Chemical Modification Of Carboxyl Groups Research Articles

Reduction of negative charge in the aspartyl proteinase from the fungus Mucor miehei by chemical modification of carboxyl groups: effect on structure-function

The study was undertaken to investigate the effect of reduction of negative charge by nonspecific chemical modification of carboxyl groups on the physicochemical properties, milk-clotting activity and structure of M. miehei proteinase

Anomalous behaviour of a protein during SDS/PAGE corrected by chemical modification of carboxylic groups

The 29,000-Mr Actinomadura R39 beta-lactamase exhibited a remarkably low electrophoretic mobility on SDS/PAGE, yielding an Mr value almost twice that computed from the corresponding gene sequence. We showed that chemical modification of the carboxylic groups of glutamic acid and aspartic acid residues restored a normal electrophoretic mobility and that the anomalous behaviour of that protein on SDS/PAGE was due to its very large negative charge at neutral pH. We also compared the behaviour of the same enzyme on gel filtration in the presence of SDS with those of other class A beta-lactamases (Mr approx. 30,000). These experiments suggested that the very low electrophoretic mobility of the Actinomadura R39 beta-lactamase upon SDS/PAGE was more probably due to a low degree of SDS binding rather than to an unusual shape of the SDS-protein complex.

Assembly properties of tubulin after carboxyl group modification.

By chemically modifying carboxyl groups we have investigated the role of the highly acidic COOH-terminal domains of alpha- and beta-tubulin in regulating microtubule assembly. Using a carbodiimide-promoted amidation reaction, as many as 25 carboxyl groups were modified by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and an amine nucleophile, [14C] glycine ethyl ester or [3H]methylamine, to assembled microtubules. Modification occurred primarily in the carboxyl-terminal region as demonstrated by limited proteolysis of modified tubulin by trypsin, chymotrypsin, subtilisin, and carboxypeptidase Y. Modified tubulin polymerized into microtubules with a critical concentration that was 15% of that for unmodified tubulin. Assembly of modified tubulin and microtubules formed from modified tubulin were less sensitive to Ca2+ and high ionic strength. Ca2+ binding studies under low ionic strength conditions indicated that modified tubulin does not contain the high affinity Ca2+ binding site. While assembly of unmodified tubulin was stimulated by Mg2+ up to 10 mM, assembly of the modified protein was inhibited by concentrations greater than 1 mM. When 24 residues were modified, polymerization was no longer stimulated by microtubule-associated proteins (MAPs) or polylysine and incorporation of high molecular weight MAPs into the polymers was reduced by about 70% compared to unmodified tubulin. These studies demonstrate that chemical modification of carboxyl groups in tubulin, most of which are localized in the COOH-terminal region, leads to an enhanced ability to polymerize and a decrease in interaction with MAPs and other positively charged species.

Irreversible thermoinactivation of glucoamylase from Aspergillus niger and thermostabilization by chemical modification of carboxyl groups

The Incubation of glucoamylase from Aspergillus niger at 70°C induced its rapid and irreversible inactivation. The covalent modifications of the protein structure involved in the thermoinactivation depended on the pH of the medium. We observed the formation of a low amount of disulfide-linked oligomers showing that disulfide exchange takes place at pH 5.5. Hydrolysis of peptide bonds at pH 3.5 and 4.5 was also detected. The chemical modification of carboxyl groups with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) decreased the rate of appearance of low-molecular-weight peptides at pH 3.5 and 4.5 upon heating at 70°C. However, the rate of inactivation at such pH values was not modified. Modification of carboxyl groups with EDC in the presence of ethylenediamine leading to the transformation of three carboxyl groups to amino groups increased the thermostability of the enzyme for temperatures above the temperature of compensation, T c, which is 60°C.

Light-induced currents from oriented purple membrane: II. Proton and cation contributions to the photocurrent

The sign of B2, the micro-second component of the photocurrent from oriented purple membrane, is that of positive charge moving away from the purple membrane in the direction of proton release. B2 could be due to internal dipole or proton movement, proton release, or metal cation release. We found that the waveform of B2 is virtually insensitive to changes in the salt concentration as long as it is >40 mM KCl, >5 mM CaCl(2), or >0.5 mM LaCl(3). However, below these limits, B2's apparent rate of decay increases as the salt concentration decreases without any change in the initial amplitude. This salt dependence suggests that B2 is due to a positive charge, either a metal cation or a proton, moving from the membrane into the solution. That the positive charge is not a metal cation is suggested by the waveform of B2 remaining unchanged upon replacing the cations both in solution and in the binding sites of the purple membrane. Direct evidence that the positive charge movement is due to protons was obtained by examining the correlation of B2 with the proton dependent processes of bacteriorhodopsin in buffers and dyes. Based on these observations, we suggest that most, if not all, of the intrinsic B2 component of the photocurrent at moderate salt concentration is due to proton release.The photocurrents from purple membranes whose surface potential has been reduced by delipidation or chemical modification of carboxyl groups with methyl esters were found to be only modestly changed. This suggests that the salt effect is not through its modulation of the surface potential. Rather, we propose that in low salt B2 represents the sum of a proton release from the surface of the purple membrane and a second current component, due to cations moving back towards the membrane, which is only important in low salt. The cation counter current is induced by proton release which creates a transient uncompensated negative charge on the membrane.

Modification of carboxyl groups in botulinum neurotoxin types A and E

M. A. Woody, A. Herian and B. R. DasGupta. Modification of carboxyl groups in botulinum neurotoxin types A and E. Toxicon 27, 1143–1150, 1989.—Effects of chemical modification of carboxyl groups of botulinum neurotoxin serotypes A and E were studied by using a water soluble carbodiimide—nucleophile reaction that is highly specific for modifying carboxyl groups of proteins. In both types A and E, increasing levels of the reagents, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and norleucine methyl ester or glycine methyl ester, at pH 4.8 caused increased loss of toxicity. More glycine could be incorporated than norleucine. Amino acid analysis did not reveal modification of any amino acid residue other than carboxyl groups (possible reaction of sulfhydryl groups was not studied). Loss of one carboxyl group did not severely affect toxicity, but modification of three carboxyl groups caused >95% detoxification in both types. Complete detoxification could not be achieved with any amount of the reagents. Modification of three to five carboxyl groups did not affect serological activity.

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Glucoamylases G1 and G2 from Aspergillus niger lost the catalytic activity by prolonged treatment with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide(EAC). A total of 23 and 16 carboxyl groups were substituted out of 71 and 54 present in the G1 and G2 enzyme, respectively. The kinetics and the pH-dependence of the inactivation were compatible with the existence of one essential carboxyl group with a pKa 5.5. The inhibitor acarbose (a pseudotetrasaccharide) and the substrate maltose prevented EAC-modification of 4 and 2 carboxyl groups, respectively, with considerable retention of enzyme activity. Following removal of these ligands, differential labelling of critical carboxyl residue(s) was achieved by means of [3H]EAC. Glucoamylase G2 with 12 EAC groups incorporated, prepared in the presence of acarbose, displayed a two-fold increase of Km and a slight reduction of Vmax for hydrolysis of starch relative to the unmodified enzyme. Both Km and Vmax for hydrolysis of maltose were almost unaffected. In addition, maltose and three inhibitors perturbed the UV absorbance spectrum of this derivative. An altered shape of the acarbose-induced difference spectrum implied substitution of a carboxyl group near a binding tryptophanyl residue at a distance from the catalytic site. Efficient protection by acarbose against only the second part of the biphasic course of inactivation similarly reflected that residues playing different roles in the mechanism of action reacted with EAC. In separate experiments, evidence was obtained for a preferred reaction of ethylenimine with enzymatically important carboxyl groups in glucoamylase.

The interaction of ferredoxin with chloroplast ferredoxin-linked enzymes

As has previously been demonstrated for ferredoxin/NADP+ oxidoreductase (NADP+ reductase) and ferredoxin/nitrite oxidoreductase (nitrite reductase), ferrodoxin-dependent glutamate synthase has been shown to form a complex with ferredoxin. The complex (Kd = 14.5 μM) is stabilized by electrostatic forces and produces changes in the chromophore(s) of at least one of the proteins. Chemical modification of carboxyl groups on ferredoxin with glycine ethyl ester in the presence of a water-soluble carbodiimide affected the binding of ferredoxin to both glutamate synthase and nitrite reductase and also inhibited the ability of reduced ferredoxin to serve as an electron donor to both enzymes.

Chemical modification of carboxyl groups on spinach ferredoxin alters its ability to interact with ferredoxin:NADP reductase

Treatment of spinach ferredoxin with glycine ethyl ester in the presence of a water soluble carbodiimide resulted in the modification of 3–4 carboxyl groups and decreased the ability of ferredoxin to participate in NADP photoreduction by chloroplast membranes by about 80%. The ability of the modified ferredoxin to receive electrons from the reducing side of Photosystem I was relatively unaffected. These findings suggest that the modified ferredoxin is unable to interact with ferredoxin:NADP reductase. This has been verified by demonstration that the modified ferredoxin fails to produce difference spectra typical of a ferredoxin-ferredoxin:NADP reductase complex when added to ferredoxin:NADP reductase.

Structure of an electron transfer complex. II. Chemical modification of carboxyl groups of cytochrome c peroxidase in presence and absence of cytochrome c.

Cytochrome c peroxidase forms an electron transfer complex with cytochrome c. The complex is governed by ionic bonds between side chain amino groups of cytochrome c and carboxyl groups of peroxidase. To localize the binding site for cytochrome c on the peroxidase, we have used the method of differential chemical modification. By this method the chemical reactivity of carboxyl groups (toward carbodiimide/aminoethane sulfonate) was compared in free and in complexed peroxidase. When ferricytochrome c was bound to cytochrome c peroxidase, acidic residues 33, 34, 35, 37, 221, 224, and 1 to 3 carboxyls at the C terminus became less reactive by a factor of approximately 4, relative to the remaining 39 carboxylates of peroxidase. Of the less reactive residues those in the 30-40 region and the 221/224 pair are on opposite sides of the surface area which contains the heme propionates. We, therefore, propose that the binding site for cytochrome c on cytochrome c peroxidase spans the area where one heme edge comes close to the molecular surface. The results are in very good agreement with chemical cross-linking studies (Waldmeyer, B., and Bosshard, H.R. (1985) J. Biol. Chem. 260, 5184-5190); they also support a hypothetical model predicted on the basis of the known crystal structures of cytochrome c and peroxidase (Poulos, T.L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330).

The Role of Surface Charge in Ionic Germination of Clostridium perfringens Spores

The surface charge against pH behaviour of spores of Clostridium perfringens type A was determined using a colloid titration method in the pH range 4-9. The native spores as well as various ionic forms of the spores loaded with divalent cations were found to be negatively charged. A positive colloid, MGCh, inhibited the ionic germination of the spores and this inhibition was highly dependent on pH. However, another positive colloid, GCh, did not inhibit the germination. Chemical modification of carboxyl groups on the spore surface by use of a water-soluble carbodiimide with two nucleophiles, glycine ethyl ester and taurine, caused spores not only to have little or no colloidal charge but also to lose the ability to germinate. The modification of carboxyl groups in the spore coat protein also resulted in elimination of, or marked reduction in its negative charge. These results suggest that carboxyl groups in the spore coat protein are the major negatively charged species on the spore surface. The role of surface charge in the ionic germination of the spores is discussed.

Effects of chemical modification of carboxyl groups on the voltage-clamped nerve fiber of the frog.

Voltage-clamped single nerve fibers of the frog Rana esculenta were treated with the carboxyl group activating reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of different primary amines and without added amine. Carboxyl groups form stable amide bonds with primary amines in the presence of EEDQ. EEDQ treatment reduced the sodium current considerably and irreversibly, regardless of the presence of a primary amine in the Ringer's solution. The potassium current was also reduced. After modification the reduced sodium currents inactivated slowly and incompletely. The descending branch of the sodium current-voltage relation, INa(E), was shifted along the voltage axis in the depolarizing direction. The size of the shift was strongly dependent on the amine present during modification with EEDQ. The voltage-dependence of sodium inactivation, h infinity (E), was shifted to more positive values of membrane potential by EEDQ in the presence of ethylenediamine (11 mV) and glucosamine (3 mV). In contrast, a small shift to more negative potentials occurred in the presence of taurine (-3 mV) or without the addition of an amine (-2 mV). A tenfold increase of the calcium concentration still shifted the INa(E) and h infinity (E) curves of the chemically modified fibers. However, these shifts were smaller than those observed on untreated fibers. The currents remaining after the modification were completely blocked by tetrodotoxin; no change of the reversal potential occurred.

Chemical modification of carboxyl groups in human fc γ fragment—II. Location of acidic residues involved in complement activation

The location of the acidic residues on human Fc γ1 which are responsible for the loss of complement-activating capacity upon chemical modification has been established. As many as 13 residues get modified at the point of functional inactivation, of which only 6 are located in the C γ2 region. Residues Glu (269) and Glu (318) are very reactive and may be extensively modified without significantly affecting anticomplementary capacity. Both Glu (333) and Asp (249) appear to be partially labelled. They are possibly involved in intramolecular ionic pairs (Deisenhofer, 1981), so that their contribution to complement activation is considered dubious. Extensive modification of Glu (258) and Glu (293) adds to the more rapid one of Glu (269) and Glu (318) resulting in abrogation of anticomplementary capacity. The data support the idea that a charge component may be an essential part of the mechanism of complement activation. They also help to define the nature of the Fc effector site that mediates this activity.

Effect of Lectins and Chemical Modification on the Hemagglutinin Activity of Human Plasma Fibronectin

Purified human plasma fibronectin has been shown to agglutinate protease-treated red cells [Vuento, Hoppe-Seyler's Z. Physiol. Chem. 360, 1327-1333, (1979)]. The present report shows that the activity is inhibited by low concentrations of lectins and by macromolecular serum factors. Chemical modification of carboxyl groups of fibronectin strongly inhibited the activity, but modification of amino groups of guanidinium groups had little effect on the activity. The results suggest that fibronectin receptors on erythrocyte surface are carbohydrate-containing molecules. Humoral macromolecular factors may control the interaction of fibronectin with cell surfaces. Chemical modification studies indicate that the parts of the fibronectin molecule responsible for the hemagglutinin activity are different from those mediating the binding of fibronectin to collagen.

Chemical modification of carboxyl groups in porcine pepsin.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTChemical modification of carboxyl groups in porcine pepsinChing-Yung Ma and Shuryo NakaiCite this: J. Agric. Food Chem. 1980, 28, 4, 834–839Publication Date (Print):July 1, 1980Publication History Published online1 May 2002Published inissue 1 July 1980https://doi.org/10.1021/jf60230a017RIGHTS & PERMISSIONSArticle Views104Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Chemical modification of carboxyl groups in human Fc γ fragment: Structural role and effect on the complement fixation

The role played by acidic residues of immunoglobulin molecules in complement activation by the classical pathway has been studied using the Fc fragment obtained from human IgG by papain digestion. Delta- and Epsilon-carboxyl groups of aspartic and glutamic acid were selectively modified by coupling to glycine-ethyl-ester through the mediation of ethyl-dimethylaminopropyl-carbodiimide (EDC). ‡ ‡ EDC, 1-ethyl-3(3-dimethyl-amino-propyl)-carbodiimide; SDS sodium dodecyl sulfate; PBS, phosphate-buffered saline (pH 7.2); CFD; complement fixation diluent; Cl, first component of complement; PAGE, polyacrylamide gel electrophoresis; Fc-1, 5, 10, 30-min, Fc fragment obtained at different times of modification; WSC, water-soluble carbodiimide. A rapid loss of anticomplementary activity was observed accompanied by small conformational changes in the Fc fragments. As shown by circular dichroism data, a gradual decreasing in the band at 225 nm (corresponding to the interaction Cγ2-Cγ3) was detected, as well as an increase of about 15% in the Stokes radius. No loss of anticomplementary activity was observed in the first minute of the reaction. However, after 5 min, when 7 out of 26 carboxyl groups per Fc chain had been modified, anticomplementary activity was completely abolished. In addition, using an immune serum to native Fc, the modified fragment was found to gradually lose its antigenicity as a function of the reaction time. These results point to an important involvement of some acidic residues of the immunoglobulin molecule in the activation of the complement sequence through the classical pathway.

Chemical modification of carboxyl groups of fibrinogen and its effect on the binding of cationic detergent.

Carboxyl groups of native human fibrinogen were modified with glycine methyl ester and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. It seemed likely that the modification occurred stepwise. Approximately 26% of the carboxyl groups of fibrinogen was modified finally. The modified fibrinogen had no interaction with cationic detergent, and did not form any complex with the detergent. In dilute acid, fibrinogen was observed to show only a slight interaction with cationic detergent. It is probable that the exposed and ionized carboxyl groups are essential for the formation of a complex between fibrinogen and cationic detergent.

The effect of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide on calcium binding and associated changes in chloroplast structure and chlorophyll a fluorescence in spinach chloroplasts

1. 1. Chemical modification of carboxyl groups on the chloroplast membrane with a water-soluble carbodiimide plus a nucleophile caused inhibition of Ca 2+ binding. 2. 2. Both binding sites were affected and showed a decrease in the number of binding sites and an increase in the dissociation constant. 3. 3. Cation-induced changes in chlorophyll a fluorescence and structural changes ( ΔA 540) were inhibited at the same carbodiimide concentrations as Ca 2+ binding, emphasizing the relationship between these processes. 4. 4. Chloroplasts that were illuminated with high intensity light for short time periods showed a decrease in the carbodiimide-mediated inhibition of Ca 2+ binding.

The role of carboxylic acid groups in the action of glucoamylase I

Received 31 October 1972 1. Introduction Carboxylic acid groups have been shown to be pre- sent in the active sites of many enzymes, including pep- sin [l], /3-D-glucosidase [2], lysozyme [3] and carboxy- peptidase A [4], and their presence has been suggested in many other cases [5]. Often the first indication that a carboxylic acid group is involved at the active site of an enzyme is obtained from the pH-rate profile, an ion- izable group with pK, about 3-5 being frequently as- sumed to be a carboxyl. Conclusions based on pH-stud- ies, however, must always be confirmed in other ways. A variety of methods have been used for the chemical modification of carboxyl groups in proteins and these can be valuable in structure-function correlations. For example, p-bromophenacyl bromide [6] and l-diazo- 6-phenyl-3-toluenesulphonamidobutan-2-one [7] inac- tivate pepsin by forming esters with the side-chain car- boxy1 of an essential aspartyl residue, and conduritol Bepoxide inactivates f?-D-glucosidase [8] by reaction with a carboxyl at its active site. Hoare and Koshland [9] developed a method for the modification of carboxyl groups in proteins, which involves the reaction of glycine methyl ester in the pres- ence of a water soluble carbodiimide. We have studied the effect of this reaction on the activity of glucoamyl- ase I, an enzyme whose pH-rate profile had suggested the possibility that a carboxyl group might be impli- cated in the enzymic process. 2. Materials and methods Crude glucoamylase from Aspergillus

Modification of carboxyl groups in the active site of trypsin

The chemical modification of carboxyl groups in chromatographically homogeneous bovine β-trypsin using the carbodiimide-nucleophile procedure resulted in complete loss of enzyme activity and the modification of 8 carboxyl groups. Under similar reaction conditions, the presence of a competitive inhibitor was found to decrease both the extent of modification and the loss of activity, giving a product with 7 carboxyls modified and 30% of the original activity. The carboxyl group protected to the largest extent was identified as Asp-177, with Asp-182 being protected to a lesser extent. Direct evidence is also presented indicating that residue 177 is indeed aspartic acid rather than asparagine.