N-Ethyl maleimide,

Dissolve the macromolecule containing sulfhydryl groups to be blocked in a buffer having a pH of 6.5-7.5. Sodium phosphate (0.01-0.1M) at pH 7.2 works well. Avoid 

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 (this chapter). 

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Figure 20-2. N-(2-Chloropropyl) maleimide, N-ethyl maleimide, N-cyclohexyl maleimide, and N-phenyl maleimide.

Sulfonated styrene-maleimide copolymers are similarly active [1073], Examples of maleimide monomers are maleimide, N-phenyl maleimide, N-ethyl maleimide, N-(2-chloropropyl) maleimide, and N-cyclohexyl maleimide. N-aryl and substituted aryl maleimide monomers are preferred. The polymers are obtained by free radical polymerization in solution, in bulk, or by suspension. 

As expected, the incorporation of pendant unsaturation in the resists greatly enhances sensitivity as demonstrated by a comparison of the contrast curves for poly(N-aiiyl maleimide-VBC) and the structurally similar poiy(N-ethyl maleimide-VBC) (Figure 4). Both polymers have similar molecular weights and nearly identical mass absorption coefficients but the allyl-containing copolymer is 5X faster. 

Signorini N, Molko D, Cadet J (1998) Polyclonal antibodies to adenine N -oxide characterization and use for the measurement of DNA damage. Chem Res Toxicol 11 1169-1175 Simic M, Hayon E (1971) Radical reactions of N-ethyl maleimide in radiation sensitization. Int J Radiat Biol 20 589-592... 

Cleavage of the carbon backbone is reported to be mediated by hydrolases [63]. However, there are no indications that this side-reaction proceeds enzymatically. Instead formation of retro-Claisen products 12 and 13 proceeds via GSH addition to the carbonyl center in 2. The active role of GSH was demonstrated with GSH-depleted cells. After treatment with N-ethyl maleimide (NEM) [64] no retro-Claisen product was detectable. The same applied for NEM-treated cell liquor. 

Inhibition. Since papain, ficin, and bromelain are all enzymes whose activity depends on a free SH group, it is to be expected that all thiol reagents act as inhibitors. Thus, a-halogen acids or amides and N-ethyl-maleimide irreversibly inhibit the thiol proteases. Heavy metal ions and organic mercurial salts inhibit in a fashion that can be reversed by low molecular weight thiols, particularly in the presence of EDTA which... 

Inhibitors reacting with SH groups depressed the reaction only at O.OIM (iodosobenzoate, N-ethyl maleimide) or 0.05M (iodoacetate, iodo-acetamide). No inhibition was obtained with lO W p-chloromercuri-benzoate (24). 

Bowen ME, Weninger K, Brunger AT, Chu S. Single molecule observation of liposome-bilayer fusion thermally induced by soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs). Biophys. J. 2004 87 3569-3584. 

Inhib. abbrev. DTT = dithioghreitol E64D = epoxysuccinyl-L-leucyl-amido-3-methyl-butane ethyl ester EDTA = ethylene-diamine-tetra-acetic acid EGTA = ethylen-glycol-tetra-acetic acid LHVS = morpholinurea-leucine-homophenyl-alanine-vinylsulfone-phenyl NEM = N-ethyl-maleimide PAI = plasminogen activator inhib. PMSF = phenil-methyl-sulfonil fluoride SBTl = soybean trypsin inhib. TIMP = tissue inhib. of metalloproteinases TPCK = tosyl-L-phenyl-alanyl-chloro-methane Al.so termed stephins. 

The investigations of Landesman et al. (1966a, b) clarify the effects of the various conditions controlling the optimum oxidation rates of ferrous iron, sulfur and reduced sulfur compounds by T. ferrooxidans. Experiments on soluble iron, sulfur and iron-containing sulfide minerals (chalcopyrite, CuFeS2, bornite, CUsFeS4, and pyrite) established that iron and sulfur can be oxidized simultaneously. With a mixed iron-sulfur substrate a rate of oxidation, equal to that of the sum of the maximum rates of oxidation of the two substrates individually was observed with both S-adapted and Fe-adapted cells. Subsequently, Duncan et al. (1967) established the differential susceptibility of the bacterial oxidation of ferrous iron and sulfur to N-ethyl maleimide and sodium azide, and determined the effect of these inhibitors on pyrite and chalcopyrite oxidation. Decreased rates... 

Scheme 65 (i) HATU, HOAc, NEM (N-ethyl maleimide), diene precursor, DMF, r.t (ii) Et3N, TBDMSOTf, CH2CI2, 0 °C ... 

C4. Cohen, H. J., and Chovaniec, M. E., Superoxide production by digitonin-stimulated guinea pig granulocytes. The effects of N-ethyl maleimide, divalent cations, and glycolytic and mitochondrial inhibitors on the activation of the superoxide generating system. J. Clin. Invest. 61, 1088-1096 (1978). 

Fig. 2. Accumulation of medicarpin in Ladino clover callus elicited with N-ethyl maleimide. Data are from three separate experiments. 1) Callus was prepared as described (19) and divided into vials containing the indicated concentrations of NEM (one vial for each NEM concentration). After 24-hour incubation period, callus was extracted with methyl ethyl ketone, and medicarpin concentrations determined in the extracts by HPLC (19) - . 2) Same as experiment 1,...

Pyrrolidine-maleimide adduct Piperidine-maleimide adduct Benzylamine-A -phenyl maleimide adduct Glycylamide-N-ethyl maleimide adduct Glycylamide-A -phenyl maleimide adduct 1715 1712 1709 1706 ) 1672J 1718] I68IJ 1634 1603 1771 1770 1779 1779 1786... 

Oxidation alone of pyridine nucleotides is not sufficient to induce Ca release. In the presence of ATP, the hydroperoxide-induced pyridine nucleotide oxidation is even accelerated, yet pyridine nucleotide hydrolysis and Ca release are inhibited [11]. Similar observations were made during the menadione-induced Ca release [10]. When liver mitochondria are treated with N-ethyl maleimide to lower intramitochondrial glutathione, both oxidation of pyridine nucleotides and Ca " release are inhibited (S. Baumhuter, C. Richter, unpubl.). Finally, both pyridine nucleotide hydrolysis and Ca release show the same sigmoidal dependence on the mitochondrial Ca load [15]. Thus, there is clear, albeit circumstantial, evidence that pyridine nucleotide hydrolysis and Ca " release are functionally related. The link between the two processes may be protein ADP-ribosylation. 

The stereochemistry of the thiol is important. Steiic effects have been used to explain the differences in rates of reactivity of RCjHiSH (R = H, 2-/-Bu, 4-/-Bu) in addition reactions with N-ethyl maleimide or displacement of 2,4-(02N)2C,H8S- from 2,4-(03N)2q,H3SSEt ... 

Abbreviations Ac-NAT, acetyl-N-acetyltransferase NEM, N-ethyl maleimide INH, isoniazid. 

Moreover, OH-Cbl binding to plasma proteins can be prevented by N-ethyl-maleimide, p-hydroxomercuribenzoate, or iodoacetamide, which are aU SH-directed agents. In addition, Cd ions not only bind to histidine in proteins, but can also block SH groups (77). This observation has been confirmed in our laboratory in 1% acetic acid and hot ethanol extracts of human plasma using protein-SH group detection with 5,5 -dithio-bis-(2-nitrobenzoic acid, DTNB) according to Sedlak and Lindsay (78). 

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