Dinitrophenyl Moiety Research Articles

Dinitrophenyl and NBD platforms based fluorescent sensors for nanomolar detection of zinc ions: Synthesis, zinc ions sensing and DFT studies

The design and synthesis of two new fluorescent probes based on dinitrophenyl (1) and 7-nitrobenz-2-oxa-1,3-diazole (2) platforms is reported. Both probes, containing bis(pyridin-2-ylmethyl)amine as a receptor unit can efficiently detect zin ions (Zn2+) in aqueous solution, with limit of detections of 124 nM and 1.06 µM, respectively. Probe 1 and 2 led to 3-fold and 2.4-fold fluorescent enhancements, respectively, with the addition of Zn2+ which suggested that working mechanism of these probes is based on the inhibition of the photoinduced electron transfer from the electron donating receptor towards the excited fluorophore. Studying binding stoichiometry by job’s plot suggested 1:1 complexation between the probe and Zn2+. Moreover, the formation of probes-Zn2+ complexes was validated by both FT-IR and 1H NMR wherein marked changes in the absorption frequencies as well as distinct downfield shifts in the resonance peaks of pyridine function along with the CH2 group linked directly to the tertiary nitrogen of the DPA unit were observed. Density functional theory (DFT) calculations suggested that the electronic density of the HOMO in 1 was concentrated on the linker between DPA and dinitrophenyl, whereas the density of the LUMO was concentrated on dinitrophenyl moiety. Upon Zn2+ binding, the electronic density in HOMO and LUMO become more delocalized. However, in case of probe 2 the delocalization of electronic density does not appreciably occur. Based on DFT simulations, both sensors form thermodynamic stable complexes with Zn2+ ions. However, sensor 2 exhibited slightly better binding preference toward Zn2+ as compared to 1. The PET based probes exhibited good selectivity to detect Zn2+ in the presence of a variety of competing metal cations. These sensors have higher sensitivity and would be able to detect chronic level of Zn2+ in the freshwater (>1.84 μM) as permitted by the US EPA.

Confining Thiolysis of Dinitrophenyl Ether to a Luminescent Metal-Organic Framework with a Large Stokes Shift for Highly Efficient Detection of Hydrogen Sulfide in Rat Brain.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that regulates various physiological and pathological processes in the central nervous system. It is vital to develop an effective method to detect H2S in vivo to elucidate its critical role. However, current fluorescent probes for accurate quantification of H2S still face big challenges due to complicated fabrication, small Stokes shift, unsatisfactory selectivity, and especially delayed response time. Herein, based on simple postsynthetic modification, we present an innovative strategy by confining H2S-triggered thiolysis of dinitrophenyl (DNP) ether within a luminescent metal-organic framework (MOF) to address those issues. Due to the cleavage of the DNP moiety by H2S, the nanoprobe gives rise to a remarkable fluorescence turn-on signal with a large Stokes shift of 190 nm and also provides high selectivity to H2S against various interferents including competing biothiols. In particular, by virtue of the unique structural property of the MOF, it exhibits an ultrafast sensing ability for H2S (only 5 s). Moreover, the fluorescence enhancement efficiency displays a good linear correlation with H2S concentration in the range of 0-160 μM with a detection limit of 0.29 μM. Importantly, these superior sensing performances enable the nanoprobe to measure the basal value and monitor the change of H2S level in the rat brain.

Synthesis, Insecticidal, Fungicidal Activities and Structure⁻Activity Relationships of Tschimganin Analogs.

For the first time, a novel series of tschimganin analogs were designed, synthesized, and evaluated for their insecticidal and fungicidal activities. Their structures were characterized by 1H-NMR, 13C-NMR and HRMS. Some of these compounds displayed excellent insecticidal and fungicidal activities, suggesting that they have potential to be used as bifunctional agrochemicals. Compound 3d and 3g with electron donating groups showed better inhibitory activity and growth inhibition activity towards Helicoverpa armigera (Hübner). The properties and positions of the substituents on the benzene ring have an important influence on the acaricidal activity of tschimganin analogs. Topomer comparative molecular field analysis (CoMFA) was employed to develop a three-dimensional quantitative structure-activity relationship model for the compounds against Tetranychus turkestani Ugarov et Nikolski. It was indicated that higher electronegativity was beneficial for acaricidal activity. Moreover, compound 3r having a 2-hydroxy-3,5- dinitrophenyl moiety displayed a fungicidal spectrum as broad as azoxystrobin against these phytopathogens.

Development of a novel europium complex-based luminescent probe for time-gated luminescence imaging of hypochlorous acid in living samples

Luminescent lanthanide complexes are key reagents used in the time-gated luminescence bioassay technique, but functional lanthanide complexes that can act as luminescent probes for specifically responding to analytes are very limited. In this work, we designed and synthesized a novel Eu3+ complex-based luminescence probe for hypochlorous acid (HOCl), NPPTTA-Eu3+, by using terpyridine polyacid-Eu3+, dinitrophenyl, and hydrazine as luminophore, quencher and HOCl-recognizer moieties, respectively. In the absence of HOCl, the probe is non-luminescent due to the strong luminescence quenching of the dinitrophenyl group in the complex. However, upon reaction with HOCl, the dinitrophenyl moiety is rapidly cleaved from the probe, which affords a strongly luminescent Eu3+ complex CPTTA-Eu3+, accompanied by a ∼900-fold luminescence enhancement with a long luminescence lifetime of 1.41 ms. This unique luminescence response of NPPTTA-Eu3+ to HOCl allowed NPPTTA-Eu3+ to be conveniently used as a probe for highly selective and sensitive detection of HOCl under the time-gated luminescence mode. In addition, by loading NPPTTA-Eu3+ into RAW 264.7 macrophage cells and Daphnia magna, the generation of endogenous HOCl in RAW 264.7 cells and the uptake of exogenous HOCl by Daphnia magna were successfully imaged on a true-color time-gated luminescence microscope. The results demonstrated the practical applicability of NPPTTA-Eu3+ as an efficient probe for time-gated luminescence imaging of HOCl in living cells and organisms.

Assessment of Protein Carbonylation and Protein Tyrosine Phosphatase (PTP) Oxidation in Vascular Smooth Muscle Cells (VSMCs) Using Immunoblotting Approaches.

Post-translational modification of proteins, such as phosphorylation and oxidation, plays a major role in cellular signaling by influencing protein structure and function. In vascular cells, in addition to influencing phosphorylation, angiotensin II (Ang II) induces oxidation of proteins, important in redox signaling in the cardiovascular and renal systems. The present chapter describes immunoblotting approaches to assess irreversible protein carbonylation and protein tyrosine phosphatase (PTPs) oxidation status in the proteome of vascular smooth muscle cells (VSMC).Protein carbonylation is generally measured using the OxyBlot™ approach, whereby derivatization of protein carbonyl groups (C = O) on oxidized amino acids by dinitrophenylhydrazine (DNPH) results in the formation of a stable dinitrophenyl (DNP) hydrazone product. The samples are analyzed by SDS-PAGE and a primary antibody raised against the DNP moiety is used to determine levels of irreversible protein carbonylation in the sample by immunoblotting.Oxidation of PTPs can be evaluated using a monoclonal antibody against the "hyperoxidized" (SO3H) catalytic site of these enzymes. The described methodology offers the ability to discriminate between irreversible (SO3H) and reversible (SOH) PTP oxidation states. Initially, the free unmodified PTP-thiols (S-) are alkylated and the sample is split into two. One part is used to assess the PTP-SO3H form. In the other part reversibly modified PTP-thiols are first reduced and then hyperoxidized by pervanadate (PV). Both untreated and PV-treated samples are analyzed by SDS-PAGE and "hyperoxidized" PTPs are detected by immunoblotting. The proportion of reversibly oxidized PTP-SOH fraction is determined by the difference between the signals in untreated and the PV-treated samples.The above immunoassays provide general approaches to detect and quantify global levels of irreversible protein oxidation and of irreversibly/reversibly oxidized PTPs in any (patho)physiological context. Characterization of the global redox status is essential to better understand the redox-sensitive mechanisms underlying chronic diseases associated with oxidative stress. This is particularly important in systems influenced by the renin angiotensin system, because Ang II is a potent inducer of oxidative stress and redox signaling.

Dynamic NMR study of dinitrophenyl derivatives of seven-membered cyclic ketals of pyridoxine.

Two pyridoxine derivatives containing a dinitrophenyl moiety were investigated by (1)H NMR spectroscopy. Conformational dynamics in solution were studied for each compound using dynamic NMR experiments. It was shown that both compounds studied are involved into two conformational exchange processes. The first process is a transformation of the seven-membered cycle conformation between the enantiomeric P-twist and M-twist forms, and the second is a rotation of the dinitrophenyl fragment of the molecules around the C-O bond. Energy barriers of both conformational transitions were determined.

D-amino carboxamide-based recruitment of dinitrophenol antibodies to bacterial surfaces via peptidoglycan remodeling.

During the past few decades there has been a rapid emergence of multidrug resistant bacteria afflicting human patients. At the same time, reduced output from pharmaceutical industry in this area precipitated a sharp decrease in the approval of new antibiotics. The combination of these factors potentially compromises the ability to effectively combat bacterial infections. While traditional drug discovery efforts continue in the pursuit of small molecule agents that disrupt bacterial growth, non-traditional efforts could serve to complement antimicrobial strategies. We recently demonstrated our ability to remodel the surface of bacterial cells using unnatural D-amino acids displaying the antigenic dinitrophenyl (DNP) handle. These immune stimulant D-amino acids derivatives were metabolically incorporated onto the peptidoglycan of bacteria via a promiscuous surface-anchored transpeptidase. The covalent modification of DNP moieties onto the peptidoglycan led to the anti-DNP antibody opsonization of the bacterial cell surface. Herein, we show that the amidation of the C-terminus to generate DNP-displaying D-amino carboxamide drastically improves antibody recruitment. Antibody opsonization using the D-amino carboxamide agent is observed at lower concentrations than the D-amino acid counterpart. In addition, the recruitment of endogenous antibodies in pooled human serum to the DNP-modified bacterial cell surface is demonstrated for the first time. We envision that the C-terminus amidation of DNP-conjugated D-amino acids could potentially facilitate translation of these results to in vivo animal disease models.

Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.

Antibodies as Drug Carriers III: Design of Oligonucleotides with Enhanced Binding Affinity for Immunoglobulin G

To understand the structural requirements in designing epitope-bearing oligonucleotides with high antibody-binding affinity. Binding affinity (KA) and stoichiometry (n) of dinitrophenyl (DNP)-derivatized model 27-mer oligonucleotides (ODNs), GGG(AAA)7GGG, to monoclonal anti-trinitrophenyl (TNP) antibodies were determined using isothermal titration calorimetry (ITC). Structural variations were made in the ODNs to assess the effects of antigenic valence, epitope density, inter-epitope linker length, and linker flexibility. Binding isotherms were fitted with a single binding-site model to obtain K(A) and n, from which changes in Gibbs free energy (deltaG(0)), entropy (deltaS(0)), and enthalpy (deltaH(0)) were derived. As expected, ligands displaying increased epitope density showed increases in K(A): for example, K(A) for (DNP)2-Cys is 3.3-fold greater than that for DNP-Lys. Introduction of multiple DNP groups via long and flexible linkers to one end of the 27-mer ODN resulted in a bivalent behavior with n value of 1. A bivalent ligand, derivatized at both ends with a long and flexible linker, failed to form an immune complex when hybridized to its antisense strand, presumably due to intercalation of the DNP moiety to the double strand. ODNs derivatized with flexible linkers exhibited a higher K(A) than those with a rigid linker. Ligands with flexible inter-epitope linkers measuring distances of 110, 60, and 40 angstroms yielded 13-, 30-, and 13-fold increases in K(A), respectively. The combination of these factors; namely, bivalence, flexible inter-epitope linkers, and optimal inter-epitope distance, resulted in an overall 66-fold increase in K(A). Thermodynamic analysis of binding indicates that the formation of high-affinity ODN-IgG complexes was a spontaneous and exothermic event, characterized by large negative deltaS degrees, deltaH degrees, and deltaG degrees values. All four strategies tested during this investigation, namely bivalence, epitope density, inter-epitope linker flexibility, and optimal inter-epitope distance, proved to be useful in improving the binding affinity of DNP-labeled ODNs to anti-TNP IgG. The final ODN design incorporating these strategies will be used in testing the systemic pharmacokinetic advantage gained from complexing such ODNs to IgG.

Rapid Induction of Cell Death by Selenium-compromised Thioredoxin Reductase 1 but Not by the Fully Active Enzyme Containing Selenocysteine

Mammalian thioredoxin reductases are selenoproteins. For native catalytic activity, these enzymes utilize a C-terminal -Gly-Cys-Sec-Gly-COOH sequence (where Sec is selenocysteine) forming a redox active selenenylsulfide/selenolthiol motif. A range of cellular systems depend upon or are regulated by thioredoxin reductase and its major protein substrate thioredoxin, including apoptosis signal-regulating kinase 1, peroxiredoxins, methionine sulfoxide reductase, and several transcription factors. Cytosolic thioredoxin reductase 1 (TrxR1) is moreover inhibited by various electrophilic anticancer compounds. TrxR1 is hence generally considered to promote cell viability. However, several recent studies have suggested that TrxR1 may promote apoptosis, and the enzyme was identified as GRIM-12 (gene associated with retinoid interferon-induced mortality 12). Transient transfection with GRIM-12/TrxR1 was also shown to directly induce cell death. To further analyze such effects, we have here employed lipid-mediated delivery of recombinant TrxR1 preparations into human A549 cells, thereby bypassing selenoprotein translation to facilitate assessment of the protein-related effects on cell viability. We found that selenium-deficient TrxR1, having a two-amino acid-truncated C-terminal -Gly-Cys-COOH motif, rapidly induced cell death (38 +/- 29% apoptotic cells after 4 h; p < 0.005 compared with controls). Cell death induction was also promoted by selenium-compromised TrxR1 derivatized with either cis-diamminedichloroplatinum (II) (cisplatin) or dinitrophenyl moieties but not by the structurally related non-selenoprotein glutathione reductase. In contrast, TrxR1 with intact selenocysteine could not promote cell death. The direct cellular effects of selenium-compromised forms of TrxR1 may be important for the pathophysiology of selenium deficiency as well as for the efficacy of antiproliferative drugs targeting the selenocysteine moiety of this enzyme.

Benzaldehyde 2,4-dinitrophenylhydrazone.

Crystals of the title compound, C(13)H(10)N(4)O(4), were obtained from a condensation reaction of benzaldehyde and 2,4-dinitrophenylhydrazine. The molecule assumes an approximately planar E configuration. Within the dinitrophenyl moiety, the average distance for the aromatic C-C bonds close to the imino group [1.417 (3) A] is appreciably longer than the average distance for the other aromatic C-C bonds in the same phenyl ring [1.373 (3) A]. This increased distance may be a result of the overlap of the non-bonding orbital of the imino N atom with the pi orbitals of the arene. It is likely that pi-pi stacking exists in the crystal structure.

An overshoot-hump in a ?-A curve and the cooperative aggregation among dinitrophenyl moieties in phospholipids at an air/water interface

An overshoot-hump in a ?-A curve and the cooperative aggregation among dinitrophenyl moieties in phospholipids at an air/water interface

Single-cell investigation by laser scanning confocal microscopy of cytochemical alterations resulting from extracellular oxidant challenge

Confocal laser scanning fluorescence microscopy coupled to image analysis was employed in order to develop and evaluate procedures for the appraisal at the single-cell level of: (1) protein-bound 4-hydroxynonenal, the specific product of membrane peroxidation (by means of immunocytochemistry with biotin-avidin revelation); (2) protein oxidation (by reaction of protein carbonyls with 2, 4-dinitrophenyl-hydrazine followed by immunocytochemistry of dinitrophenyl moieties); and (3) cellular protein thiols (by direct alkylation of sulfhydryl groups with thiol-specific fluorescent reagents possessing different cell permeabilities). The procedures proved able to reveal the subcellular distribution of cytochemical parameters useful as indices of oxidative stress conditions, and may allow "redox phenotyping" of isolated cells, which would provide an efficient tool in selected experimental models.

New probes for angiotensin II receptors. Synthesis, radioiodination and biological properties of biotinylated and haptenated angiotensin derivatives.

The present work delineates the basis for chemical modifications which can be introduced on the angiotensin II (AII) molecule to design probes suitable for indirect affinity techniques, especially for receptor purification. Using the solid-phase synthesis strategy, biotin or dinitrophenyl moieties have been added at the N-terminus of AII, with aminohexanoic acid as spacer arm. The resulting probes, (6-biotinylamido)hexanoyl-AII (Bio-Ahx-AII) and dinitrophenylaminohexanoyl-AII (Dnp-Ahx-AII), were prepared in their monoiodinated and highly labelled radioiodinated forms, with possible sulphoxidation of biotin. In addition to their ability to interact with streptavidin and anti-Dnp antibodies respectively, the two ligands displayed almost unchanged affinities for hepatic AII receptors as compared with AII. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. The potential applications of these probes, receptor purification, cell labelling and sorting and histochemical receptor visualization, are discussed.

Design of Angiotensin II Derivatives Suitable for Indirect Affinity Techniques: Potential Applications to Receptor Studies

The design of angiotensin II (A II)-derived probes suitable for indirect affinity techniques is presented. Biotin or dinitrophenyl moieties have been added at the N-terminus of A II, through aminohexanoic acid as spacer arm, to generate (6-biotinylamido)-hexanoyl-AII (Bio-Ahx-AII) and dinitrophenyl- aminohexanoyl-AII (Dnp-Ahx-AII). Monoiodinated and highly labeled radioiodinated forms of these probes have been prepared. The two bifunctional ligands displayed high affinities for rat liver A II receptors (Kd values in the nanomolar range) and their secondary acceptors: streptavidin and monoclonal anti-Dnp antibodies respectively. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. Based on these structural assessments, the parent photoactivable azido probe: Bio-Ahx-(Ala1,Phe(4N3)8)A II. A II was synthesized and proved to possess similar biological properties than the non-azido compound. The hepatic A II receptor could be covalently labeled by the radioiodinated probe, with a particularly high yield (15-20%); SDS-polyacrylamide gel electrophoresis of solubilized complexes revealed specific labeling of a 65 Kdaltons binding unit, in agreement with previous data obtained with other azido AII-derived compounds. The potential applications of these probes are: i) receptor purification by combination of its photoaffinity labeling and adsorption of biotin-tagged solubilized hormone-receptor complexes on avidin gels. ii) cell labeling and sorting. iii) histochemical receptor visualization.

PREFERENTIAL EXPRESSION OF CATFISH LIGHT CHAIN IMMUNOGLOBULIN ISOTYPES IN ANTI‐DINITROPHENYL ANTIBODIES

Previous studies indicated that immunoglobulins of the channel catfish, Ictalurus punctatus, contained two distinct isotypes of light chains. These different light chains, designated F and G, are reminiscent of kappa and lambda light chains of higher animals. This study was undertaken to establish whether or not there was preferential expression of one of the light chains in antibodies induced to the dinitrophenyl moiety during the course of the catfish humoral antibody response. The results indicated that, although antibody produced very early (1-2 weeks) after primary immunization contained significant amounts (approximately 20%) of G light chains, the vast majority (approximately 90%) of antibody produced later (3 weeks through 1 year) was of the F isotype. Thus fish, as well as higher vertebrates, can express different isotypes of antibody during the immune response.

Dinitrophenyl-pepstatins as active-site-directed localization reagents for cathepsin D.

1. N-Pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane, a potential active-site-directed localization reagent for cathepsin D, was found to bind non-specifically to immuno-precipitates containing cathepsin D. 2. Three new water-soluble localization reagents were synthesized, by using NN'-bis-(3-aminopropyl)piperazine, 3-oxa-1,5-diamino-pentane or 3,6-dioxa-1,8-diamino-octane, as spacer arms between the pepstatin and dinitrophenyl moieties. 3. The hydrophilic dinitrophenyl-pepstatins were all tight-binding inhibitors of cathepsin D at pH 3.5, but showed little or no binding to immuno-precipitates containing the inactive enzyme at pH 7.4. 4. Gel-chromatographic experiments showed that, at pH 5.0, all the dinitrophenyl-pepstatins were bifunctional reagents able to bind cathepsin D and anti-dinitrophenyl antibody at the same time. Enzyme-inhibitor-antibody complexes were not formed at pH 7.4, thus confirming that the reagents were active-site-directed. 5. Cultured human synovial cells were fixed and incubated with the dinitrophenyl-pepstatins at pH 5.0 or pH 7.4. After washing briefly, the cells were incubated at the appropriate pH value with anti-dinitrophenyl antibody labelled with fluorescein. When examined by fluorescence microscopy the cells stained at pH 5.0 showed fluorescent perinuclear granules, which were not seen in the cells treated at pH 7.4. The distribution of cathepsin D, determined by indirect immuno-fluorescence at pH 7.4, closely resembled that revealed by the dinitrophenyl-pepstatins at pH 5.0. 7. NN'-(3-Pepstatinylaminopropyl-3'-dinitrophenylaminopropyl)piperazine gave the most intense lysosomal staining and showed no non-specific binding. We conclude that this reagent is suitable for the subcellular localization of the active conformation of cathepsin D.

Inhibition of tubulin self-assembly in vitro by fluorodinitrobenzene

The self-assembly of bovine brain tubulin into microtubules is inhibited by low molar ratios of 1-fluoro-2,4-dinitrobezene (FDNB). Binding studies using [ 14C]FDNB indicate that the incorporation of between one and two dinitrophenyl groups is sufficient to inhibit assembly completely, although more dinitrophenyl groups can be incorporated using higher FDNB/tubulin ratios. Dinitrophenyltubulin, under assembly conditions, tends to assemble into amorphous aggregates. Thiolysis by 2-mercaptoethanol removes the dinitrophenyl moieties. Paper chromatography and high-voltage electrophoresis of acid-hydrolyzed modified tubulin containing one dinitrophenyl group identified the residue as S- dinitrophenylcysteine . The β-monomer of tubulin is preferentially modified at low FDNB/tubulin ratios. The reaction with FDNB is greatly reduced when tubulin is in polymerized form. FDNB reduces the colchicine binding activity but does not affect the Mg(II) content of the protein.

Immunological properties of dinitrophenyl derivatives of beta-subunit of the human chorionic gonadotropin

Four different derivatives with varying proportions of dinitrophenyl (DNP) groups tagged covalently to β-subunit of HCG (DNP (1)-β-HCG, DNP (3.3)-β-HCG, DNP (6)-β-HCG and DNP (10)-β-HCG) have been prepared. Their immunogenicity as well as the reactivity of antibodies formed in mice, rabbit and rhesus monkey have been studied. The derivatives give rise to the formation of antibodies in all species tested which react with HCG and in some species with DNP moieties. The anti-DNP response increases with derivatives bearing more than 3–4 DNP groups per molecule of β-HCG. On the other hand the anti-HCG response diminishes with derivatives bearing higher than 3–4 DNP groups per β-HCG molecule. The antibody response in monkeys is of long duration (> 14 months). The association constants for binding with HCG are of the order of 4 × 10 9 to 6 × 10 10 L/mole. the affinity of the antibodies tends to increase with time.

Affinity labelling to - SH groups in adenosine - triphosphate - phosphoribosyl transferase with the dinitrophenyl group from S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate.

Adenosine-triphosphate-phosphoribosyl transferase from Escherichia coli reacts with S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate. In this reaction the dinitrophenyl group becomes attached to the enzyme, while the nucleotide is split off. Most aliphatic high and low-molecular-weight-SH compounds react with the thioether in the opposite way, i.e. bind the nucleotide and split off dinitrothiophenol. It appears that the dinitrophenyl moiety of the thioether interacts with the enzyme in a specific way, and that this interaction activates the bond between the dinitrophenyl group and the sulfur atom. In support of this it was found that dinitrophenol inhibits the transferase reaction with half maximal effect at 0.4 mM. The inhibition is competitive with ATP. Dinitrophenol also competes with ATP in binding studies.