N-ethylmaleimide

Dissolve the macromolecule containing sulfhydryl groups to be blocked in a buffer having a pH of 6.5—7.5. Sodium phosphate (0.01—0.1 M) at pH 7.2 works well. Avoid amine-containing buffers, since an excess of amines may cause some reactivity with the maleimide groups. Also, avoid the presence of sulfhydryl-containing disulfide reductants such as DTT or 2-mercaptoethanol, which will rapidly react with NEM. 

Add at least a 10-fold molar excess of NEM over the amount of sulfhydryls present in the reaction. Alternatively, add an equal mass of NEM to the amount of macromolecule present. To facilitate the addition of a small quantity of reagent, a more concentrated stock solution may be prepared in buffer and an aliquot added to the reaction medium. Make the stock solution up fresh, and use it immediately to prevent loss of activity due to maleimide group breakdown. 

iodoacetamide has the highest reactivity toward cysteine sulfhydryl residues and may be directed specifically for —SH blocking. If iodoacetamide is present in limiting quantities (relative to the number of sulfhydryl groups present) and at slightly alkaline pH, cysteine modification will be the exclusive reaction. For additional information on a-haloacetate reactivities and a protocol for blocking, see Section 4.2. 

---

Aqueous solutions of aequorin also emit light upon the addition of various thiol-modification reagents, such as p-quinone, Br2, I2, N-bromosuccinimide, N-ethylmaleimide, iodoacetic acid, and p-hydroxymercuribenzoate (Shimomura et al., 1974b). The luminescence is weak and long-lasting ( 1 hour). The quantum yield varies with the conditions, but seldom exceeds 0.02 at 23-25°C. The luminescence is presumably due to destabilization of the functional moiety caused by the modification of thiol and other groups on the aequorin molecule. 

Lipid phosphate phosphohydrolases (LPPs), formerly called type 2 phosphatidate phosphohydrolases (PAP-2), catalyse the dephosphorylation of bioactive phospholipids (phosphatidic acid, ceramide-1-phosphate) and lysophospholipids (lysophosphatidic acid, sphingosine-1-phosphate). The substrate selectivity of individual LPPs is broad in contrast to the related sphingosine-1-phosphate phosphatase. LPPs are characterized by a lack of requirement for Mg2+ and insensitivity to N-ethylmaleimide. Three subtypes (LPP-1, LPP-2, LPP-3) have been identified in mammals. These enzymes have six putative transmembrane domains and three highly conserved domains that are characteristic of a phosphatase superfamily. Whether LPPs cleave extracellular mediators or rather have an influence on intracellular lipid phosphate concentrations is still a matter of debate. 

Catalytic antibody 1E9, the first catalytic antibody discovered for Diels-Alder reaction, catalyzes the cycloaddition between tetrachlorothiophene dioxide and N-ethylmaleimide (Equation 4.15) [86]. 

Rideout and Breslow first reported [2a] the kinetic data for the accelerating effect of water, for the Diels Alder reactions of cyclopentadiene with methyl vinyl ketone and acrylonitrile and the cycloaddition of anthracene-9-carbinol with N-ethylmaleimide, giving impetus to research in this area (Table 6.1). The reaction in water is 28 to 740 times faster than in the apolar hydrocarbon isooctane. By adding lithium chloride (salting-out agent) the reaction rate increases 2.5 times further, while the presence of guanidinium chloride decreases it. The authors suggested that this exceptional effect of water is the result of a combination of two factors the polarity of the medium and the... 

Table 6.2 Sodium and guanidinium salt effects (relative reaction rates) of Diels-Alder reaction of anthracene-9-carbinol and N-ethylmaleimide...

NEM N-Ethylmaleimide, a chemical that alkylates sulfhy-dryl groups... 

Step 7 Hydrolysis of ATP by NSF is essential for fusion, a process that can be inhibited by NEM N-ethylmaleimide). Gertain other proteins and calcium are also required. 

Identification of cysteine residues that react with N-ethylmaleimide (MalNEt)... 

On the other hand, the use of a-cyclodextrin decreased the rate of the reaction. This inhibition was explained by the fact that the relatively smaller cavity can only accommodate the binding of cyclopentadiene, leaving no room for the dienophile. Similar results were observed between the reaction of cyclopentadiene and acrylonitrile. The reaction between hydroxymethylanthracene and N-ethylmaleimide in water at 45°C has a second-order rate constant over 200 times larger than in acetonitrile (Eq. 12.2). In this case, the P-cyclodextrin became an inhibitor rather than an activator due to the even larger transition state, which cannot fit into its cavity. A slight deactivation was also observed with a salting-in salt solution (e.g., quanidinium chloride aqueous solution). 

ND = Not determined DTNB 2,2 -Dithiobis(5-nitropyridine) DTNP 2,2 -Dithiobis(5-nitrobenzoate) DEDC Diethyldithiocarbamate IA Iodoacetate NEM N-ethylmaleimide PCMB p-chloromercuribenzoate 1, 10-PT 1, 10-phenanthroline 8-Q 8-Quinolinol 2, 2 -BP 2, 2 -Bipyridyl. 

Gorin, G., Martin, P.A., and Doughty, G. (1966) Kinetics of the reaction of N-ethylmaleimide with cysteine and some congeners. Arch. Biochem. Biophys. 115, 593. 

Haugaard, N., Cutler, J., and Ruggieri, M.R. (1981) Use of N-ethylmaleimide to prevent interference by sulfhydryl reagents with the glucose oxidase assay for glucose. Anal. Biochem. 116, 341-343. 

Smyth, D.G. (1967) Acetylation of amino and tyrosine hydroxyl groups./. Biol. Chem. 242, 1592-1598. Smyth, D.G., Blumenfeld, O.O., and Konigsberg, W. (1964) Reaction of N-ethylmaleimide with peptides and amino acids. Biochem. J. 91, 589. 

N-ethylmaleimide-sensitive Trimeric ATPase required for in vitro membrane fusion during vesicular transport. Probably function as factor (NSF) chaperones in synaptic vesicle recycling. 

NSF N-ethylmaleimide-sensitive factor PNUTS phosphatase 1 nuclear targeting subunit... 

The extent of formation of protein disulfides with time was determined by withdrawing aliquots which were acidified to pH 5.5 and alkylated with N-ethylmaleimide. The disulfide content of the peptide was determined after its isolation. Formation of two intrapeptide disulfide bonds proceeded at the same rate (within experimental error) as formation of the first two disulfides in reduced lysozyme. The first-order rate constant for these two processes (0.5 min-1) was eight times that describing the rate of oxidation of reduced lysozyme in the presence of 6 M guanidinium chloride, suggesting substantial specificity in the process in absence of denaturant. An additional indication of specificity was the finding that 13-105 reached its maximum of two —S—S— bonds in less than 20 minutes, retaining one reduced thiol from 20 to 240 minutes. For subsequent studies this material was S-alkylated with N-ethylmaleimide. 

Moriyama, Y., and Nelson, N., 1988, Purification and properties of a vanadate- and N-ethylmaleimide- sensitive ATPase from chromaffin granule membranes. J. Biol. Chem., 263 8521-8527. 

Table II shows that UDP-pyridoxal had a similar inhibitory effect on red beet glucan synthase. It inhibited activity at much lower concentrations than other covalent modification reagents, such as N-ethylmaleimide (cysteine), phenylglyoxal (arginine) and formaldehyde (lysine). UDP-pyridoxal had an I50 that is 62-fold lower than formaldehyde.

Another successful example of such guest design is the Diels-Alder reaction markedly accelerated by the cyclodextrin inclusion. As shown in Table XXIV, /J-cyclodextrin accelerates the addition of a small dienophile to cyclopentadiene, but inhibits that of N-ethylmaleimide to anthracene-9-carbinol (117). Thus, the guest design is a really helpful concept for the remarkable catalysis. However, there seems to be some limitation in the choice of reactions, if cyclodextrins have no special functional group for the... 

---