the silver-staining techniques for nucleolar organizing regions (NORs) was used to stain selectively the macronuclear replication bands (RBs) and nucleoli in hypotrichous ciliated protozoa (Euplotes, Stylonychia, and Oxytricha). Silver staining of both types of structures was trypsin-sensitive and DNase I-insensitive, suggesting the involvement of proteins. Silver-staining proteins in the RB were differentially extracted with acid, without any decrease in nucleolar staining. Triton-acid-urea gel electrophoresis of an acid extract of Euplotes macronuclei revealed enhanced silver reaction with a single protein upon selective silver staining. An abundance of thiol groups was also demonstrated in the RBs and nucleoli by the fluorochrome 3-(4-maleimidylphenyl)-7-diethylamino-4-methyl coumarin (coumarin maleimide). Histochemical studies, including blocking thiols with N-ethyl maleimide (NEM), indicated that thiols were not necessary for silver staining, and that proteins in the RBs and nucleoli reacting with coumarin maleimide were not acid extractable.

Animals↗
PubMed2006

A retro-Diels-Alder reaction to uncover maleimide-modified surfaces on monolayer-protected nanoparticles for reversible covalent assembly.

Maleimide-modified monolayer-protected gold nanoparticles (MPGN) are prepared from the protected furan-maleimide via the thermally reversible Diels-Adler reaction when required. These maleimide-MPGNs serve as a general platform allowing for a Diels-Alder reaction with furan-modified MPN to prepare larger 3D networks reversibly. [reaction: see text]

Journal Article↗
PubMed1981

Fluorescence titration and fluorescence stopped-flow studies on skeletal troponin C labeled with fluorescent maleimide reagent or dansylaziridine.

Skeletal muscle troponin C (TN-C) labeled with N-(p-(2-benzimidazolyl)phenyl)-maleimide (BIPM) shows about 5% fluorescence increase and 82% fluorescence increase upon Ca2+ binding and Mg2+ binding to the high affinity Ca2+-binding sites (sites III and IV) of TN-C, respectively. TN-C labeled with N-(1-anilinonaphthyl-4)maleimide (ANM) shows about 26% fluorescence decrease and 22% fluorescence increase upon Ca2+ binding and Mg2+ binding to the high affinity Ca2+-binding sites, respectively. These findings indicate that environmental change around Cys-98, where the maleimide reagent bind, induced by Ca2+ binding to the high affinity Ca2+-binding sites is very different from that induced by Mg2+ binding to the same sites. These dye-protein conjugates do not show fluorescence intensity change upon Ca2+ binding to the low affinity Ca2+-binding sites (sites I and II). Dansylaziridine (DANZ)-labeled TN-C shows more than 100% fluorescence increase upon Ca2+ binding to the low affinity Ca2+-binding sites of TN-C. Hence, we can observe the kinetic processes which TN-C undergoes upon Ca2+ binding to or removal from the high affinity Ca2+-binding sites by following the fluorescence intensity change of ANM-labeled TN-C, respectively. THe kinetic processes of the fluorescence intensity change associated with the Ca2+ binding and removal reactions with the high affinty Ca2+-binding sites (rate constants, 3.7-157 s-1) are slower than the kinetic processes associated with the low affinity Ca2+-binding sites (rate constants, equal to or higher than 230 s-1) in the absence of MgCl2. In the presence of 2 mM MgCl2, a new slow phase (rate constant, 10-16 s-1) appears in the kinetic processes associated with the low affinity Ca2+-binding sites, in addition to the rapid phase which is already observed in the absence of MgCl2. Kinetic properties associated with the high affinity Ca2+-binding sites are not essentially altered by addition of 2 mM MgCl2 to the system, but ANM-labeled TN-C shows lower rate constants (0.67-26 s-1).

Animals↗