Reaction Of N-ethylmaleimide Research Articles

Kinetics of Diels–Alder reactions in critical binary solutions

The kinetics of the Diels–Alder (D–A) reactions of N-ethylmaleimide with 2,3-dimethyl-1,3-butadiene and of 9,10-dimethyl-anthracene with diethyl maleate in two critical binary solutions n-tetradecane+dimethyl carbonate and n-nonane+diethyl maleate have been studied by the three-wavelength UV-spectrophotometry. It was found that neither speeding up nor slowing down effect for the first reaction was observed, which obeyed the Griffiths–Wheeler rule; while a very weak slowing down effect was detected for the second reaction, but significantly weaker as compared with the prediction by the Griffiths–Wheeler rule. It was attributed to that only less than 0.002% of diethyl maleate changed to the products during the reaction, hence the concentration of diethyl maleate may be considered as a fixed thermodynamic density in the critical reaction system, resulting in no thermodynamic slowing down being detected.

Periplasmic Loops of Osmosensory Transporter ProP in Escherichia coli Are Sensitive to Osmolality

ProP is an osmosensory transporter. The activities of ProP and ProP*, a cysteine-less, His(6)-tagged ProP variant, increase with osmotic pressure in cells and proteoliposomes. In proteoliposomes, ProP activity is osmolality-dependent only if the magnitude of the membrane potential (DeltaPsi) exceeds 100 mV. Some amino acid replacements rendered ProP activity osmolality-insensitive [e.g., Y44M in transmembrane segment 1 (TMI); S62C in periplasmic loop 1 (loop P1)], whereas others elevated the osmolality at which ProP activates (e.g., A59C). This suggested that the environments and/or conformations of TMI and loop P1 might be osmolality-dependent. This report correlates structural dynamics of ProP with osmoregulation of its transport activity. Residues in periplasmic loops were replaced with Cys, and changes in their environments were detected by monitoring their reactivities with N-ethylmaleimide (NEM). Increasing osmolality markedly increased the NEM reactivity of some Cys residues (e.g., C59, loop P1; C415-C418, loop P6) but not others (e.g., C293, loop P4; C348, loop P5). The NEM reactivity of C62 was insensitive to osmolality, as expected. Substitution Y44M rendered the transport activities of ProP*-A59C and ProP*-Q415C, and the NEM reactivities of the introduced Cys, osmolality-insensitive. Furthermore, osmolality did not affect the reactivity of C59 in cells lacking a protonmotive force, consistent with evidence that DeltaPsi is required for osmosensing by ProP. These results indicate that the osmotically induced increases in NEM reactivity of C59 and C415 in energized bacteria are due to a conformational change of ProP in response to osmolality. They therefore constitute the first direct evidence of an osmotically induced conformational change associated with osmosensing by a transporter.

Characterization of the Sulfhydryl-Sensitive Site in the Enzyme Responsible for Hydrolysis of 2- Arachidonoyl-Glycerol in Rat Cerebellar Membranes

We have previously reported that the endocannabinoid, 2-arachidonoyl-glycerol (2-AG), is hydrolyzed in rat cerebellar membranes by monoglyceride lipase (MGL)-like enzymatic activity. The present study shows that, like MGL, 2-AG-degrading enzymatic activity is sensitive to inhibition by sulfhydryl-specific reagents. Inhibition studies of this enzymatic activity by N-ethylmaleimide analogs revealed that analogs with bulky hydrophobic N-substitution were more potent inhibitors than hydrophilic or less bulky agents. Interestingly, the substrate analog N-arachidonylmaleimide was found to be the most potent inhibitor. A comparison model of MGL was constructed to get a view on the cysteine residues located near the binding site. These findings support our previous conclusion that the 2-AG-degrading enzymatic activity in rat cerebellar membranes corresponds to MGL or MGL-like enzyme and should facilitate further efforts to develop potent and more selective MGL inhibitors.

The Fourth Transmembrane Domain of the Helicobacter pylori Na+/H+ Antiporter NhaA Faces a Water-filled Channel Required for Ion Transport

Cysteine-scanning mutagenesis was performed from Ser-130 to Leu-160 in the fourth transmembrane domain (TM4) of the Na+/H+ antiporter NhaA from Helicobacter pylori to determine the topology of each residue and to identify functionally important residues. All of the mutants were based on cysteine-less NhaA (Cys-less NhaA), which functions very similarly to the wild-type protein, and were expressed at a level similar to Cys-less NhaA. Discontinuity of [14C]N-ethylmaleimide (NEM)-reactive residues suggested that TM4 comprises residues Gly-135 to Val-156. Even within TM4, NEM reactivity was high for I136C, D141C to A143C, L146C, M150C, and G153C to R155C. These residues are thought to be located on one side of the -helical structure of TM4 and to face a putative water-filled channel. Pretreatment of intact cells with membrane-impermeable maleimide did not inhibit [14C]NEM binding to the NEM-reactive residues within TM4, suggesting that the putative channel opens toward the cytoplasm. NEM reactivity of the A143C mutant was significantly inhibited by Li+. The T140C and D141C mutants showed lower affinity for Na+ and Li+ as transport substrates, but their maximal antiporter velocities (Vmax) were relatively unaffected. Whereas the I142C and F144C mutants completely lost their Li+/H+ antiporter activity, I142C had a lower Vmax for the Na+/H+ antiporter. F144C exhibited a markedly lower Vmax and a partially reduced affinity for Na+. These results suggest that Thr-140, Asp-141, and Phe-144 are located in the end portion of a putative water-filled channel and may provide the binding site for Na+, Li+, and/or H+. Furthermore, residues Ile-142 to Phe-144 may be important for the conformational change that accompanies ion transport in NhaA.

Binding of ligand or monoclonal antibody 4B1 induces discrete structural changes in the lactose permease of Escherichia coli.

By using Cys-scanning mutagenesis with site-directed sulfhydryl modification in situ [Frillingos, S., & Kaback, H. R. (1996) Biochemistry 35, 3950-3956], conformational changes induced by binding of ligand or monoclonal antibody (mAb) 4B1 in the lactose permease of Escherichia coli were studied. Out of 31 single-Cys replacement mutants in helices I, V, VII, VIII, X, or XI, 4B1 binding alters the reactivity of Val238-->Cys (helix VII), Val331-->Cys (helix X), or single-Cys355 (helix XI) permease with N-ethylmaleimide (NEM) in right-side-out membrane vesicles. In addition, site-directed fluorescence spectroscopy shows that mAb 4B1 binding causes position 331 (helix X) in the permease to experience a more hydrophobic environment. In contrast, ligand binding elicits more widespread changes, as evidenced by enhancement of the NEM reactivity of Ala244-->Cys, Thr248-->Cys (helix VII), Thr265-->Cys (helix VIII), Val315-->Cys (helix X), Gln359-->Cys, or Met362-->Cys (helix XI) permease, none of which are altered by 4B1 binding. Furthermore, no effect of 4B1 is observed on the reactivity of Cys148 (helix V), Val264-->Cys, Gly268-->Cys, or Asn272-->Cys (helix VIII), positions which probably make direct contact with substrate. With respect to the N-terminal half of the permease, 4B1 binding causes a small increase in the reactivity of mutants Pro28-->Cys or Pro31-->Cys (helix I), while ligand binding causes much greater increases in reactivity. The findings indicate that 4B1 binding induces a structural change in the permease that is much less widespread than that induced by ligand binding.

The role of helix VIII in the lactose permease of Escherichia coli: II. Site-directed sulfhydryl modification.

Cys-scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S. Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431-437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N-ethylmaleimide (NEM)-sensitive mutant is examined in the absence or presence of beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264-->Cys, Gly 268-->Cys, and Asn 272-->Cys, and alkylation of these single-Cys mutants in right-side-out membrane vesicles with [14C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265-->Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM-labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262-->Cys, Glu 269-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, or Ala 279-->Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand-induced conformational change.

Identification and Characterization of the N-Ethylmaleimide-sensitive Site in λ-Integrase

Integrase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and a type I topoisomerase. Upon modification with N-ethylmaleimide (NEM), a sulfhydryl-directed reagent, Int loses its capacity to bind "arm-type" DNA sequences and, consequently, to carry out recombination; however, its ability to bind "core-type" sequences and its topoisomerase activity are unaffected. In this report, the NEM-sensitive site was identified by modifying Int with [14C]NEM. Following cleavage by formic acid, which cleaves Asp-Pro bonds, and fractionation on a Fractogel HW-50 (F) sizing column, the fragment containing the primary site of [14C]NEM incorporation was subjected to amino acid sequencing. The results indicate that the primary site of [14C]NEM incorporation is in the peptide-spanning amino acid residues 1-28, which contains a cysteine at position 25. To confirm that Cys-25 is the target of NEM reactivity, site-directed mutagenesis was used to change this cysteine to alanine or serine. The mutant protein is not chemically modified by NEM and shows no loss of activity after NEM treatment. The fact that C25A and C25S both retain full recombination activity indicates that the SH group of Cys-25 does not provide any critical contacts, either with arm-type DNA or with other parts of the Int protein to form the arm-type recognition pocket. The loss of arm-type DNA binding and the concomitant loss of recombination function as a result of NEM modification must be due to the presence of the maleimide moiety and not due to loss of a critical cysteine contact.

Dynamics of Lactose Permease of Escherichia coli Determined by Site-Directed Chemical Labeling and Fluorescence Spectroscopy

Mutants with a single Cys residue in place of Phe27, Pro28, Phe29, Phe30, or Pro31 at the periplasmic end of putative transmembrane helix I were used to study the interaction of lactose permease with ligand by site-directed chemical modification or fluorescence spectroscopy. With permease embedded in the native membrane, mutant Phe27-->Cys or Phe28-->Cys is readily labeled with [14C]-N-ethylmaleimide (NEM), while mutant Phe29-->Cys, Phe30-->Cys, or Phe31-->Cys reacts less effectively. beta,D-Galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) has little or no effect on the reactivity of Phe27-->Cys, Phe29-->Cys, or Phe30-->Cys permease. Remarkably, however, Pro31-->Cys permease which is essentially unreactive in the absence of ligand becomes highly reactive in the presence of TDG. Ligand also enhances the NEM reactivity of the mutant with Cys in place of Pro28 which is presumably on the same face of helix I as position 31. The five single-Cys mutants which also contain a biotin acceptor domain in the middle cytoplasmic loop were purified by monomeric avidin-affinity chromatography in dodecyl beta,D-maltoside and subjected to site-directed fluorescence spectroscopy. Mutants Phe27-->Cys, Phe29-->Cys, and Phe30-->Cys react rapidly with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS), and reactivity is not altered in the presence of TDG. In striking contrast, mutants Pro28-->Cys and Pro31-->Cys react extremely slowly with MIANS in the absent of ligand, and TDG dramatically enhances reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate-induced acceleration of N-ethylmaleimide reaction with the Cys-65 mutant of the transposon Tn 10-encoded metal-tetracycline/H + antiporter depends on the interaction of Asp-66 with the substrate

We previously reported that the reaction of [ 14C] N-ethylmaleimide (NEM) with the S65C mutant of the transposon Tn10-encoded metal-tetracycline/H + antiporter (TetA(B)) is competitively inhibited by tetracycline [Yamaguchi, A. et al., FEBS Lett. 322 (1993) 201–204]. However, this observation has been revealed to be a mistake. The reaction of [ 14C]NEM with S65C TetA(B) was significantly and reproducibly accelerated by tetracycline, i.e. not inhibited. When Asp-66 was replaced by Ala, the reaction of NEM with the Cys-65 residue was no longer affected by tetracycline. In contrast, when Arg-70 was replaced by Ala, the acceleration of the reaction was unaltered. The tetracycline acceleration of the reaction to the Cys-65 residue was further stimulated with energization of the membrane on the addition of NADH. On the other hand, the tetracycline-induced acceleration was not observed in the absence of a divalent cation. These observations indicated that the Cys-65 locus is exposed to the medium according to the interaction of a divalent cationtetracycline chelation complex with Asp-66.

A structural change in the Neurospora plasma membrane [H+]ATPase induced by N-ethylmaleimide.

The reaction of N-ethylmaleimide (NEM) with Cys-532 of the Neurospora plasma membrane [H+]ATPase results in inhibition of ATP hydrolysis which is protected by MgADP (Pardo, J. P., and Slayman, C. W. (1989) J. Biol. Chem. 264, 9373-9379). To examine the conformational state of the ATPase upon NEM modification, we have used limited trypsinolysis and domain-specific antibodies. The NEM-reacted ATPase shows increased sensitivity to trypsin, particularly in the central hydrophilic region of the polypeptide thought to contain the ATP binding and phosphorylation sites. In addition, competitive enzyme-linked immunosorbent assays indicate that the C-terminal domain of the ATPase becomes more accessible to antibody binding while the N-terminal region becomes more protected. The NEM-induced structural change is accompanied by loss of the ability to form a phosphoenzyme intermediate. The change in tertiary conformation occurs specifically upon NEM reaction with Cys-532 since neither NEM modification of Cys-545 nor fluorescein 5'-isothiocyanate modification of Lys-474 alters the tryptic digestion pattern of the ATPase. Furthermore, modification of Cys-532 with the less bulky sulfhydryl reagent methyl methanethiosulfonate does not result in a detectable structural change or loss of enzymatic activity. Thus, the introduction of a relatively bulky maleimide group at Cys-532 has specific and far-reaching effects upon the structure and function of the ATPase.

Essential residues in lysolecithin:lysolecithin acyltransferase from rabbit lung: Assessment by chemical modification

The inhibition of lysolecithin:lysolecithin acyltransferase by several specific reagents was studied. Diisopropyl fluorophosphate (DFP) completely inhibited both activities at a concentration of 4 m m. Activity was not protected by substrate and the enzyme showed a change in circular dichroism spectrum upon treatment with inhibitor. Phenylmethanesulfonyl fluoride, another serine-specific reagent, did not inhibit either hydrolysis or transacylation. Therefore, we suggest that DFP does not modify an active serine in the catalytic site. p-Hydroxymercury benzoate and N-ethylmaleimide (NEM) abolished both activities of the enzyme. The presence of substrate partially protected against inactivation. Far-uv CD spectrum of NEM-modified enzyme revealed no changes in protein structure. The existence of two classes of essential cysteine residues was deduced from kinetics of NEM inactivation. Both classes differ in NEM reactivity and also in their participation in the catalytic mechanism. A tyrosine-specific reagent, tetranitromethane, also inhibited hydrolysis and transacylation, following first-order kinetics. The partial protection by substrate suggested the possible existence of essential tyrosines near the active site. At pH 5.0 N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline inactivated hydrolysis but not transacylation. However, both of them remained unchanged at pH 6.5. The substrate prevented the loss of hydrolytic ability. Therefore, a carboxyl residue participating just in the catalytic mechanism of hydrolysis is proposed.

Some properties of diastereomers formed in the reactions of N-ethylmaleimide with biological thiols.

Some properties of diastereomers formed in the reactions of N-ethylmaleimide with biological thiols.

The reactivity of the sulphydryl groups of chorismate mutase/prephenate dehydratase - a bifunctional enzyme of phynylalanine biosynthesis in Escherichia coli K12

The reaction of N-ethylmaleimide with chorismate mutase/prephenate dehydratase (chorismate pyruvatemutase/prephenate hydrolyase (decarboxylating) EC 5.4.99.5/EC 4.2.1.51) from Escherichia coli K12, which leads to the preferential inactivation of the prephenate dehydratase activity (Gething, M-J.H. and Davidson B.E. (1977) Eur. J. Biochem. 78, 111–117), was found to involve only the sulphydryl groups of the enzyme. Determination of the reactivities of the four different cysteine residues indicated that the reaction was not specific for a single residue, although two residues (Cys-216 and Cys-374) were more reactive than the others. The amount of inhibition of the prephenate dehydratase activity approximated in extent to the sum of the stoichiometries of the individual reactions of N-ethylmaleinide with these two cysteine residues. In the presence of either phenylpyruvate, the product of the prephenate dehydratase activity, or cis-aconitate, a competitive inhibitor with respect to prephenate, the prephenate dehydratase activity was substantially protected from inactivation. This protection was concomitant with a significant decline in the reactivities of both Cys-216 and Cys-374. These results are interpreted as indicating that both of these cysteine residues are at, or near to, the prephenate dehydratase active site and are possibly essential for the prephenate dehydratase activity of the enzyme.

14C]N-ethylmaleimide labeling of the plasma membrane [H+]-ATPase of Neurospora crassa.

Treatment of the purified plasma membrane [H+]-ATPase of Neurospora crassa with the sulfhydryl reagent N-ethylmaleimide (NEM) leads to a marked inhibition of ATPase activity. Several lines of evidence indicate that inhibition is caused by the reaction of NEM with a single cysteine residue on the Mr = 104,000 polypeptide. (1) Inhibition by NEM follows pseudo-first order kinetics. (2) MgADP protects against NEM inactivation and at the same time decreases the incorporation of [14C]NEM into the Mr = 104,000 polypeptide. When "protectable" [14C]NEM incorporation is plotted as a function of inhibition of ATPase activity, a linear relationship is observed with a slope of 0.8. (3) Labeling of the ATPase with [14C]NEM can be restricted to the protectable site by pretreatment with cold NEM in the presence of MgADP, followed by an incubation with [14C]NEM in the absence of nucleotides. When this is done, and the enzyme is subjected to tryptic mapping and autoradiography, a single radioactive peptide fragment is found. The protectable site may yield information about the role of cysteine in the ATPase mechanism, and should also serve as a useful point of reference in enzyme mapping studies.

Phosphate transport in rat liver mitochondria. Membrane components labeled by N-ethylmaleimide during inhibition of transport.

N-ethylmaleimide (NEM) inhibits the transport of phosphate in mitochondria but is without effect on permeation of other metabolities. In spite of its specificity for inhibition of phosphate transport, NEM reacts in an unspecific manner with inner membrane proteins in general. Treatment of mitochondria with [3H]NEM just sufficient to produce inhibition of phosphate transport results in labeling of at least 10 polypeptide components of the inner membrane. A marked increase in the specificity of reaction of NEM for components of the phosphate transport system is attained by first protecting the transport system with p-mercuribenzoate (p-MB) and then by irreversibly blocking reactive sulfhydryl groups unassociated with transport by the addition of unlabeled NEM. Subsequent addition of dithiothreitol removes p-MB and restores 65 to 75 percent of the original phosphate transport activity. Reinhibition of transport with [3H]NEM results in both a 6-fold decrease in the amount of [3H]NEM bound by purified inner membrane vesicles and a substantial reduction in the number of labeled polypeptide components. Five distinct labeled species are detected by this method, one of which is a 32,000 molecular weight protein containing 40 percent of the bound radioactivity, or approximately 160 pmol/mg of inner membrane protein. Correlation of binding of [3H]NEM by inner membrane proteins with inhibition of phosphate transport suggests that the maximum concentration of the NEM-sensitive component of the phosphate transport system is 60 pmol/mg of mitochondrial protein. This value, when combined with V-max of NEM-sensitive transport of 205 nmol times min-1 times mg-1 at O degrees (Coty, W. A., and Pedersen, P. L. (1974) J. Biol. Chem. 249, 2593) yields an approximate minimum turnover for this process of 3500 min-1 at 0 degrees. This turnover number is at least 20-fold greater than similarly calculated values for adenine nucleotide transport and succinate oxidation in rat liver mitochondria at this temperature. Taken together these results suggest that the NEM-sensitive phosphate transport system in rat liver mitochondria has an unusually high catalytic activity compared to other mitochondrial processes, and that at least one of the five NEM-binding proteins is likely to be an essential component of this transport system.

The reaction of N-ethylmaleimide at the active site of succinate dehydrogenase.

Since 1938 mammalian succinate dehydrogenase has been thought to contain thiol groups at the active site. This hypothesis was questioned recently, because irreversible inhibition by bromopyruvate and N-ethylmaleimide appeared not to satisfy the requisite criteria for reaction at the active site. These recent observations of incomplete inactivation of succinate dehydrogenase by N-ethylmaleimide and incomplete protection by substrates can, however, be explained adequately by the presence of oxalacetate and other strong competitors of the inactivation process in the enzyme used in these studies. Substrates, competitive inhibitors, and anions which activate succinate dehydrogenase protect the enzyme from inhibition by N-ethylmaleimide. Inhibition of succinate dehydrogenase by N-ethylmaleimide involves at least two second order reactions which are pH dependent, with pKa values of 8.0 to 8.2. This pH dependence, the known reactivity of N-ethylmaleimide toward thiols, and the protection by substrate and competitive inhibitors indicate that sulfhydryl residues are required for catalytic activity and perform an essential, not secondary, role in the catalysis. Just as the presence of tightly bound oxalacetate prevents inhibition by N-ethylmaleimide, alkylation of the sulfhydryl residue(s) at the active site prevents the binding of [14C]oxalacetate. Thus, these thiol groups at the active site also may be the site of tight binding of oxalacetate during the activation-deactivation cycle.

The reaction of N-ethylmaleimide with different forms of cytochrome oxidase.

The reaction of N-ethylmaleimide (NEM) with three different forms of beef heart cytochrome oxidase was studied. Whereas denatured oxidase binds 4–5 mol NEM per heme a, native oxidase and a new form of the oxidase prepared by extensive dialysis against water each binds 0.8–1.0 mol NEM per heme a. Oxidase in the form of coupled vesicles binds only half as much NEM as the native molecule. Six different polypeptides are associated with cytochrome oxidase. They have molecular weights of (A) 45 000; (B) 25 000; (C) 16 000; (D) 12 500; (E) 10 500; and (F) 8500. Four of the six different subunits appear to contain groups that bind NEM. In the native oxidase subunits A, B, D, and F react with NEM, subunit B being the most reactive, subunit F the least. Water-dialyzed oxidase binds NEM to subunits A, B, and F, the binding to D being greatly diminished. Vesicular oxidase binds NEM to only subunit B.

Radiation Sensitization Reactions of N-Ethylmaleimide with Model Compounds

The reaction of $e_{{\rm aq}}^{-}$ , OH radicals and H atoms with N-ethylmaleimide (NEM) and N-ethylmaleamic acid (NEMA, which is produced from the hydrolysis of NEM at pH > 7) were investigated in aqueous solutions. The reaction rate constants, the transient optical absorption spectra and the decay kinetics of the resulting intermediates were obtained. The free radical sensitization reactions of NEM with model compounds were followed by pulse radiolysis. A number of organic free radicals were found to react with NEM by an electron transfer process (oxidation of free radicals) to produce $\cdot {\rm NEM}^{-}$ , and/or by addition to NEM. NEMA, being a poorer oxidizing agent, does not appear to undergo most of these reactions. Electron adducts (e.g., radical anions of pyrimidine bases) were shown to transfer an electron to NEM. Solutes with redox potentials higher than that of NEM, such as menadione and oxygen, were found to be good electron acceptors from ...

Reaction of N-ethyl maleimide with the ribosomes of Escherichia coli

The ribosomal proteins which react when 70 s ribosomes are exposed to N-ethyl maleimide are identified. The reactivities of 30 s components change when the 70 s complex dissociates, while the reactivities of the 50 s components remain constant. The uptake of one 30 s component, 2b, parallels the N-ethyl maleimide-induced loss of ribosome activity. When ribosomes are exposed to iodoacetamide, only slight changes in incorporating activity are seen, even though 2b reacts extensively. However, iodoacetamide-treated ribosomes become insensitive to N-ethyl maleimide inactivation when the iodoacetamide reaction has proceeded to the point where 2b is no longer available for reaction. It is suggested that 2b is the N-ethyl maleimide-sensitive protein in the ribosome.

Influence of chemical modifications of the reactive SH groups on the proton binding behaviour of human and horse hemoglobin

From a proton-binding study of human HbCO with the reactive β93 SH groups substituted by iodoacetamide, we found that in this derivative two imidazole groups more appear to be titratable than in HbCO. In order to investigate the specificity of iodoacetamide in this respect, we have studied the influence of two other commonly used SH reagents, viz. N-ethylmaleimide and p-mercuribenzoate. 1. 1. Reaction of N-ethylmaleimide and p-mercuribenzoate with human HbCO does not lead to the unmasking of two imidazole groups. 2. 2. Upon blocking the SH groups with p-mercuribenzoate, two groups from the neutral region change their p K from 7 to 8. 3. 3. Horse HbCO behaves analogously to human HbCO. The p K SH however seems to be higher than in human HbCO.