Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.     

Mapping of acyl carrier domain within the subunit of type I bacterial fatty acid synthetase

A fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, was used to label the acyl carrier site of the bacterial fatty acid synthetase from Brevibacterium ammoniagenes. The reagent bound preferentially to the 4'-phosphopantetheine thiol group of the acyl carrier domain and irreversively inactivated the enzyme. The modified enzyme was cleaved by proteinases for the mapping of the labeled site. The fluorescent fragment was readily detected on a polyacrylamide gel after electrophoresis. The region of 45 kDa containing the 4'-phosphopantetheine was located on the polypeptide at around two-thirds of the full length from the N-terminal.     

Synthesis of N-(9-Acridinyl)maleimide,a Fluorometrical Reagent for Thiol Compounds

The authors have reported¹⁾ a new maleimide type fluorescent thiol reagent, N-(9-acridinyl)- maleimide (NAM). In this paper the syntheses of NAM and its coupling products with thiol compounds are presented. NAM was synthesized from 9-aminoacridine and maleic anhydride through dehydratic cyclization in polyphosphoric acid. NAM showed no substantial fluorescence but its coupling products with thiol compounds exhibited strong blue fluorescence. Application of NAM for the fluorometrical analysis of cysteine and glutathione are suggested.     

The reaction of rabbit muscle creatine kinase with some derivatives of iodoacetamide.

The dimeric enzyme creatine kinase from rabbit muscle was treated with three derivatives of iodoacetamide that are capable of introducing fluorescent groups into the enzyme. All the three reagents (4-iodoacetamidosalicylate (IAS), 5-[N-(iodoacetamidoethyl)amino]-naphthalene-1-sulphonate (IAEDANS) and 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate (IAANS)) were shown to react at the same single thiol group on each enzyme subunit, leading to complete inactivation of the enzyme. The reaction with IAS was extremely rapid by comparison with the reaction with iodoacetamide or iodoacetate, but various lines of evidence suggest that IAS is not a true affinity label. However, kinetic and binding studies indicate that salicylate itself probably binds at the nucleotide-binding site on the enzyme. As the size of the modifying reagent increased, the first thiol group reacted more rapidly than the second; this trend was more pronounced at 0 degree C than at 25 degree C. With the largest modifying reagent used (IAANS), the pronounced biphasic nature of the modification reaction permitted the preparation of a hybrid enzyme in which only one subunit was modified, but a study of the thiol-group reactivity showed that this hybrid enzyme preparation underwent subunit rearrangement.     

N-(3-Triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC): a heterobifunctional reagent for immobilization of oligonucleotides on glass surface

A new heterobifunctional reagent, namely, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC) was developed and its potentiality for fixing of thiol (-SH) modified oligonucleotides were tested. The covalent attachment of oligonucleotides with the reagent was achieved through its maleimide functionality at one end via stable thioether linkage while the other end bearing triethoxysilyl functionality has been utilized for coupling with the virgin glass surface with simplified methodologies. Immobilization of oligonucleotides was achieved by two alternating ways. The PATH-1 involves formation of conjugate of reagent and SH-modified oligonucleotides through thioether linkage and was subsequently immobilized on unmodified glass surface through triethoxysilyl group and alternatively, PATH-2 involves reaction of reagent first with unmodified glass surface to get maleimide functionality on the surface and then the SH-modified oligonucleotides were immobilized via thioether linkage. The specificity of immobilization was tested by hybridization study with complementary fluorescein labeled oligonucleotide strand.     

Purification of human cytidine deaminase: molecular and enzymatic characterization and inhibition by synthetic pyrimidine analogs

Cytidine deaminase has been purified to homogeneity from human placenta by a rapid and efficient procedure consisting of affinity chromatography followed by hydrophobic interaction chromatography. The final enzyme preparation showed a specific activity of 64.1 units/mg, corresponding to about 46,000-fold purification with respect to the crude extract. The enzyme is a 52-kDa oligomeric protein composed of four apparently identical subunits. The acidic isoelectric point is 4.5. The enzyme's stability is strictly dependent on the presence of reducing agents. Amino acid analysis reveals the presence of five thiol groups per monomer which cannot be titrated by Ellman's reagent in the native enzyme. However, the presence of sulfhydryl groups involved in the catalytic activity was evidenced by the inhibition exerted by p-chloromercuribenzoate and heavy metal ions. In addition, the protection effected by the substrate against the p-chloromercuribenzoate inhibition and the competitive inhibition exerted by 5-(chloromercuri)cytidine suggest the presence of a thiol group(s) in the catalytic site of the enzyme. pH studies have shown that the rapid decline of activity occurring at pH 4.5 might result from the protonation of the pyrimidine ring at the N-3 position. The enzyme catalyzes the deamination of cytidine, deoxycytidine, and several analogs, including antineoplastic agents, thus abolishing their pharmacological activity. Therefore, several pyrimidine nucleoside analogs have been tested as potential inhibitors of the enzyme. The competitive inhibition exerted by cytidine analogs having the ribose moiety replaced by aliphatic chains is interesting.     

Modification of Escherichia coli ribosomes with the fluorescent reagent N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid. Identification of derivatized L31' and studies on its intraribosomal properties

N-[[(Iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS) is a fluorescent reagent which reacts covalently with the free thiol groups of proteins. When the reagent is reacted with the Escherichia coli ribosome under mild conditions, gel electrophoresis shows modification of predominantly two proteins, S18 and L31', which become labeled to an equal extent. When the native (i.e., untreated) ribosome is dissociated into 30S and 50S subunits, only the 30S ribosomal protein S18 reacts with IAEDANS despite the fact that L31' is still present on the large subunit. Upon heat activation of the subunits, a procedure which alters subunit conformation, S18 plus a number of higher molecular weight proteins is modified, but not L31'; the latter reacts with IAEDANS only in the 70S ribosome or when it is free. In contrast to the relatively stable association of L31' with native or with dissociated ribosomes, dissociation of N-[(acetylamino)ethyl]-5-naphthylaminesulfonic acid (AEDANS)-treated ribosomes weakens the AEDANS-L31'/ribosome interaction, resulting, upon gel filtration analysis, in ribosomes devoid of this derivatized protein.     

Organothallium(III) reagents for modification of biomacromolecules: irreversible labelling of phosphoglycerate kinase from rabbit muscle

Organothallium(III) reagents, by analogy with organomercurials, have been found to rapidly label phosphoglycerate kinase from rabbit muscle. By use of a radio-labelled version of p-methylphenylthallium(III) bis-trifluoroacetate (MPT) the inhibition was shown to be irreversible by the criterion of gel filtration desalting. The rate of labelling was shown to depend on the temperature, enzyme and thallium reagent concentrations, and the presence or absence of the various substrates of the enzyme. The structure and oxidation state of the thallium reagent used affected the extent of modification by the compounds MPT, o-carboxyphenylthallium(III) bis-trifluoroacetate, thallic trifluoroacetate and thallous acetate. A number of pieces of evidence implicate cysteine residues in the labelling, including changes in the free thiol titre of the enzyme on thalliation, model studies on the interaction of thiols (e.g. glutathione) with thallium(III) and thallous materials, the lack of inactivation of phosphoglycerate kinase from yeast (which has only one thiol residue distant from the active site), and the partial restoration of enzymic activity by treatment of thalliated enzyme with sulphydryl reducing agents. Substrate protection studies showed that modification of rabbit muscle phosphoglycerate kinase by MPT was fully prevented by 3-phosphoglycerate and partially by MgATP. The latter protected only against the fast phase of thallic modification, the slower phase being unaffected. The presence of MgADP potentiated the labelling by MPT. No evidence of an MgADP-induced conformational change in the enzyme could be obtained from fluorescence or circular dichroic spectroscopies, although changes of the native spectra were noted on thalliation by MPT alone. The cross-linking potential of these arylthallium(III) reagents is discussed along with conformational changes required to trigger the hinge-movement between the N- and C-domains of the protein.     

Evidence for histidine in the active sites of ficin and stem-bromelain

1. Ficin and stem-bromelain are irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. Evidence is presented that establishes that a histidine residue is within a 5A locus of the active-site cysteine residue in both enzymes. The histidine residue in both enzymes is alkylated at N-1 by dibromoacetone. It is suggested that, as with papain, the thiol and imidazole groups act in concert in the hydrolysis of substrates by these enzymes. 2. The inhibition of thiol-subtilisin with 1,3-dibromoacetone is shown to be due to the alkylation of a cysteine residue only.     

2,4-Dinitrophenyl [14C]cysteinyl disulfide allows selective radioactive labeling of protein thiols under spectrophotometric control

2,4-Dinitrophenyl [1-14C]cysteinyl disulfide readily introduces by disulfide exchange [14C]cysteine as a label into proteins with exposed thiols. The release of an equivalent amount of colored 2,4-dinitrothiophenolate allows the labeling reaction to be followed spectrophotometrically. In reaction with two cysteine residues of rabbit skeletal muscle actin, the thiol selectivity of the reagent corresponded to that of 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) and was superior to that of N-[14C]ethylmaleimide. Labeling of single SH groups of actin and papain proceeded faster than titration with Ellman's reagent under the same conditions. The [14C]cysteine label could be removed under mild conditions, e.g., with dithiothreitol, but proved to be stable during cyanogen bromide degradation of the protein and peptide purification. 2,4-Dinitrophenyl cysteinyl disulfide can be easily prepared within a few hours.     

The effect of thiol reagents on GABA transport in rat brain synaptosomes

The nature of gamma-aminobutyric acid (GABA) transport has been investigated in preparations of rat brain synaptosomes using a number of thiol reagents with varying membrane permeabilities. N-Ethylmaleimide, p-chloromercuribenzoate and p-chloromercuriphenylsulfonate effectively inhibited GABA transport in both directions (i.e., uptake and release) whereas 5,5'-dithiobis-2-nitrobenzoate, mercaptopropionate and N- nitroethylenediamine were much less effective, or ineffective, even at millimolar concentrations. For each of the thiol reagents, the inhibition profile for GABA uptake was approximately the same as that for its release. The effectiveness of the reagents indicates that there is an external, reactable SH-group on the transporter, that the thiol reagent must be somewhat lipophilic for it to react with the SH-group(s), and that the same synaptosomal transport system is responsible for both uptake and release of GABA.     

Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine

Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure.     

Identification of residues contributing to the ATP binding site of Kir6.2

The ATP-sensitive potassium (K(ATP)) channel links cell metabolism to membrane excitability. Intracellular ATP inhibits channel activity by binding to the Kir6.2 subunit of the channel, but the ATP binding site is unknown. Using cysteine-scanning mutagenesis and charged thiol-modifying reagents, we identified two amino acids in Kir6.2 that appear to interact directly with ATP: R50 in the N-terminus, and K185 in the C-terminus. The ATP sensitivity of the R50C and K185C mutant channels was increased by a positively charged thiol reagent (MTSEA), and was reduced by the negatively charged reagent MTSES. Comparison of the inhibitory effects of ATP, ADP and AMP after thiol modification suggests that K185 interacts primarily with the beta-phosphate, and R50 with the gamma-phosphate, of ATP. A molecular model of the C-terminus of Kir6.2 (based on the crystal structure of Kir3.1) was constructed and automated docking was used to identify residues interacting with ATP. These results support the idea that K185 interacts with the beta-phosphate of ATP. Thus both N- and C-termini may contribute to the ATP binding site.     

Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from salmon liver

Salmon liver was chosen for the isolation of 6-pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500-fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The enzyme is heat-stable up to 80 degrees C, has an isoelectric point of 6.0-6.3, and a pH optimum at 7.5. The enzyme is Mg2+ -dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 microM. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues. Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6-pyruvoyl tetrahydropterin synthase, nor was any cross-reaction in ELISA tests seen.     

Enzyme immunoassay of viomycin. New cross-linking reagent for enzyme labelling and a preparation method for antiserum to viomycin.

A new cross-linking reagent of the hetero-bisfunctional type, a N-(maleimidobenzoyloxy)-succinimide (MBS) was prepared and used for enzyme labelling of viomycin under mild aqueous conditions by a two-step process. In the first step a maleimide residue was selectively introduced onto the N1-amino group of viomycin with a limited amount of MBS. The second step consisted of thioether formation between the maleimide residue and free thiol groups of beta-D-galactosidase. An antiserum to viomycin was raised in rabbit by immunization with a viomycin-BSA conjugate. The conjugate was prepared by protecting N6-amino group of viomycin with an acetyl group and succinylating the N1-amino group, activating the carboxyl group by a mixed anhydride method and coupling it with the amino groups of bovine serum albumin (BSA). The specificity of the antiserum was proved by an enzyme immunoassay based on the competition between viomycin and its enzyme conjugate toward diluted solutions of the antiserum. By use of the viomycin-enzyme conjugate and the antiserum to viomycin, enzyme immunoassay of viomycin was successfully performed by the competitive binding procedure with the double-antibody method, and 0.1 to 4 ng of the antibiotic could be detected.     

Spectrophotometric measurement of mercaptans with 4,4'-dithiodipyridine

In a preceding study, 4,4'-dithiodipyridine (DTDP) was shown to be superior to 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent) for spectrophotometric measurement of thiol groups in aqueous solution. (i) Sensitivity is higher because a larger absorbance increase is seen at a given thiol concentration. (ii) The intrinsic reactivity of thiolate anions for DTDP is much higher than for Ellman's reagent; thus, the reaction can be carried out at pH > or = 4.5 instead of at pH 8.0. In the present study, these advantages of DTDP were exploited for spectrophotometric measurement of thiols in organic solvent. DTDP was found to quantitatively react with nonpolar thiols when triethylamine was used as catalyst, intense light absorption (between 344 and 360 nm) was seen when the reaction was terminated with acetic acid, and the spectrophotometric responses were independent of the nonthiol portions of the mercaptans. The determination limit (10x the standard deviation of the reagent blank) was 3 microM, and the upper limit was approximately 40 microM on a typical spectrophotometer. The thiol contents of the mercaptans were independently verified by a modification of standard iodometry in which toluene/butanol or chloroform/butanol was included to dissolve nonpolar mercaptans.     

Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins

This protocol describes a detailed method to study the static and dynamic features of membrane proteins, as well as solvent accessibility, by utilizing the lactose permease of Escherichia coli (LacY) as a model. The method relies on the use of functional single-Cys mutants, an affinity tag and a PhosphoImager. The membrane-permeant, radioactive thiol reagent N-[ethyl-1-14C]ethylmaleimide ([14C]NEM) is used to detect site-directed alkylation of engineered single-Cys mutants in situ. The solvent accessibility of the Cys residues is also determined by blockage of [14C]NEM labeling with membrane-impermeant thiol reagents such as methanethiosulfonate ethylsulfonate (MTSES). The labeled proteins are purified by mini-scale affinity chromatography and analyzed by gel electrophoresis. Gels are dried and exposed to a PhosphoImager screen for 1-5 d, and incorporation of radioactivity is visualized. Initial results can be obtained in 24 h.     

A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay

A novel optically active thiol compound, N-(tert-butylthiocarbamoyl)-L-cysteine ethyl ester (BTCC), is synthesized as a chiral derivatization reagent. This compound and o-phthalaldehyde react with amino acid enantiomers to produce fluorescent diastereomers that are readily separable on a reverse-phase column by HPLC. Enantioseparation of acidic amino acids in particular is markedly improved using BTCC. In this study, the HPLC method for enantioseparation with the novel compound is applied to the aspartate (Asp) racemase assay. Derivatized D-Asp is eluted before the L-Asp derivative. Consequently, a small amount of D-Asp produced by the activity of racemase on a large quantity of L-Asp substrate may be quantified accurately, even at very low activity. Since the derivatization reaction proceeds rapidly at room temperature, a fully automated system is established for derivatization and sample injection. The automated method is practical and successfully applied to the archaeal Asp racemase assay. We presume that the procedure is additionally applicable to the enantioseparation of other amino acids, amino alcohols, and catecholamines.     

N-(Iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED): a new heterobifunctional reagent for the preparation of biochips

Design and synthesis of a new heterobifunctional reagent, N-(iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED), have been described for the preparation of oligonucleotide-based biochips. The performance of the featured reagent is probed by the immobilization of thiolated and thiophosphorylated oligonucleotides on modified glass microslides via two routes (routes A and B). The immobilization procedure was accelerated by performing a chemical reaction between thiolated oligomers and the iodoacetyl moiety of the reagent under microwaves (MW), where it is completed in just 10 min. The quality of the constructed oligonucleotide microarrays was tested by performing a hybridization assay with a complementary target and subsequently used for the detection of base mismatches. The immobilized probes were found to be thermally stable.     

Nucleotide in monomeric actin regulates the reactivity of the thiol groups

A new thiol reagent, 2,4-dinitrophenyl glutathionyl disulfide, allowed the characterization of four thiol groups in monomeric actin by stoichiometric reaction. The number of thiol groups exposed to the reagent was found to depend on the nucleotide bound. In the absence of ATP, G-actin exposed four thiol groups ( G4s ). On the addition of ATP (1 equiv), three of them were shielded. The resulting actin with one thiol group exposed ( G1s ) is the form of monomeric actin normally produced by depolymerization of F-actin in buffers containing ATP. G1s is stable over hours, while G4s , i.e., monomeric actin in ATP-free solution, is not. This must be concluded from the fact that the shielding effect of thiol groups induced by addition of ATP was lost within ca. 30 min probably due to denaturation of G4s to G4s *. Therefore, denaturation of monomeric actin must be understood in terms of loss of thiol shielding, rather than by oxidation of the thiol groups. Addition of equimolar amounts of Ca2+ significantly retarded the denaturation process. ADP (50 equiv) shielded only ca. two of the four thiol groups but, similar to ATP, protected actin from denaturation. Three ATP analogues (10 equiv) were tested but had no shielding effect. In the presence of these analogues actin ( G4s ) rapidly denatured (to G4s *) as in the absence of added nucleotides. It was shown that the thiol-shielding activity and the protective capacity of a nucleotide are interrelated with its binding capability to monomeric actin. G1s was found to be polymerizable as was G approximately 2s on the addition of ATP. No polymerization could be detected for G4s or G4s *.(ABSTRACT TRUNCATED AT 250 WORDS)