Reactive Side Chains Research Articles

Subunit association and side-chain reactivities of bovine erythrocyte superoxide dismutase in denaturing solvents.

The copper- and zinc-containing superoxide dismutase of bovine erythrocytes retains its native molecular weight of 32 000 in 8.0 M urea for at least 72 h at 25 degrees C, as evidenced by sedimentation equilibrium analysis. Subsequent to prolonged exposure to urea, the dimeric enzyme could be dissociated by sodium dodecyl sulfate in the absence of reductants, indicating the absence of unnatural disulfide cross-links. The sulfhydryl group of cysteine-6 was unreactive toward 5,5'-dithiobis(2-nitrobenzoic acid) or bromoacetic acid in both neutral buffer and 8.0 M urea. The histidine residues of the enzyme were resistant to carboxymethylation in neutral buffer and 8.0 M urea. However, when the enzyme was exposed to bromoacetic acid in the presence of 6.0 M guanidinium chloride and 1 mM (ethylenedinitriol)tetraacetic acid (EDTA), both sulfhydryl and histidine alkylation were observed. Guanidinium chloride (6.0 M) increased the reactivity of the sulfhydryl group of cysteine-6 and allowed the oxidative formation of disulfide-bridged dimers. This was prevented by 1 mM EDTA. It follows that 8.0 M urea neither dissociates the native enzyme into subunits nor produces a conformation detectably different than that possessed under native conditions.

MINIMAL SIDE‐CHAIN PROTECTION CAN BE A SUCCESSFUL STRATEGY IN SOLID‐PHASE PEPTIDE SYNTHESIS

The N-terminal tyrosine residue of Met-enkephalin could be readily incorporated without protection of its phenolic hydroxylgroup. Furthermore, the HF-cleaved product contained fewer impurities than that derived from hydroxyl-protected material. Despite the presence of Tyr in the center of the chain, an LH-RH antagonist, [D-Phe2, D-Trp3, D-Phe6]-LH-RH, could also be made in normal yield by incorporation of free Boc-Tyr. Syntheses of the same model peptide without protection of the Ser residue and protection of the Arg residue as the guanidine HCl salt also gave excellent yields of analog. Finally, the LH-RH inhibitor and a highly active agonist, [D-Leu6, desGly-NH2(10)]-LH-RH ethylamide, were synthesized without protection of Tyr, Ser and Arg, which enabled free peptides to be generated directly by ammonolysis and ethylaminolysis, respectively, without HF treatment. In all examples, no evidence emerged to suggest reaction of side-chains during synthesis.

Model elastomeric networks prepared by selectively cross-linking polydimethylsiloxane chains having known amounts of reactive side chains

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Reactivity of amino groups in various complexes of the peptide chain elongation factor EF-Tu from Escherichia coli a new method of competitive labelling using reductive methylation

The protein modification technique of reductive methylation (Means & Feeney, 1968) was investigated on its merits for use in the method of competitive labelling (Kaplan et al., 1971) . It turned out to be a valuable extension of the method for an efficient analysis of changes in reactivity and p K values of amino groups due to conformational changes of the protein molecule. The new method was applied to each of the elongation factor complexes EF-Tu · GDP, EF-Tu · GTP and EF-Tu · GTP · Phe-tRNA in the presence of free Phe as an internal standard of reactivity. Complexes were first labelled with trace amounts of formaldehyde and tritiated sodium borohydride, amino groups being competitively methylated according to their reactivity. Thereafter, they were fully methylated using nonradioactive reagents. The preparations were mixed with standard amounts of fully [ 14C]methyl-labelled EF-Tu and subsequently subjected to fragmentation and peptide analysis. The final determination of 3H/ 14C ratios revealed a few remarkable changes in reactivity of lysine side-chains in EF · Tu · GTP · Phe-tRNA as compared with the corresponding ones in EF-Tu · GTP. On the other hand, no large difference in reactivity was found by comparing EF-Tu · GDP and EF-Tu · GTP.

Calcium-induced conformational changes in a cyanogen bromide fragment of troponin C that contains one of the binding sites.

The increase observed in alpha helix from 20 to 38%, the spectral red shift of the absorption bands of the side chain chromophores, the increase of phenylalanine side chain optical activity and a decrease of cysteinyl side chain reactivity with 5,5'-dithiobis(2-nitrobenzoic acid) are consistent with a coil to helix change in the segment containing Cys-98 and Phe-99 and 102 when the CB9 fragment of troponin C (TnC) with an intact binding site III binds Ca2+. Since similar spectrophotometric observations have been made on the whole molecule the data with CB9 confirm our previous suggestion that binding site III of TnC is one of the strong binding sites for Ca2+. The estimate of the binding constant of Ca2+ for CB9 is about 2 orders of magnitude lower than in whole TnC, indicating further stabilization in the intact molecule.

On the molecular conformation of human haemopexin: I. Reactivity of the tyrosine and tryptophan side chains

The reactivity of the aromatic side chains of Tyr and Trp in human haemopexin were studied by chemical modifications and analysis of spectrophotometric titration curves. It has turned out that: 1. 1. Under non-denaturing conditions the aromatic rings of Tyr resisted both acetylation and nitration. 2. 2. Three indole groups of Trp reacted with the Koshland agent, without the native conformation of the protein being markedly affected (CD spectra). 3. 3. Oxidation by N-bromosuccinimide split the peptide chain and the molecular conformation collapsed. 4. 4. The Tyr residues could be placed into three classes, according to their p K values: 2 (or 1 in the haem·haemopexin complex) were normally accessible to titration, 5 were masked and the remaining 7 (or 8) were buried. 5. 5. The spectrophotometric titration curve could not be analysed in terms of the Linderstrøm-Lang equation. The findings 1 to 3 refer to both haemopexin and its complex with haem; the spectrophotometric titration curves of the two molecules are very similar too. Consequently, the binding of haem is not associated with a profound alteration of the molecular architecture. The generally low reactivity of the side chains studied indicates that the hydrophobic peptide core of this glycoprotein is a compact one, very restricted in its contacts with the environment.

Side Reactions in Peptide Synthesis: tert‐Butylation of Tryptophan

The amino acid tryptophan contains a reactive side chain which can be attacked, e.g. on tert-butylation of peptides containing tryptophan residues. Model reactions showed (1) to be the main product.

Photochemical coupling of enzymes to mammalian cells

Summary A thermochemical-photochemical bifunctional reagent has been prepared and used for the stepwise coupling of catalase to Chinese hamster ovary cells, to polystyrene beads, and to glucose oxidase. N-(4-Azido-2-nitrophenyl)-4-aminobutryl N'-oxysuccinimide was reacted in the dark with catalase to prepare a catalytically active enzyme derivative containing dark-stable photoreactive side chains. This enzyme derivative was then photocoupled with visible light to tissue culture cells to yield predominantly intact, viable cells “armed” with an average activity equivalent of ∼ 106 enzyme molecules per cell. The photoactivated enzyme derivative containing highly reactive aryl nitrene side chains was coupled under similar reaction conditions to uniform spherical polystyrene latex beads and, simultaneously, to native glucose oxidase.

19] Peptide aldehydes: Potent inhibitors of serine and cysteine proteases

Publisher Summary The peptide acids generated by protease-catalyzed hydrolysis of proteins are generally good inhibitors of the enzymes that produce them. In the past few years it has become clear that derivatives of peptide acids in which the carboxylic acid group has been replaced by an aldehyde group are even more potent inhibitors of certain proteases than are the original acids. This chapter explains the procedure used successfully to synthesize peptides containing phenylalaninal (Pheal), alaninal (Alaal), or glycinal (Glyal). It should be applicable to the preparation of peptides containing other a-amino aldehydes, including those with reactive side chains, provided that suitable acid-labile protecting groups are available for those side chains. For a list of acid-labile protecting groups for amino acids, the reader should consult a text or recent review of the synthesis of peptides, for example, “Peptide Synthesis.”

A structural study of pig liver glyceraldehyde-3-phosphate dehydrogenase

A substantial portion of the primary structure of pig liver glyceraldehyde-3-phosphate dehydrogenase has been investigated and the results compared with those previously reported for the pig muscle enzyme. Liver and muscle glyceraldehyde-3-phosphate dehydrogenases show the same amino acid content, and the first N-terminal 13 residues occur in the same sequence. No differences in N-terminal residues and amino acid composition have been evidenced by analysis of several tryptic peptides, which account for about 50% of the total amino acid sequence. From the electrophoretic mobilities of peptides T 8 T 9 and T 25 it is concluded that residues Asp 60, Asp 67 and Glu 220 in the reported sequence of the pig muscle enzyme must be present as amides in the liver enzyme. The NAD + content was found to be 2 mol per tetramer, while higher values have been reported for the muscle enzyme from various mammalian sources. The reactivity of lysyl side chains towards pyridoxal 5′-phosphate has been examined: the results indicate that Lys 212 is the main site reacted in fully inactivated pig liver holoenzyme. A similar result has been found for rabbit muscle apoenzyme, whereas rabbit muscle holoenzyme reacts at Lys 212 and 191.

38] Immobilized proteins in single crystals

Publisher Summary This chapter describes the technique of immobilizing single protein crystals, presents data on the properties of such crystals, and discusses the uses of insolubilized protein crystals in X-ray diffraction analysis and in studies of the physicochemical properties of enzymes in the solid state. It is suggested that insolubilization of protein crystals could be easily accomplished because the molecules within a crystal are ordered in three dimensions and any reactive amino acid side chain between molecules, which would be few, would be effectively juxtaposed for cross-linking with bifunctional reagents. X-ray structure analysis of a number of proteins indicates that only a few polar side chains (i.e., lysine) at the surface of protein molecule would have any appreciable flexibility in the crystalline state. Crystallographic analysis of glutaraldehyde-treated carboxypeptidase Aα-crystals indicates that intermolecular cross-links are most likely found between lysine residues of different molecule and are few in number.

The Covalent Reactions of Primary Amino Side‐chains and Disulphide Bonds in Wool Keratin

Previous work on the reactions of different types of reactive dyes with wool is reassessed and new jindings presented. The decrease in reactivity of basic wool side chains to reaction with 1:fluoro:2:4 dinitrobenzene after wool has been dyed with acidic or reactive dyes is further investigated. New and previous correction factors measuring the extent of this inactivation are compared. Using such corrections, the extent of actual covalent binding of reactive dyes to basic wool side chains is determined. Applying these corrections gives reasonable stoichiometry in the dye‐fibre reaction. The extent of reaction of thiols, basic and hydroxyl groups in wool with five reactive dyes of different classes has been measured, and compared for two dyeing methods (long‐liquor and urea pad‐batch). The differences and similarities in the extent and type of reaction of the dyes with the fibre sites, using the two methods of application, is discussed. The role of disulphide degradation in reactive dyeing is further investigated. The effect of alkaline treatments on the fixation of reactive dyes, and the availability of wool lysine side‐chains for reaction (in the urea pad‐batch dyeing system) is reported upon.

The origin of proteins: Heteropolypeptides from hydrogen cyanide and water

Evidence from laboratory and extraterrestrial chemistry is presented consistent with the hypothesis that the original heteropolypeptides on Earth were synthesized spontaneously from hydrogen cyanide and water without the intervening formation of chi-amino acids, a key step being the direct polymerization of atmospheric hydrogen cyanide to polyaminomalononitrile (IV) via dimeric HCN. Molecular orbital calculations (INDO) show that the most probable structure for (HCN)2 is azacyclopropenylidenimine. Successive reactions of hydrogen cyanide with the reactive nitrile side chains of IV then yield heteropolyamidines which are converted by water to heteropolypeptides. To study this postulated modification of a homopolymer to a heteropolymer, poly-chi-cyanoglycine (IX) was prepared from the N-carboxyanhydride of chi-cyanoglycine. Hydrolysis of IX, a polyamide analog of the polyamidine IV, yielded glycine. However, when IX was hydrolysed after being treated with hydrogen cyanide, other chi-amino acids were also obtained including alanine, serine, aspartic acid and glutamic acid, suggesting that the nitrile groups of IX (and therfore of IV) are indeed readily attacked by hydrogen cyanide as predicted. Further theoretical and experimental studies support the view that hydrogen cyanide polymerization along these lines is a universal process that accounts not only for the past formation of primitive proteins on Earth, but also for the yellow-brown-orange colors of Jupiter today and for the presence of water-soluble compounds hydrolyzable to chi-amino acids in materials obtained from environments as diverse as the moon, carbonaceous chondrites and the reaction chambers used to simulate organic synthesis in planetary atmospheres.

Accumulation of a sterol intermediate during reaction in the presence of homocysteine with cell-free extract of yeast

D,L-Homocysteine, at the concentration of 10 mM, inhibited the methylation reaction of sterol side chain in cell-free extract of yeast, but did not inhibit 14C-incorporation from [ 14C]mevalonate into nonsaponifiable lipids. Under this condition, a radioactive C 27-sterol was accumulated. Examination by gas-chromatography on a DEGS column, partial hydrogenation, side chain cleavage, and by methylation with crude methyl transferase preparation, suggested the accumulated sterol to be 5α-cholesta-7, 24-diene-3 β-ol. The possible role of this sterol as a natural acceptor of the methyl group in ergosterol biosynthesis of yeast was discussed.

Epoxides of carcinogenic polycyclic hydrocarbons are frameshift mutagens.

K-region epoxides of the carcinogens benz[a] anthracene, dibenz[a,h]anthracene, and 7-methylbenz[a] anthracene are mutagenic in strains of Salmonella typhimurium designed to detect frameshift mutagens. Parent hydrocarbons, K-region diols and phenols and some other epoxides are inactive as mutagens in these tests. Polycyclic hydrocarbon epoxides, and other presumed proximal carcinogens, are discussed as examples of intercalating agents with reactive side chains. It has been shown previously that intercalating agents with reactive side chains are potent frameshift mutagens.

N-acylsuccinimides as acylating agents for proteins: the selective acylation of lysine residues.

The suitability of N‐acylsuccinimides as reagents for the selective acylation of reactive side chains in proteins has been examined. A detailed study of the reaction of N‐acetylsuccinimide with ribonuclease and bovine serum albumin showed that acetylation occurred in the pH range 3 to 8. Lysine side chains were acetylated selectively, with little or no modification of tyrosine side chains. There was no evidence for acetylation of serine, threonine or histidine side chains. The analogous N‐propionyl, N‐n‐butyryl, N‐i‐butyryl and N‐n‐valerylsuccinimides also selectively acylate lysine side chains in proteins. This series of reagents, capable of changing the charge of lysine side chains and adding acyl groups of increasing hydrocarbon chain length, should prove a useful tool in studies of protein structure.

On the theory of the effects of ortho substituents on the side-chain reactivity of benzene derivatives

On the theory of the effects of ortho substituents on the side-chain reactivity of benzene derivatives

Membrane structure: The reactivity of tryptophan, tyrosine and lysine in proteins of the microsomal membrane

1. 1. A microsomal subfraction devoid of adsorbed soluble proteins has been prepared from rat liver by density gradient centrifugation. Through the use of group-specific reagents the reactivity of tryptophanyl, tyrosyl, and lysyl side chains of the membrane has been examined. Significant differences are noted when the results are compared to literature values for non-membrane proteins. 2. 2. In the native microsomal membrane, tryptophan was unreactive toward N- bromosuccinimide . The results suggest that strong tryptophan interactions may exist which could likely have an important role in the maintenance of normal membrane structure. Furthermore, tryptophan appears to be involved at different levels of structural organization since extremely sharp transitions in reactivity were noted when sodium dodecyl sulfate was used as a perturbant. 3. 3. 85% of the tyrosyl residues occupied an exposed position on the membrane surface and were characterized by their ease of nitration in the absence of denaturing agents. The unusual accessibility of tyrosine suggests that side chain interactions involving tyrosyl residues do not play a prominent structural role. 4. 4. Carbamylation studies revealed that only 56% of the lysyl residues in the native membrane were available for reaction with cyanate while an additional 20–25% became reactive in the presence of urea. The remaining lysyl residues were unreactive under a wide variety of denaturing conditions. 5. 5. Sodium dodecyl sulfate interacted with the membrane in such a way as to markedly decrease the apparent accessibility of tyrosyl and lysyl residues while substantially increasing the reactivity of tryptophan.

Inter- and Intramolecular Interactions of α-Lactalbumin: X. EFFECT OF ACYLATION OF TYROSYL AND LYSYL SIDE CHAINS ON MOLECULAR CONFORMATIONS

Abstract Of the 4 tyrosyl residues of bovine α-lactalbumin, 2 were found to be quite reactive with N-acetylimidazole; the third group is less so, and the fourth is only weakly reactive. Acylation of the two most reactive groups results in a reversible increase in the quantum yield of a tryptophan residue or residues which emit maximally at 350 mµ. At relatively high concentrations of reagent, in addition to acylation of tyrosyl residues, six amino groups react giving rise to quenching of the fluorescence of tryptophan emitting maximally below 320 mµ and enhancement of fluorescence from groups which emit maximally at 350 mµ. Thus, acylation of the two tyrosyl groups perturbs tryptophan residues which are freely in contact with the medium, whereas acylation of amino groups, by contrast, appears to involve tryptophan residues which are not freely in contact with the solvent. Changes in circular dichroism were observed on acylation of bovine α-lactalbumin in the 250 to 300 mµ side chain band system, in the 208 mµ band, and in the 212 to 230 mµ region. These circular dichroism changes, which depend in a complex way on the nature of the groups acylated, appear to be due primarily to alteration of the environment of the side chains in the protein. Changes in fluorescence and circular dichroism properties observed on acylation of amino groups were shown to be similar to those observed on acid denaturation. It is likely that those processes, as well as alkaline denaturation, bring about essentially the same local conformational change which gives rise to increased freedom of rotation of tyrosyl and tryptophyl side chains. The pH dependence of the conformational transitions, as well as the chemical reactivity of side chains, suggests that breaking of charge pairs (amino and carboxylic acid groups) may initiate the structural change. Inspection of a recently proposed model for bovine α-lactalbumin indicates the proximity of a number of lysyl, glutamyl, and aspartyl side chains, in accord with the above hypothesis. The model further indicates that Tyr-36 and Trp-118, and Tyr-103 and Trp-104 are sufficiently close, respectively, to explain the changes in fluorescence on acylation of tyrosyl groups. The environments of Trp-60 and perhaps Trp-104, located in a crevice-like region of the molecule, are such that they may be the low wave length emitting groups normalized on acylation of amino groups.

Active-site-directed phenacyl halides inhibitory to trypsin

p-Amidino-phenacyl bromide (I) and p-guanidino-phenacyl bromide (II) have been synthesized in the expectation that they would act as site-specific inactivators of trypsin. Like TLCK 3 3 Abbreviations used: APB, p-amidinophenacyl bromide; BAPA, dl-benzoylarginine p-nitroanilide; GPB, p-guanidinophenacyl bromide; GPC, p-guanidinophenacyl chloride; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone. they were found to form a substrate-like complex with trypsin leading to irreversible inactivation of the enzyme in a subsequent, stoichiometric alkylation step. However, histidine was not involved. Instead, it was concluded that p-guanidino-phenacyl bromide (II) formed an ether linkage with the active center serine. Since the hydrolytic region of the active center contains two nucleophilic groups, Ser-183 and His-46, the exclusive alkylation of one or the other by reagents of the same chemical class, i.e., halo-ketones, cannot reflect the relative reactivity of these amino acid side chains in trypsin, since the probable ranges of their p K values are greatly diverse. It is concluded that the geometry within the complex formed by trypsin with substrate-like halo-ketones results in small differences that determine which of two nearby side-chains become modified. This is in contrast to chemical modification by ordinary alkylating agents for which reactivity and accessibility are the determining features of the protein structure under modification.