Introduction of sulfhydryl groups into proteins at carboxyl sites

A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. Then the disulfide bond is reduced with dithiothreitol, yielding the amide of 2-aminoethanethiol. This procedure was used to incorporate sulfhydryl groups into carbonic anhydrase and adrenocorticotropic hormone. The effect of carbodiimide concentration and pH of the coupling reaction on stoichiometry of sulfhydryl group incorporation was examined. The method was used to prepare bovine carbonic anhydrase containing up to nine sulfhydryl groups per molecule with no loss of enzymatic activity and biologically active adrenocorticotropic hormone containing one sulfhydryl group per molecule.     

Synthesis of some long-chain acylamidoalkyl glucosides

4-O-β-D-Galactopyranosyl-α,β-D-glucopyranosylamine (lactosylamine), β-D-gluco-, α- and β-D-manno-pyranosylamines were bound to the carbodiimide-activated groups of lysozyme. Of the 11 free carboxyl groups of the protein, ≈3 were substituted by α,β-6-lactosylamine, and ≈2 by the monohexo-sylamines. One of the 4 glycopeptides isolated from the tryptic digest of the lysozyme-lactosylamine conjugate was identical to synthetic l-N-L-leucinoyl-4-O-β-D-galactopyranosyl-β-D-glucopyranosylamine, indicating the substitution of the carboxyl group of the C-terminal leucine residue. The isolation of a glycopeptide containing the aspartic acid residue in position 117 indicates that the second α,β-lactosylamine residue is linked to the carboxyl group of this amino acid. Both of the 2 other glycopeptides contain the same free carboxyl groups (one glutamic and two aspartic acid residues in positions 35, 48, and 52, respectively). The third α,β-lactosylamine residue seems to be linked to one of these carboxyl groups.     

A new approach for detecting C‐terminal amidation of proteins and peptides by mass spectrometry in conjunction with chemical derivatization

We describe a mass spectrometric method for distinguishing between free and modified forms of the C‐terminal carboxyl group of peptides and proteins, in combination with chemical approaches for the isolation of C‐terminal peptides and site‐specific derivatization of the C‐terminal carboxyl group. This method could most advantageously be exploited to discriminate between peptides having C‐terminal carboxyl groups in the free (COOH) and amide (CONH₂) forms by increasing their mass difference from 1 to 14 Da by selectively converting the free carboxyl group into methylamide (CONHCH₃). This method has been proven to be applicable to peptides containing aspartic and glutamic acids, because all the carboxyl groups except the C‐terminal one are inert to derivatization, according to oxazolone chemistry. The efficiency of the method is illustrated by a comparison of the peaks of processed peptides obtained from a mixture of adrenomedullin, calcitonin, and BSA. Among these components of the mixture, only the C‐terminal peptide of BSA exhibited the mass shift of 13 Da upon treatment, eventually unambiguously validating the C‐terminal amide structures of adrenomedullin and calcitonin. The possibility of extending this method for the analysis of C‐terminal PTMs is also discussed.     

Chemical modification of horseradish peroxidase. Preparation and characterization of tracer enzymes with different isoelectric points.

Horseradish peroxidase (HRP), a plant glycoprotein with a molecular weight of 40,000 D and a molecular radius (ae) of 30 A, has been modified chemically to prepare tracer molecules with different molecular charge. Modification of free carboxyl groups on the enzyme is achieved by carbodiimide activation and subsequent reaction of activated carboxyl groups with a nucleophile; uncharged groups or radicals containing additional positively charged moieties are introduced into the protein molecule resulting in an increased net positive charge of the tracer. Amino groups in the protein molecule are modified by acetylation or succinylation; this reaction will increase the net negative charge of the enzyme by either introducing an uncharged group or an additional carboxyl radical. The tracer molecules so obtained are then characterized in terms of molecular size and charge by column chromatography and isoelectric focusing respectively. The enzymatic activity as measured by 3,3'-diaminobenzidine reaction, the pH optimum and the absorption spectra for the modified enzymes remain virtually unchanged.     

The role of the carboxyl and amino groups of polyene macrolides in their interactions with sterols and their selective toxicity. A P-NMR study

The permeability induced by amphotericin B and vacidin A derivatives in large unilamellar lipidic vesicles containing various sterols has been studied using the proton-cation exchange method and ³¹P-NMR spectroscopy. Derivatives which have a free ionizable carboxyl group induce biphasic ‘all or none’ permeability typical of channel-forming ionophores, whatever the sterol present. In sterol-free membranes, they have no significant activity. Derivatives which lack a free ionizable carboxyl group exhibit this channel-like mode of action only in membranes containing ergosterol or sterols with an alkyl side like that of ergosterol. In membranes containing cholesterol or sterol whose side-chain is alike, a slow and progressive permeability is observed at high concentrations. This activity is observed in sterol-free membranes as well. Derivatives containing sugars with substituted amino groups always have lower ionophoric activity than those which are unsubstituted. The greatest decrease in activity was observed for N-acetyl derivatives. Substitution of the amino groups has no effect on the mode of action. A model of interaction of polyenes with sterols is presented accounting for the data obtained on vesicles and the observed selective toxicity of polyene derivatives in biological membranes.     

Potentiometric studies of hydrophobic effect on ion binding of ionic dextran derivatives

In order to clarify the effect of degree of substitution of ionic and hydrophobic group on the polyelectrolytic behavior of polysaccharides, potentiometric titration and activity measurement of counterions were made for carboxymethyldextran (Cm-dextran) having various degrees of substituted carboxyl group and for carboxymethylbenzyldextran (Cm-Bzl-dextran) containing various degrees of substituted benzyl group. From the shape of titration curve, no conformational change was observed for both Cm-dextran and Cm-Bzl-dextran. The pK₀ value of Cm-dextran was independent of the degree of the degree of substituted carboxy group. However, the pK₀ of Cm-Bzl-dextran increased with an increasing degree of substituted benzyl group. The suppression of dissociation of a carboxyl group, caused by the surrounding hydrophobic groups, was discussed mainly in terms of the change of water structure around such groups. From the results of activity measurement for counterions of these dextran derivatives, we proposed the possibility of ion selectivity based on the hydrophobicity.     

Inhibition of the alternative pathway of human complement by structural analogues of sialic acid

Liposomes were used to determine whether gangliosides containing certain structurally defined analogues of sialic acid could inhibit activation of the alternative pathway of human C. Gangliosides containing sialic acid residues with modifications in the N-acetyl group, carboxyl group, or polyhydroxylated tail were either isolated from natural sources or prepared by chemical modification of the native sialic acid structure. Sialic acid lost more than 90% of its inhibitory activity after removal of just the C9 carbon from the polyhydroxylated tail. Sialic acid was also unable to inhibit activation after converting the carboxyl group to a hydroxymethyl group. Galactose oxidase/NaB3H4 treatment of liposomes containing gangliosides with native or modified sialic acid residues confirmed that neither modification altered the amount of gangliosides exposed at the liposome surface. Changing the N-linked acetyl group to a glycolyl group had no effect on the inhibitory activity of sialic acid. These data further define the structural features of sialic acid that are important in regulation of alternative pathway activation. Both the C9 carbon of the polyhydroxylated tail and the carboxyl group are essential for this function; whereas, the N-linked acetyl group may be modified without loss of activity.     

The essential activated carboxyl group of inorganic pyrophosphatase.

1. A carboxyl group of high reactivity has been found in inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from yeast. This group interacts with agents which react neither with carboxyl groups of low molecular weight compounds nor with other carboxyl groups of the protein. 2. The reaction of this activated carboxyl group with inorganic phosphate, hydroxylamine, N-methyl- and O-methylhydroxylamines, and glycine methyl ester has been studied. 3. Homoserine and homoserine lactone were found in the hydrolyzate of phosphorylated and NaBH4-reduced pyrophosphatase, indicating that an aspartyl residue is phosphorylated. 4. Hydroxylamine and other nucleophilic agents cause inactivation of pyrophosphatase as a result of interaction with a carboxyl group. Both diaminobutyric and diaminopropionic acids were seen in the acid hydrolyzate of the protein treated with hydroxylamine and subjected to rearrangement in the presence of carbodiimide. 5. The ways in which the activation of a carboxyl group in the enzyme is achieved and the presumed mechanism of action of inorganic pyrophosphatase are discussed.     

Role of charged groups in factor XI/XIa activity

To elucidate the role of charged groups in expression of factor XI coagulant activity, the charged groups of purified human blood coagulation factor XI/XIa containing 125I-XI/XIa were derivatized: free amino groups by succinylation, guanido groups of arginine by reaction with phenylglyoxal hydrate, and free carboxyl groups by reaction with ethylenediamine. The modified proteins were tested for: 1) ability to adsorb to glass, 2) ability to be cleaved by trypsin or factor XII-high molecular weight kininogen, 3) coagulant activity. The amino group-modified factor XI had a significantly decreased ability to bind to glass; modification of arginine or carboxyl groups did not affect adsorption. Trypsin cleaved factor XI with modified free amino, guanido, or carboxyl groups. Factor XII-high molecular weight kininogen could cleave only the arginine-modified factor XI. Amino group-modified factor XI and carboxyl group-modified factor XI lost all their factor XI assay activity, whereas arginine-modified factor XI retained 50% of the original activity. Amino group-modified factor XI could not be activated by trypsin, but arginine-modified and carboxyl group-modified factor XI could be activated by trypsin to 50% of the original activity. Succinylation of the amino groups of factor XIa destroyed all its factor XIa activity. Arginine-modified and carboxyl group-modified factor XIa retained 50% of their factor XIa activity. We conclude that epsilon-amino groups are essential for adsorption; activation by factor XII-high molecular weight kininogen requires free amino and carboxyl but not guanido groups; free amino, carboxyl, and guanido groups in factor XIa all appear to be critical for interaction of factor XIa with factor IX.     

The phenylic hydroxyl group is essential for the induction of stress response by sodium salicylate

We have shown that sodium salicylate (SA) activates the heat shock promoter and induces the expression of heat shock proteins (Hsps) with a concomitant increase in the thermotolerance of cells. To identify the functional groups of SA necessary for the induction of Hsps, we evaluated the effect of various derivatives of SA using a mammalian cell line containing a reporter gene downstream of an hsp105 promoter. Among the derivatives, the compounds in which the carboxyl group of SA was substituted activated the hsp105 promoter at 37 degrees C as SA did, but the compounds in which the hydroxyl group was substituted did not. Thus, the phenylic hydroxyl group but not the carboxyl group of SA seemed to be necessary for a stress-induced response. In addition, the orientation of two functional groups on the benzene ring of SA derivatives was also important for the induction of a response. Among these compounds, salicylalcohol which strongly induced the expression of Hsps suppressed the protein aggregation and apoptosis caused by an expanded polyglutamine tract in a cellular model of polyglutamine disease. These findings may aid in the development of novel effective Hsp-inducers.     

Structural, dynamic, and metal-ion binding studies of the core glycopeptides beta-D-Gal-(1----3)-alpha-D-GalNAc----Ser, Thr by 13C-N.m.r. spectroscopy

13C-N.m.r. spectral data as well as spin-lattice relaxation times (T1 values) are presented for the core glycopeptides beta-D-Gal-(1----3)-alpha-D-GalNAc----Ser, Thr. The binding of Gd3+ to these model compounds containing N-terminal blocking groups and esterified carboxyl groups indicates that the disaccharide contains a rather weak, but unique, binding-site in the vicinity of C-2 of alpha-D-GalNAc (possibly involving N-2', the acetamido carbonyl group, O-3' and/or possibly the glycosidic oxygen atom (O-3)).     

The use of cycloamylose to probe the “charge-relay” system

The effect of the combination of imidazolyl and carboxyl groups on the cleavage of m-t-butylphenyl acetate in the presence of α-cyclodextrin was examined to shed light on the role of the “charge-relay” system in serine esterases. 2-Benzimidazole-acetic acid, which has both the imidazolyl and carboxyl groups in the same molecule, accelerates the cleavage of m-t-butylphenyl acetate in the presence of α-cyclodextrin. On the other hand, neither benzimidazole (which has only an imidazolyl group) nor 2-naphthaleneacetic acid (which has only a carboxyl group) exhibited measurable acceleration. The cleavage of m-t-butylphenyl acetate by the α-cyclodextrin-2-benzimidazolecetic acid system takes place through inclusion complex formation between m-t-butylphenyl acetate and α-cyclodextrin, followed by catalysis associated with the combination of the carboxyl anion, the neutral imidazolyl group, and the alkoxide anion. The most probable explanation for the combination of the three groups in the catalysis involves nucleophilic attack by the imidazolyl group, assisted by the carboxyl and alkoxide anions. The mechanism of the combination of the imidazolyl, carboxyl, and hydroxyl groups is apparently different from those shown by the “charge-relay” system in enzymatic reactions.     

Effect of modification of heme propionate groups on the reactivity of horseradish peroxidase

Artificial horseradish peroxidases were prepared containing hemin in which propionate groups at the 6,7-positions were modified. All of the unnatural molecules had the chemical and enzymic properties of the native enzymes but not to the same extent. This finding eliminates the possibility that a propionate group in the 6- or 7-position of the hemin plays a catalytic role in compound I formation. The main effect of modifications at the positions of heme carboxyl groups may be caused by changes in the electric charge at the periphery of the hemin.     

Synthesis and structural analysis of a series of d-glucose derivatives as low molecular weight gelators

Low molecular weight gelators are an interesting new type of compounds that are important in supramolecular chemistry and advanced materials. Previously, we had synthesized several acyl derivatives of methyl 4,6-O-benzylidene-α-d-glucopyranoside and found that a number of terminal acetylene-containing esters are good gelators. To understand the structure requirement of the acyl chains, we synthesized a series of analogs containing different functional groups including aryl, alkenyl, and halogen derivatives. X-ray crystal structures of a monoester and a diester derivative were also obtained to help understand the relationship between structure and gelation. For good gelation properties, the carboxyl derivatives should possess alkyl groups containing a terminal acetylene group and aryl derivatives.     

Novel substrates for angiotensin I converting enzyme

Homogenous human angiotensin converting enzyme (EC 3.4.15.1) cleaves dipeptides from the C-terminus of substrates containing a free carboxyl group. In this study we demonstrate that peptides containing a C-terminal nitrobenzylamine are also cleaved by the enzyme. The hydrolysis of these substrates is inhibited by the specific converting enzyme inhibitors captopril and MK421 as well as by anti-converting enzyme antibody. Sodium chloride accelerates the rate of hydrolysis forty-fold. The product of the reaction, an amino acid nitrobenzylamide, was identified by thin layer chromatography and high performance liquid chromatography. These results suggest that the carboxyl group is not an absolute requirement for substrate hydrolysis.     

Sbustrate specificity of the elastase and the chymotrypsin-like enzyme of the human granulocyte.

Human granulocyte elastase (EC 3.4.21.11) differs from hog pancreatic elastase in its specificity for synthetic substrates. Although hydrolyzing peptide bonds adjacent to the carboxyl group of alanine, the granulocyte enzyme prefers valine at the cleaved bond, in contrast to the pancreatic enzyme which prefers alanine. Peptide bonds involving the carboxyl group of isoleucine can be hydrolyzed by the granulocyte enzyme but are not hydrolyzed to any significant extent extent by pancreatic elastase. This difference in specificty could explain the lower sensitivity of the granulocyte enzyme to inhibitors containing alanine analogs, such as the peptide chloromethyl ketones and elastatinal. The human granulocyte chymotrypsin-like enzyme differs from pancreatic chymotrypsin by being able to cleave substrates containing leucine in addition to those containing the aromatic amino acids.     

Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues

Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.     

Polyene--sterol interaction and selective toxicity

From permeability experiments carried out with series of amphotericin B derivatives in both biological and model membranes, it was concluded that derivatives, whose carboxyl group at the C18 position is blocked by substitution, are much more efficient at inducing permeability in ergosterol-containing than in cholesterol-containing membranes, whereas derivatives whose carboxyl group is free and ionizable are equally efficient in both membranes types. Binding measurements on erythrocyte membranes showed that all amphotericin B derivatives simply partition between membrane lipids and aqueous medium, according to their lipid solubility. There is no relationship between binding and efficiency in inducing permeability. Permeability studies carried out on lipidic vesicles containing various sterols showed that: 1) derivatives having their carboxyl free induced permeability of the 'channel' type, regardless of the sterol present, and no detectable permeability in sterol-free membranes; 2) derivatives whose carboxyl group is blocked induce channels only in membranes containing ergosterol or sterols having an alkyl side chain identical to that of ergosterol. In the presence of other sterols or in sterol-free membranes, their ionophoric activity is poor and always of the 'mobile-carrier' type. A model of polyene-sterol interaction is proposed, accounting for the data obtained with both biological and model membranes.     

Preparation, properties and antileukemic activity of arabinosylcytosine polysaccharide conjugates

1. Conjugates of 1-beta-D-arabinofuranosylcytosine (araC) with polysaccharides containing carboxyl groups, such as polygalacturonic acid (PGA) and carboxymethylated yeast beta-D-glucan (CMG) were prepared. 2. Activation of the polysaccharidic carboxyl group by isobutylchloroformiate and formation of a peptide bond via 4-NH2 group of araC was used for a coupling reaction. 3. Elementary analysis, u.v. and i.r. spectra confirmed the structures of the conjugates. 4. The conjugates were most stable against the hydrolysis under the mild acid conditions. 5. It was also shown that under the physiological conditions trypsin catalyze the conjugate hydrolysis and the catalytic effect is higher than that of chymotrypsine. 6. It is suggested that trypsin or trypsin-like proteases could participate in the hydrolysis of the conjugates in vivo. PGA-araC and CMG-araC showed 1.5- or 2.5-times higher antileukemic activity than both free araC or polysaccharides.     

Water-soluble fluorescent diblock nanospheres

The hydroxyl groups of poly(tert-butyl acrylate)-block-poly(2-hydroxyethyl methacrylate) or PtBA-b-PHEMA were reacted with succinic anhydride to introduce some carboxyl groups into the PHEMA block. Such carboxyl groups were then reacted with Texas-red cadverine (TX-NH(2)) to incorporate dye molecules. The TX-bearing diblocks formed probably spherical micelles in block-selective solvent DMF/toluene containing 2% DMF. "Permanent" micelles or nanospheres were prepared after cross-linking the TX-bearing PHEMA core block. Such nanospheres were made water soluble by cleaving the tert-butyl groups from the PtBA coronas. Water-soluble nanospheres with high TX numbers and fluorescence quantum yields may find applications in fluorescence in situ hybridization assays.