Inhibition of pigeon liver fatty acid synthetase by specific modification of lysine residues with 2,4,6-trinitrobenzenesulphonic acid

Pigeon liver fatty acid synthetase was inactivated irreversibly by 2,4,6-trinitrobenzenesulphonic acid (TNBS). Biphasic inactivation of the enzyme was observed with the inhibitor. NADPH provided protection to the enzyme against inactivation by TNBS and the extent of protection increased with NADPH concentration indicating that the essential lysine residues are present at the NADPH binding site. The stoichiometric results with TNBS showed that 4 mol of lysine residues are modified per mole of fatty acid synthetase upon complete inactivation. The rapid reaction of two amino groups per enzyme molecule led to the loss of 60% of the enzyme activity. These approaches suggested that two lysine residues present at the active site are essential for the enzymatic activity of fatty acid synthetase.     

Differential reaction of cell membrane phospholipids and proteins with chemical probes.

The major aims of this study were to determine the degree of phospholipid asymmetry and the neighbor analysis of phospholipids in different types of cell membranes. For this study a penetrating probe (FDNB), a non-penetrating probe (TNBS) and a cross-linking probe (DFDNB) were used. The reaction of hemoglobin, membrane protein and membrane PE and PS of erythrocytes with DFNB and TNBS was studied over a concentration range of 0.5 to 10 mM probe. TNBS reacts to an extremely small extend with hemoglobin over the concentration range 0.4 to 4 mM whereas FDNB reacts with hemoglobin to a very large extent (50 fold more than TNBS). The reaction of membrane protein of intact erythrocytes reaches a sharp plateau at 1 mM TNBS whereas the reaction of membrane protein goes to a much larger extent with FDNB with no plateau seen up to 4 mM FDNB. This data shows that TNBS does not significantly penetrate into the cell under our conditions whereas FDNB does penetrate into the cell. The results show that there are four fold more reactive sites on proteins localized on the inner surface of the erythrocyte membrane as compared to the outer surface. TNBS at 0.5 to 2 mM concentration does not label membrane PS and labels membrane PE to a small extent. The reaction of PE with TNBS shows an initial plateau at 2 mM probe and a second slightly higher plateau between 4 to 10 mM probe. TNBS from 0.5-2.0 mM does not react with PS, but between 3 to 10 mM concentration, a very small amount of PS reacts with TNBS. Hence above 2 mM TNBS or FDNB a perturbation occurs in the membrane such that more PE and PS are exposed and react with these probes. These results demonstrate that essentially no PS is localized on the outer surface of the membrane and only 5% of the total membrane PE is localized on the outer surface of the erythrocyte membrane. TNBS and FDNB were reacted with yeast, E. coli, and Acholeplasma cells. With yeast cells, FDNB reacts to a much larger extent with PE than does TNBS, indicating that FDNB penetrates into the cell and labels more PE molecules. With E. coli, but not with erythrocytes or yeast cells, phospholipase A activity was very pronounced at pH 8.5 giving rise to a large amount of DNP-GPE from DNP-PE. A phosphodiesterase was also present which hydrolyized DNP-GPE to DNP-ethanolamine. The multilayered structure of the E. coli cell envelop did not permit a definitive interpretation of the results. It is clear, however, that TNBS and FDNB react to a different extent with PE in this cell. The Acholeplasma membrane had no detectable PE or PS but contains amino acid esters of phosphatidylglycerol. The reaction of these components with TNBS and FDNB indicate that these aminoacyl-PG are localized on both surfaces of the membrane, with 31% being on the outer surface and 69% on the inner surface...     

Compartmentation of membrane phosphatidylethanolamine formed by base-exchange reaction in rat brain microsomes

The compartmentation of membrane phosphatidylethanolamine (PE) formed by base-exchange reaction in rat brain microsomal vesicles has been investigated. After labelling membrane PE by base-exchange in vitro, microsomal vesicles were treated with trinitrobenzenesulfonic acid (TNBS). The amount of membrane PE reacting with TNBS depends on the duration and the temperature of the reaction as well as on the TNBS concentration. It was found that almost all of the labelled PE molecules, but only about 24% of membrane PE, were accessible to TNBS, under very mild reaction conditions. It is concluded that PE labelled by base-exchange is completely localized in the cytoplasmic leaflet of microsomal vesicles.     

Desulfonation of a colitis inducer 2,4,6-trinitrobenzene sulfonic acid produces sulfite radical

2,4,6-Trinitrobenzene sulfonic acid (TNBS) has been used in vivo to induce colitis. With the nitroreductase of intestinal cells, TNBS underwent redox cycling to produce TNBS-nitro and superoxide radical anions which are thought to be involved in initial oxidative reactions that lead to colonic injury. In this study, we demonstrated that the TNBS desulfonative reaction with tissue amino acids produces sulfite which is subsequently oxidized to sulfite radical. Sulfite radical was measured using a spin trapping methodology. Sulfite radical adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) were detected in a mixture of TNBS and lysine, xanthine oxidase, red blood cells, colonic mucosal or submucosal muscle tissues. TNBS alone did not produce sulfite radical, indicating that its formation required the presence of amino acids. Because sulfite radical is the precursor of highly reactive sulfiteperoxyl and sulfate radicals, our data imply that these sulfite-derived free radicals may also contribute to oxidative reactions leading to colonic injury in TNBS-induced colitis.     

A convenient manual trinitrobenzenesulfonic acid method for monitoring amino acids and peptides in chromatographic column effluents.

The trinitrobenzenesulfonic acid (TNBS) method of R. Fields (1971, Biochem. J., 124, 581–590) has been modified for the manual detection of amino acids and peptides in chromatographic column effluent by changing the reaction conditions to 1 mm TNBS in 0.4 m potassium borate buffer, pH 9.2, at room temperature for 30 to 50 min. The reaction with amines and the spontaneous hydrolysis of TNBS are stopped by neutralization to pH 6.25 with sodium monobasic phosphate (0.33 m). Sodium sulfite (3 mm) is added to increase the absorptivity of the product. The TNBS reagent blank is less than 0.100 A₄₂₀ after 50 min of reaction. Since the ΔA₄₂₀ of the reagent blank is ～0.002/min before quenching the reaction, and zero afterward, the time required for reaction and for absorbance measurements need not be controlled precisely. Alkaline hydrolysis of peptides is carried out prior to detection to increase the sensitivity of the method. This procedure is convenient for the manual determination of 5 to 100 nmol of amino acids in the 50–100 samples required to define a chromatographic elution profile.     

Identification of lysine residue at or near active site of luffin-a, a ribosome-inactivating protein from seeds of Luffa cylindrica

Luffin-a, a ribosome-inactivating protein from the seeds of sponge gourd (Luffa cylindrica), was modified with 2,4,6-trinitrobenzenesulfonic acid (TNBS) at pH 8.0 and 20 degrees C. The inhibitory activity of the modified luffin-a on protein synthesis using rabbit reticulocyte lysate was lost rapidly at a rate compatible with that of the modification of a single highly reactive amino group in the initial stage of the reaction. By cation-exchange FPLC of the products of 5-min modification, TNP-luffin-a containing one modified amino group was obtained and shown to have only 6.7% of the activity of native luffin-a without any gross conformational change. The amino acid composition and sequence of the TNP-peptide, isolated by reverse-phase HPLC of the tryptic digest of the TNP-luffin-a, unambiguously demonstrate the trinitrophenylation of lysine residue at position 231. From these results, it was concluded that Lys231 of luffin-a is highly reactive to TNBS and is located at or near the active site of luffin-a.     

Trinitrophenylation of the bilirubin binding site of human serum albumin.

Human serum albumin has been modified with 2,4,6-trinitrobenzenesulphonic acid and picryl chloride in low ratios of reagents/albumin. The derivatives have been investigated by spectrophotometry and by thin layer chromatography of the hydrolysates in order to assess the specificity of the reagents. The same reaction conditions were used to modify albumin previously complexed with bilirubin in the ratio of 1:1. The affinity of bilirubin to the modified albumins was estimated by an improved perozidase method. It is concluded that TNBS and picryl chloride react almost quantity with epsilon-amino groups of lysine on the albumin molecule. The results also suggest that at least on TNBS reactive amino group and at least one picryl chloride reactive amino group are located in or near the high-affinity bilirubin binding site.     

An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines.

The use of 2,4,6-trinitrobenzenesulfonic acid (TNBS) as a reagent for determining the concentrations of amines has been widely accepted (1–3) since its introduction in 1960 by Satakeet al. (4). The original procedure has since been modified by Mokrasch (5) to permit the determination of amines, amino acids, and proteins in mixtures. In both procedures the trinitrophenylation reaction is followed by a quenching step, after which the amino content is related to the increase in absorbance at 340 nm (4) or 420 nm (5). We have studied the trinitrophenylation reaction and have found that amino content can be related directly to the absorbance of the trinitrophenylation reaction mixture after a relatively short incubation period (15–30 min). Therefore, it is unnecessary to quench this reaction. We describe herein an extremely convenient procedure for the determination of amines, amino acids, and proteins where the quenching step employed by previous investigators has been eliminated. The proposed method has a greater sensitivity than previously described techniques employing TNBS.     

Inactivation of rat testicular NADPH-cytochrome P-450 reductase by 2,4,6-trinitrobenzenesulfonate

Rat testicular NADPH-cytochrome P-450 reductase was inactivated by treatment with 2,4,6-trinitrobenzene sulfonate (TNBS) or with 2',3'-dialdehyde derivatives of 5'-ATP and NADP+. The inactivation rates were dependent on reaction time and followed pseudo-first order kinetics. The rate of inactivation of cytochrome c reducing activity by TNBS was faster than that of reducing activities for K3Fe(CN)6 and for dichlorophenol indophenol (DCPIP). Cytochrome c and DCPIP prevented NADPH-cytochrome P-450 reductase from inactivation by TNBS, but NADP(H) protected to a lesser extent. Stoichiometry indicated that two residues of amino acid modified with TNBS were essential for the enzyme activity. The 2',3'-dialdehyde derivatives of 5'-ATP and NADP+ were specific ligands for the modification of lysine residues, whereas TNBS would possibly modify residues of lysine and/or cysteine. By differential and sequential modification by 5,5'-dithio-bis(2-nitrobenzoic acid), TNBS and dithiothreitol, the residues of lysine and cysteine were identified in the active site of NADPH-cytochrome P-450 reductase. These results suggest that lysyl and cysteinyl residues are located at or near the active region of NADPH-cytochrome P-450 reductase from the rat testicular microsomal fraction.     

Intestinal inflammation in TNBS sensitized rats as a model of chronic inflammatory bowel disease

An enteritis, based on a delayed-type hypersensitivity reaction, was induced in TNBS (2,4,4-trinitrobenzenesulphonic acid) sensitized rats by multiple intrajejunal challenge with TNBS via an implanted catheter. This treatment induced chronic inflammation of the distal small intestine characterized by intense hyperaemia, oedema and gut wall thickening as assessed by macroscopic scoring and weighing a defined part of the dissected intestine. Histologically, the inflammatory response included mucosal and submucosal cell infiltration by lymphocytes and histiocytes, transmural granulomatous inflammation with multinucleated cells and activated mesenteric lymph nodes. Ex vivo stimulated release of the inflammatory mediator LTB(4) in the dissected part of the intestine was increased following TNBS treatment. Drug treatment with sulphasalazine or 5-aminosalicylic acid improved the enteritis score and attenuated TNBS induced oedema formation and LTB(4) production. The applicability and relevance of this new model are discussed with respect to drug development and basic research of inflammatory bowel diseases.     

Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase]

Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.     

Study of amino and sulfhydryl sites in the sodium pathway in dog red blood cell membranes

Summary Amino reactive TNBS (2,4,6-trinitrobenzene sulfonic acid), SITS (4-acetamido-4-isothiocyano-stilbene-2-2-disulfonic acid), and Zn⁺⁺, and SH reactive Hg⁺⁺ were employed to study sodium channels in dog red blood cells. Simultaneous modification of the membrane with both a SH and an amino modifier results in an increase in Na⁺ permeability which is equal to the sum of their individual effects. This indicates that SH and amino sites are separate units. Three lines of evidence indicate that the amino sites are more superficial than the SH sites. (1) Pretreatment with an amino modifier decreases the effectiveness of subsequent SH modification. (2) SITS, a nonpenetrating amino reagent, enhances Na⁺ permeability while DTNB, a nonpenetrating SH modifier, is ineffective. (3) Pretreatment of amino sites decreases the apparent affinity of Hg⁺⁺ for SH sites. In addition, three lines of evidence indicate that TNBS and Zn⁺⁺ modify different amino sites. First, simultaneous modification with TNBS and Zn⁺⁺ results in an increase in Na⁺ permeability equal to the sum of their individual effects. Secondly, Zn⁺⁺ causes an increase in Na⁺ permeability in cells previously treated with TNBS. Finally, the pH dependence of Zn⁺⁺ modification is opposite that for TNBS modification. These pH experiments suggest that Zn⁺⁺ enhances Na⁺ permeability by reacting with unprotonated amino sites while TNBS modifies protonated amino sites. It is concluded that the sodium permeability of dog red blood cells is normally limited by superficial amino sites and deeper sulfhydryl sites in the sodium channels.     

The determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid.

A procedure using 2,4,6-trinitrobenzenesulfonic acid (TNBS) for the determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials is presented. The major modification from previous procedures is an extended TNBS reaction time to allow a stoichiometric reaction with amino groups. In addition, autoclave hydrolysis is used to assure sample dissolution for spectrophotometric measurements. The assay accuracy was evaluated by determining epsilon-amino groups of insulin and bovine albumin. The determinations differed from literature values by < or = 3.3%. The epsilon-amino group content of Type B gelatin was found to be 33.0 mol/gelatin molecule of 1000 residues and is in agreement with similar source gelatins and collagen. The coefficient of variation for determinations on all three materials was < or = 5.3%. The assay should be applicable to a broad range of proteinaceous materials.     

Interaction of chemical probes with sarcoplasmic reticulum membranes

The labelling of the sarcoplasmic reticulum membranes by the chemical probes, trinitrobenzenesulfonate (TNBS) and fluorodinitrobenzene (FDNB) has been investigated. The incorporation of TNBS, but not of FDNB, depends on the binding of Ca2+ or Mg2+ to the membranes. The labelling of lipids and of the various reticulum proteins by TNBS is increased by those agents, but the effect is not uniform for all membrane proteins. The Ca2+ -ATPase contributes only 2.2% for the total labelling of the sarcoplasmic reticulum proteins, whereas the proteins of molecular weight 90 000 and 30 000 contribute about 34 and 56%, respectively. However, the Ca2+-ATPase isolated from the membrane reacts with an amount of TNBS 5-fold higher than that which reacts with the enzyme in situ. Both probes, TNBS and FDNB, inhibit the Ca2+-ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum, whereas the Mg2+-ATPase remains unaffected. The results indicate that FDNB is maximally incorporated into the sarcoplasmic reticulum membrane, whereas only some of the membrane amino groups are accessible to TNBS in the absence of Ca2+, Mg2+ or ATP which, when present, make additional amino groups available to TNBS. The highest degree of TNBS incorporation takes place into proteins, other than the ATPase, but sufficient reaction occurs with the enzyme to inhibit its activity.     

Structure-function relationship of NAD(P)H:quinone reductase: characterization of NH2-terminal blocking group and essential tyrosine and lysine residues

The amino terminal blocked peptide of rat liver NAD(P)H:quinone reductase (DT-diaphorase) was determined by amino acid sequence analysis and by mass spectrometry. The mature protein is composed of 273 amino acids and contains an acetylated amino terminus, which was not identified by previous cDNA analysis. The enzyme was inactivated by p-nitrobenzenesulfonyl fluoride (NBSF) or 2,4,6-trinitrobenzenesulfonate (TNBS) with pseudo-first-order kinetics. These studies suggest that essential tyrosine and lysine may be present in the active site of this enzyme. The NBSF inhibition was protected by 1-naphthol and 1-naphthylamine, but not by NAD+. However, TNBS inhibition was not prevented by the naphthalene derivatives or NAD+. Specific peptides labeled with NBSF or TNBS were isolated by high-performance liquid chromatography and were sequenced. These analyses revealed that the NBSF-labeled tyrosine resides in a predominantly hydrophobic region and TNBS-labeled lysine in a predominantly hydrophilic region.     

Functional groups and quaternary structure of chicken liver xanthine dehydrogenase

Xanthine dehydrogenase from chicken liver is a dimeric enzyme, each hemimolecule containing one FAD and two Fe/S groups. Determination of sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuribenzoic acid (PMB) showed a variable number of sulfhydryl groups depending onpH, ionic strength, and nature of the reaction medium and buffer. The number of disulfide bonds was determined with DTNB and reducing conditions. Amino groups were determined with 2,4,6,-trinitrobencensulfonic acid (TNBS). At constant temperature andpH the reaction of DTNB and TNBS with native xanthine dehydrogenase showed an exponential dependence on time. From the obtained parameters the number of available sulfhydryl and amino groups at infinite concentration of enzyme and the rate constant of the equation were determined. The absorption spectrum of the enzyme changed with time when a chaotropic agent (1 M sodium nitrate) was added to the medium. This difference was detected by measuring the absorbance in the range 450–550 nm. The absorption spectrum (between 350 and 600 nm) also changed when a denaturating agent (sodium dodecyl sulfate) was added. This modification increased with time and depended on the medium.     

A spin-label study on human high density lipoprotein

Human plasma high density lipoproteins (HDL) have been labeled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (NEM-TEMPO). The spin-labeled HDL exhibited an ESR spectrum containing signals of both strongly immobilized and weakly immobilized components by the reaction with a high concentration of NEM-TEMPO, while an ESR spectrum containing only signals of a strongly immobilized component range between 4 degrees C and 37 degrees C, the signal height of the strongly immobilized component exhibited reversible temperature-dependent changes, whereas that of the weakly immobilized component changed irreversibly at temperatures above 25 degrees C. The activation energy of the irreversible change was estimated to be 26 kcal per mol. The strongly immobilized component was derived from NEM-TEMPO which modified apolipoprotein A-I covalently, while the weakly immobilized component was derived from NEM-TEMPO noncovalently bound to HDL. The rate of binding of NEM-TEMPO to either the strongly binding or weakly binding sites and the number of the strongly binding sites in apolipoprotein A-I were estimated to be 125 M-1.day-1 and 1.78, respectively. The binding of NEM-TEMPO to the strongly binding sites was suppressed greatly by pretreatment of HDL with 2,4,6-trinitrobenzene sulfonic acid (TNBS). The slow reaction and suppression with TNBS suggest that NEM-TEMPO binds to some amino acid residue, probably a lysine residue, in apoprotein A-I. The strongly immobilized and weakly immobilized components were reduced almost completely by ascorbate at the same rate, 0.048 min-1 at pH 7.4 and at 4 degrees C.     

An o-phthalaldehyde spectrophotometric assay for proteinases

A rapid and convenient spectrophotometric assay has been devised to measure proteolysis. The assay is based on the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol with amino groups released during proteolysis of a protein substrate. The reaction is specific for primary amines in amino acids, peptides, and proteins, approaches completion within 1 to 2 min at 25 degrees C (half-times of approx 10-15 s), and requires no preliminary heating or separation of the hydrolyzed products from the undegraded protein substrate prior to performing the assay. The OPA assay was relatively as successful as a 2,4,6-trinitrobenzenesulfonic acid (TNBS) procedure in predicting the extent of hydrolysis of a protein substrate. The utility of the OPA method was demonstrated by measuring the degree of proteolytic degradation caused by trypsin, subtilisin, Pronase, and chymotrypsin of various soluble protein substrates. Ethanethiol (instead of 2-mercaptoethanol) or 50% of dimethyl sulfoxide can be included in the assay solution to stabilize certain OPA-amine products. The present method approaches the sensitivity of ninhydrin and TNBS procedures, is more convenient and rapid, and could substitute for these reagents in most assay systems.     

The functional involvement of Lys-38 in the heavy subunit of rat kidney γ-glutamylcysteine synthetase: Chemical modification and mutagenesis studies

Rat kidneyγ-glutamylcysteine synthetase (γGCS) was inactivated by reaction with trinitrobenzene sulfonate (TNBS), and the reaction followed pseudo-first-order kinetics. Inactivation kinetics revealed that only one of the amino acid residues modified by TNBS was essential for-γGCS activity. The addition of 10 mM Mg²⁺ to the TNBS inactivation reaction resulted in a 16-fold increase in the rate of inactivation. Chromatographic analysis on the tryptic hydrolyzates of trinitrophenylated (TNP) derivatives showed that Lys-38 in theγGCS heavy subunit was significantly modified in the presence of Mg²⁺. In contrast to small changes in the catalytic properties observed by mutation of Lys-38 to Arg, the mutants K38N and K38E had a marked decrease in enzymatic activity and about twofold increase inK _m for glutamate. These results suggest that the positively charged Lys-38 may sbe involved in the binding of glutamate toγGCS.     

Monitoring biotransformations in polyamide fibres

The enzymatic hydrolysis of polyamide fibres yields amino and carboxylic groups. These groups can be found in solution treatments as polyamide monomers and soluble oligomers. The amino groups can also be found at the surface of the fibres as end group chains. In this paper we report two methods to quantify the formation of these groups as a result of the enzymatic action. Soluble amino groups can be quantified with 2,4,6-trinitrobenzenesulfonic acid (TNBS), which yields a coloured complex which can be determined spectrophotometrically. The amino groups on the fibre surface can be quantified by reaction with a wool reactive dye and determination of colour intensities after a dyeing procedure below the glass transition temperature of polyamide.