Thiol Binding Site Research Articles

Analysis of total thiol concentrations in environmental samples using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)

In this study, we developed an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) approach to measure the concentration of total reactive thiol binding sites in a range of types of environmental samples by monitoring the decrease of monobromo-(trimethylammonio)-bimane (qBBr) caused by thiol-qBBr binding reactions. We first examined the stability of qBBr and potential side reactions that could reduce the accuracy of the UHPLC-MS approach. We found that significant loss of qBBr occurs in the absence of thiols in aqueous solutions after 2–4 h due to qBBr degradation and other side reactions, such as a chloride-for-bromide substitution reaction. To address these issues, in our approach we conduct the thiol-qBBr binding reaction in the presence of excessive qBBr for only 1 h, and we compare the MS signal of unbound qBBr in a thiol-free qBBr standard with that in a thiol-bearing qBBr standard in order to calculate the decrease in the concentration of free qBBr in solution that is caused by thiol-qBBr binding. Using 100–600 nM of N-Acetyl-L-cysteine (ACYS) as a model thiol in a 2 μM qBBr standard, we found that the measured decrease in the MS signal of unbound qBBr linearly correlates to the concentration of added ACYS, with a R2 of 0.988, demonstrating that monitoring the decrease in unbound qBBr can be used to calculate the concentration of total thiol sites in a sample. We demonstrate that the new UHPLC-MS approach yields accurate results when measuring a series of single and mixed thiol standards in the 200–500 nM range, with measured values ranging from 86% to 109% of the known concentration of thiols. In addition to the above control tests, we also used the UHPLC-MS approach to measure dissolved thiol concentrations in several lake water samples. These measurements revealed that each lake contained low μM levels of total thiols, not only demonstrating the ability of the analytical approach to determine thiol site concentrations in natural waters, but also suggesting the widespread importance of these sites in controlling chalcophile element behavior in these systems. The UHPLC-MS approach not only can be used to determine the concentration of dissolved thiol sites at nM levels, but can also be adapted to measure the concentration of thiol binding sites on other environmental samples such as soil, sediment, and solid-phase organic matter surfaces.

Effect of deacetylated konjac glucomannan on heat-induced structural changes and flavor binding ability of fish myosin

This work investigated the effects of deacetylated konjac glucomannan (DKGM) on heat-induced structural changes and flavor binding in bighead carp myosin. DKGM could cross-link with fish myosin to form a thermostable complex and improve the gel strength of myosin. The incorporation of DKGM increased the surface hydrophobicity and total sulfhydryl content of heat-induced myosin. Increasing DKGM concentrations resulted in a decrease in the absolute zeta potential and a continuous increase in particle size. DKGM addition significantly reduced the α-helical content of myosin with a concomitant increase in β-sheet, β-turn, and random coil content. The binding abilities of myosin to flavors were significantly enhanced by increasing amounts of DKGM, attributing to the accelerative unfolding of myosin secondary structures and the exposure of additional hydrophobic and thiol binding sites. Increased numbers of available hydroxyl groups after DKGM treatment could also cause an increase of flavor adsorption by hydrogen bonding.

Electrodeposited MoS2 Thin Films for Stable Immobilization of Thiolated Biomolecules

We report a robust new combination of substrate materials and immobilization chemistry for biosensing that addresses one of the oldest challenges in this field, the stability and reproducibility of biomolecule immobilization. The substrate materials are electrodeposited MoS2 and Cu-doped MoS2 thin films atop glassy carbon electrodes (GCE) created by a two-step process of cathodic electrodeposition, followed by high temperature annealing. Electrodeposition atop a GCE is preceded by an electrochemical pre-treatment process to increase the surface roughness and introduce oxygen functional groups, dramatically improving film adhesion. MoS2 thin films are electrodeposited from 10 mM (NH4)2MoS4 and 0.2 M KCl at pH 6.8, while Cu-doped MoS2 thin films are electrodeposited from 10.0 mM (NH4)2MoS4, 5.0 mM CuSO4, 0.10 M KCl, and 0.50 M KSCN at pH 6.95. Since MoS2 is an active hydrodesulfurization (HDS) catalyst for sulfur removal from hydrocarbons, both thin film materials immediately react with and desulfurize the electrolyte from which they are deposited. The desired MoS2 stoichiometry, which includes coordinately unsaturated Mo sites, can be recovered by high temperature annealing in a reducing environment. High temperature annealing removes the excess sulfur from the as-deposited film, and also partially recrystallizes and partially orients the MoS2 thin films, as suggested by x-ray diffraction (XRD) studies. This annealing procedure regenerates the coordinately unsaturated and highly sulfiphilic Mo sites atop HDS catalyst materials, which can then form strong bonds to thiolated biomolecules. We demonstrate this by disulfide bond reduction at the hinge region of polyclonal antibodies utilizing tris (2-carboxyethyl) phosphine (TCEP) as the reducing agent. Unlike other reducing agents for disulfide bond cleavage, which contain thiol groups, TCEP probably does not compete with antibody fragments for thiol binding sites atop the MoS2 surface. The attached figure illustrates the biosensing platform described here. This new biosensing platform is demonstrated by disulfide bond reduction within polyclonal antibodies to 3-phenoxybenzoic acid (3-PBA), binding of these antibody fragments to newly created MoS2 and Cu-doped MoS2 thin films, and detection of the 3-PBA antigen using electrochemical impedance spectroscopy (EIS). Control experiments demonstrate that only the annealed MoS2 and Cu-doped MoS2 thin films are active towards binding of antibody fragments. In both cases, the charge transfer resistance (Rct) is the equivalent circuit element from the Randles circuit that is most sensitive to antigen binding. Atop un-doped MoS2, the sensitivity for 3-PBA detection by EIS is 2.7x108 Ω-cm2-M-1, and the detection limit is 2.5x10-6 M. Atop Cu-doped MoS2, the sensitivity for 3-PBA detection is 5.9x108 Ω-cm2-M-1, and the detection limit is 3.8x10-6 M. The variation of Rct with antigen concentration atop MoS2 is consistent with the Langmuir adsorption isotherm, with an initial linear relationship, and Rct eventually saturating at high concentrations when all binding sites are occupied. However, two plateau regions are reproducibly observed atop Cu-doped MoS2. The rms surface roughness obtained by atomic force microscopy (AFM) measurements atop MoS2 and Cu-doped MoS2 substrates ranges from 60–140 nm, so these methods are not limited to ultra-smooth substrates. Figure 1

Structural Characterization of the Xi Class Glutathione Transferase From the Haloalkaliphilic Archaeon Natrialba magadii.

Xi class glutathione transferases (GSTs) are a recently identified group, within this large superfamily of enzymes, specifically endowed with glutathione-dependent reductase activity on glutathionyl-hydroquinone. Enzymes belonging to this group are widely distributed in bacteria, fungi, and plants but not in higher eukaryotes. Xi class GSTs are also frequently found in archaea and here we focus on the enzyme produced by the extreme haloalkaliphilic archaeon Natrialba magadii (NmGHR). We investigated its function and stability and determined its 3D structure in the apo form by X-ray crystallography. NmGHR displays the same fold of its mesophilic counterparts, is enriched in negatively charged residues, which are evenly distributed along the surface of the protein, and is characterized by a peculiar distribution of hydrophobic residues. A distinctive feature of haloalkaliphilic archaea is their preference for γ-glutamyl-cysteine over glutathione as a reducing thiol. Indeed we found that the N. magadii genome lacks a gene coding for glutathione synthase. Analysis of NmGHR structure suggests that the thiol binding site (G-site) of the enzyme is well suited for hosting γ-glutamyl-cysteine.

Rational design of fluorescent probe for Cys dynamics imaging in living cell

We report the first recyclable fluorescent probe for rapid and specific fluorescent detection of Cys. We utilize unsaturated olefinic bond as thiol binding site and carboxyl group as terminal anchor to fix the amino group of Cys to synthesize probe 1 featured reversible and specificity toward Cys probes through nucleophilic addition reaction. The detection limit of probe 1 for Cys was found to be ∼0.122 μM. What's more, this probe can be recycled five times or more in the presence of NEM or hydrogen peroxide, indicating that probe 1 could reflect Cys dynamics in the detection system on line, especially for living cells applications.

Homocysteine Editing, Thioester Chemistry, Coenzyme A, and the Origin of Coded Peptide Synthesis †.

Aminoacyl-tRNA synthetases (AARSs) have evolved “quality control” mechanisms which prevent tRNA aminoacylation with non-protein amino acids, such as homocysteine, homoserine, and ornithine, and thus their access to the Genetic Code. Of the ten AARSs that possess editing function, five edit homocysteine: Class I MetRS, ValRS, IleRS, LeuRS, and Class II LysRS. Studies of their editing function reveal that catalytic modules of these AARSs have a thiol-binding site that confers the ability to catalyze the aminoacylation of coenzyme A, pantetheine, and other thiols. Other AARSs also catalyze aminoacyl-thioester synthesis. Amino acid selectivity of AARSs in the aminoacyl thioesters formation reaction is relaxed, characteristic of primitive amino acid activation systems that may have originated in the Thioester World. With homocysteine and cysteine as thiol substrates, AARSs support peptide bond synthesis. Evolutionary origin of these activities is revealed by genomic comparisons, which show that AARSs are structurally related to proteins involved in coenzyme A/sulfur metabolism and non-coded peptide bond synthesis. These findings suggest that the extant AARSs descended from ancestral forms that were involved in non-coded Thioester-dependent peptide synthesis, functionally similar to the present-day non-ribosomal peptide synthetases.

Aminoacyl-tRNA synthetases and the evolution of coded peptide synthesis: the Thioester World.

Coded peptide synthesis must have been preceded by a prebiotic stage, in which thioesters played key roles. Fossils of the Thioester World are found in extant aminoacyl-tRNA synthetases (AARSs). Indeed, studies of the editing function reveal that AARSs have a thiol-binding site in their catalytic modules. The thiol-binding site confers the ability to catalyze aminoacyl~coenzyme A thioester synthesis and peptide bond formation. Genomic comparisons show that AARSs are structurally related to proteins involved in sulfur and coenzyme A metabolisms and peptide bond synthesis. These findings point to the origin of the amino acid activation and peptide bond synthesis functions in the Thioester World and suggest that the present-day AARSs had originated from ancestral forms that were involved in noncoded thioester-dependent peptide synthesis.

Gold-phosphine binding to de novo designed coiled coil peptides

The coordination of the therapeutically interesting [AuCl(PEt3)] to the de novo designed peptide, TRIL23C, under aqueous conditions, is reported here. TRIL23C represents an ideal model to investigate the binding of [AuCl(PEt3)] to small proteins in an effort to develop novel gold(I) phosphine peptide adducts capable of mimicking biological recognition and targeting. This is due to the small size of TRIL23C (30 amino acids), yet stable secondary and tertiary fold, symmetric nature and the availability of only one thiol binding site. [AuCl(PEt3)] was found to react readily with the Cys side chain in a 1:1 ratio as confirmed by UV-visible, 31P NMR and mass spectrometry. Circular dichroism confirmed that the coiled coil structure was retained on coordination of the {Au(PEt3)}+ unit. Redesign of the exterior of TRIL23C based on a biologically relevant recognition sequence found in GCN4, did not alter the coordination chemistry of [AuCl(PEt3)]. To the best of our knowledge, this represents the first report on the coordination of gold(I) phosphine compounds to de novo designed peptides, and could lead to the generation of novel gold(I) phosphine peptide therapeutics in the future.

Synthesis of Dense Poly(acrylic acid) Brushes and Their Interaction with Amine-Functional Silsesquioxane Nanoparticles

Poly(acrylic acid) polyelectrolyte brushes were synthesized by surface-initiated atom transfer radical polymerization (SI-ATRP) of tert-butyl acrylate on planar gold surfaces and subsequent hydrolysis. Three types of monolayers with different numbers of thiol binding sites per initiating unit were used. The binding strength to the gold surface turned out to be of crucial importance for the formation of uniform brush layers after acidic hydrolysis. The monolayers and polymer brushes were characterized by ellipsometry, infrared spectroscopy, water contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. Their interaction with [(diglycidylamino)propyl]silsesquioxane nanoparticles at various pH values was studied by surface plasmon resonance.

Automated Simultaneous Analysis of Monomethyl and Mercuric Hg in Biotic Samples by Hg-Thiourea Complex Liquid Chromatography Following Acidic Thiourea Leaching

A simple leaching procedure has been validated for quantitative isolation of both monomethyl (CH3Hg+) and inorganic (Hg(II)) mercuryfrom fresh or dried biotic tissue for simultaneous analysis via separation and quantification with Hg-thiourea complex liquid chromatography cold vapor atomic fluorescence spectrometry (HgTU/LC-CVAFS). The leaching solution comprises thiourea, hydrochloric acid, and glacial acetic acid and works by protonating thiol binding sites and forming water-soluble cationic CH3HgS=C(NH2)2+ and Hg[S=C(NH2)2]2(2+) complexes, which are easily separated from the solid matrix. The isolated complexes are preconcentrated online by either thiol resin trapping or a new iodide-complex polydivinylbenzene resin trapping (I-PDVB). The I-PDVB trapping involves only one reagent addition, requires no pH adjustments, and is quantitative over a large range of volumes and flow rates. The chromatography system can use either ion chromatography or a new ion-pairing reversed phase separation coupled to cold vapor generation and atomic fluorescence detection. The system allows quantitative sample introduction and yields absolute detection limits of 0.4 pg and 0.7 pg, for CH3Hg+ and Hg(II) respectively, enabling relative detection limits as low as 4 and 7 pg g(-1) with 100 mg samples, and yields % CV routinely less than 5% with well homogenized samples. Accuracy for both forms of mercury has been validated with multiple biotic reference materials and by comparison of the sum of CH3Hg+ and Hg(II) with total Hg on a variety of different biotic sample types (n = 49). The system can be calibrated with either aqueous standards or leached reference materials.

Quantitative Study of the Effects of Surface Ligand Concentration on CdSe Nanocrystal Photoluminescence

We study the impact of surface chemistry on the photoluminescence (PL) quantum yield of CdSe core, CdSe/CdS core/shell, and CdSe/CdZnS/ZnS core/shell/shell nanocrystals prepared by multiple synthetic routes. We expose as-synthesized particles to varying concentrations of n-alkylamine and n-alkanethiol ligands and verify that the addition of n-alkanethiols to CdSe and CdSe/CdS nanocrystal solutions quenches their PL. We also show that the addition of n-alkylamines to nanocrystal solutions can increase or decrease nanocrystal PL, an effect that depends on the concentration of both nanocrystals and ligands. We demonstrate the importance of considering the nanocrystal concentration when fitting ligand binding curves, and show that common solvent impurities can affect the PL and ligand binding data. While alkanethiols quench CdSe nanocrystals prepared using multiple synthetic procedures, we find the exact shape of the quenching curve depends on the synthetic route chosen. We emphasize that the ligand binding data extracted from PL quenching curves are contingent on the assumptions made during fitting. By fitting our PL quenching curves to a Langmuir isotherm and accounting for the particle surface sites, we estimate a lower limit for the equilibrium CdSe−alkanethiol binding constant on the order of 109 M-1 with different numbers of thiol binding sites depending on the method of nanocrystal synthesis.

Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by β-mercaptoethanol for specific staining of tetracysteine-tagged proteins

Recent advances in the field of small molecule labels for live cell imaging promise to overcome some of the limitations set by the size of fluorescent proteins. We tested the tetracysteine-biarsenical labeling system in live cell fluorescence microscopy of reggie-1/flotillin-2 in HeLa and N2a cells. In both cell types, the biarsenical staining reagent FlAsH/Lumio Green accumulated in active mitochondria and led to mitochondrial swelling. This is indicative of toxic side effects caused by arsenic, which should be considered when this labeling system is to be used in live cell imaging. Mitochondrial accumulation of FlAsH/Lumio Green was reversed by addition of low concentrations of thiol-containing reagents during labeling and a subsequent high stringency thiol wash. Both ethanedithiol and beta-mercaptoethanol proved to be effective. We therefore established a staining protocol using beta-mercaptoethanol as thiol binding site competitor resulting in a specific staining of tetracysteine-tagged reggie-1/flotillin-2 of adequate signal to noise ratio, so that the more toxic and inconvenient ethanedithiol could be avoided. Furthermore, we show that staining efficiency was greatly enhanced by introducing a second tetracysteine sequence in tandem.

Screening of Raw Coffee for Thiol Binding Site Precursors Using “In Bean” Model Roasting Experiments

The purpose of the following study was to investigate the influence of coffee roasting on the thiol-binding activity of coffee beverages, and to investigate the potential of various green bean compounds as precursors of thiol-binding sites by using promising "in bean" model roast experiments. Headspace gas chromatographic analysis on coffee brews incubated in the presence of the roasty-sulfury smelling 2-furfurylthiol for 20 min at 30 degrees C in septum-closed vessels revealed that the amounts of "free" thiol decreased drastically with increasing the roasting degree of the beans used for preparation of the brews. A half-maximal binding capacity (BC(50)) of 183 mg of 2-furfurylthiol per liter of standard coffee beverage was determined for a roasted coffee (CTN value of 67), thus demonstrating that enormous amounts of the odor-active thiol are "bound" by the coffee. Furthermore, biomimetic "in bean" precursor experiments have been performed in order to elucidate the precursor for the thiol-binding sites in the raw coffee bean. These experiments opened the possibility of studying coffee model reactions under quasi-natural roasting conditions and undoubtedly identified chlorogenic acids as well as thermal degradation products caffeic acid and quinic acid as important precursors for low-molecular-weight thiol-binding sites. In particular, when roasted in the presence of transition metal ions, chlorogenic acids and even more caffeic acid showed thiol-binding activity which was comparable to the activity measured for the authentic coffee brew.

Characterization of metal ion-exchange on modified surfaces of porous carbon

Two biologically based ligands, cysteine (Cys) and poly-l-cysteine (PLCys), were coupled with Carbopack-X, a commercially available porous carbon surface after acid activation, through simple cross-linking techniques. The resulting Cys-X and PLCys-X systems are effective for metal binding with capacities in the range of 1–25μmol metal/g column, depending on the metal tested. Both columns demonstrate similar selectivity for the soft and moderately soft acid metals such as Cd2+, Cu+, and Pb2+, probably as a result of the soft basic character of the thiol ligand side chains, and exhibit minimal binding of the harder groups IA and IIA metals. Even though PLCys-X and Cys-X displayed similar strong and weak binding characteristics, the PLCys-X system contained stronger binding sites than those possessed by Cys-X. The strongest site displayed by Cys-X has a binding constant of 1×109, with the ability to compete for metal with moderately strong ligands such as ethylenediamine. However, the strongest PLCys-X site possessed a binding constant of about 1013, with the ability to compete for metals bound by very strong ligands such as EDTA. Both systems gave quantitative release of bound metals upon exposure to acid. Exposing the thiol-rich ligands to chemical reducing or oxidizing environments altered the metal binding characteristics of both columns, with at least a 50% decrease in metal binding on the oxidized columns due to thiol binding sites because of disulfide bond formation.

Determination of ligand binding constants for the iron-molybdenum cofactor of nitrogenase: monomers, multimers, and cooperative behavior.

Equilibrium titrations in N-methylformamide (NMF) of G-25 gel filtered (ox)-state FeMo cofactor [FeMoco(ox)] from Azotobacter vinelandii nitrogenase were carried out using sodium ethanethiolate and followed using UV/Vis absorption spectroscopy. For Fe-Moco(ox), a non-linear least squares (NLLSQ) fit to the data indicated a strong equilibrium thiolate-binding step with Keq = 1.3+/-0.2x10(6) M(-1). With 245 molar excess imidazole, cooperative binding of three ethanethiolates was observed. The best NLLSQ fit gave Keq=2.0+/-0.1x10(5) M(-2) and a Hill coefficient n=2.0+/-0.3. A Scatchard plot of these data was concave upward, indicating positive cooperativity. The fit to previously published data involving benzenethiol titration of the one-electron reduced (semi-reduced) cofactor, FeMoco(sr), as followed by EPR required a model that included both a sub-stoichiometric ratio of thiol to FeMoco(sr) and about five cooperative ligand binding sites. These constraints were met by modeling FeMoco(sr) as an aggregate, with fewer thiol binding sites than FeMoco(sr) units. The best fit model was that of FeMoco(sr) as a dodecamer with five cooperative benzenethiol binding sites, yielding a thiol binding constant of 3.32+/-0.09x10(4) M(-4.8) and a Hill coefficient n=4.8+/-0.6. The results of all the other published ligand titrations of FeMoco(sr) were similarly analyzed successfully in terms of equilibrium models that include both cooperative ligand binding and dimer-level aggregation. A possible structural model for FeMoco aggregation in NMF solution is proposed.

Uptake of Choline and Its Conversion to Glycine Betaine by Bacteria in Estuarine Waters

The uptake and degradation of nanomolar levels of [methyl-C]choline in estuarine water samples and in seawater filtrate cultures composed mainly of natural free-living bacteria was studied. Uptake of [C]choline exhibited Michaelis-Menten kinetics, with K(t) + S(n) values of 1.7 to 2.9 nM in filtrate cultures and 1.7 to 4.1 nM in estuarine-water samples. V(max) values ranged from 0.5 to 3.3 nM . h. The uptake system for choline in natural microbial assemblages therefore displays very high affinity and appears able to scavenge this compound at the concentrations expected in seawater. Uptake of choline was inhibited by some natural structural analogs and p-chloromercuribenzoate, indicating that the transporter may be multifunctional and may involve a thiol binding site. When 11 nM [C]choline was added to water samples, a significant fraction (>50%) of the methyl carbon was respired to CO(2) in incubations lasting 10 to 53 h. Cells taking up [C]choline produced [C]glycine betaine ([C]GBT), and up to 80% of the radioactivity retained by cells was in the form of GBT, a well-known osmolyte. Alteration of the salinity in filtrate cultures affected the relative proportion of [C]choline degraded or converted to [C]GBT, without substantially affecting the total metabolism of choline. Increasing the salinity from 14 to 25 or 35 ppt caused more [C]GBT to be produced from choline but less CO(2) to be produced than in the controls. Lowering the salinity to 7 ppt decreased [C]GBT production and increased CO(2) production slightly. Intracellular accumulations of [C]GBT in the salt-stressed cultures were osmotically significant (34 mM). Choline may be used as an energy substrate by estuarine bacteria and may also serve as a precursor of the osmoprotectant GBT, particularly as bacteria are mixed into higher-salinity waters.

Aminoacyl thioester chemistry of class II aminoacyl-tRNA synthetases.

Lysyl-tRNA synthetase, a class II enzyme, edits homocysteine by converting it into homocysteine thiolactone. In a similar reaction, the enzyme converts homoserine into homoserine lactone. Other class II enzymes, aspartyl-tRNA synthetase and seryl-tRNA synthetase, do not edit any of the amino acids tested. However, all three class II aminoacyl-tRNA synthetases catalyze AMP- and pyrophosphate-independent deacylation of cognate aminoacyl-tRNA in the presence of thiols, mimicking editing of homocysteine. Thiol-dependent deacylations exhibit saturation kinetics with respect to concentration of thiols, suggesting the presence of a thiol binding site on each enzyme. 3-Mercaptopropionate-, N-acetyl-L-cysteine-, and dithiothreitol-dependent deacylations of aminoacyl-tRNA yield corresponding aminoacyl thioesters. Cysteine-dependent enzymatic deacylations of aminoacyl-tRNA by these class II enzymes yield dipeptides, N-(aminoacyl)cysteine. The formation of N-(aminoacyl)cysteine involves thioester intermediates S-(aminoacyl)-L-cysteine, which are not observed because of the facile transacylation of the aminoacyl residue from the sulfur to the alpha-amino group of cysteine to form a stable peptide bond. These data indicate that class II aminoacyl-tRNA synthetases possess unique thiol-binding subsites within their active sites. That the thiol-binding subsite exists also in AspRS and SerRS, which do not need editing function, suggests that these class II enzymes possess vestigial editing functions.

Bihaptens with 5- and 6-methyl-substituted alkylcatechols and methylene lactone functional groups: tools for hapten (allergen or tolerogen)-protein interaction studies.

In order to investigate hapten-protein interactions in vivo, potential skin sensitizers 1-3, with two different haptenic ends, (a) a catechol, a 5-methylcatechol, and a 6-methylcatechol respectively, and (b) an alpha-methylene-gamma-butyrolactone moiety, separated by a straight 10-carbon chain, have been prepared and used to sensitize guinea pigs. Related "monohapten lactones" 19-21, containing an electrophilic alpha-methylene-gamma-butyrolactone moiety connected to the protected catechol structures, and "monohapten catechols" 25-27, containing a reduced alpha-methyl-gamma-butyrolactone linked to the catechol, also have been prepared. Bihaptens 1 and 2 which were found to have very close biological activities appear to react with proteins through the catechol ring, indicating a low importance of the C-6 position in the binding of catechols with amino acids. In contrast, bihapten 3 was found to react through both the catechol and lactone ring. The influence of amino and thiol binding sites on the biological activity (allergy or tolerance) as well as the influence of the sensitizing method are discussed.

Spatial relationship between SH 1 and the actin binding site on myosin subfragment-1 surface

To examine the spatial relationship between SH 1 thiol and actin binding site on subfragment-1 surface, we studied the interaction with actin of subfragment-1 whose SH 1 was labeled with an iodoacetate derivative of biotin and covered with avidin. Subfragment-1-avidin complex bound F-actin and its Mg 2+ ATPase activity was activated by actin. Considering the size and the location of biotin binding site on avidin, our results suggest that SH 1 is separated from the actin binding site on subfragment-1 surface by at least 17–20 Å.

Studies on the Mechanism of Fatty Acid Synthesis: XXIII. THE ACYL BINDING SITES OF THE PIGEON LIVER FATTY ACID SYNTHETASE

Abstract The covalent binding of acetyl, malonyl, and hexanoyl groups to the pigeon liver fatty acid synthetase has been investigated. Performic acid oxidation of the acyl synthetases has revealed that the synthetase possesses two types of acyl binding sites, these being thiol and nonthiol in nature. From studies on radioactive peptic peptides obtained from 14C-acetyl synthetase and 14C-malonyl synthetase, it has been established that there are two thiol binding sites. One of these thiol sites is the sulfhydryl group of the 4'-phosphopantetheine moiety of the synthetase. Both acetyl and malonyl groups bind to this sulfhydryl group. The second thiol binding site is the sulfhydryl group of a cysteine residue. This sulfhydryl binds acetyl groups but not malonyl groups. The nonthiol binding site or sites has been tentatively identified as being the hydroxyl group of a serine residue. Prior treatment of the synthetase with high concentrations of N-ethylmaleimide inhibits all binding of acetyl and malonyl groups, while prior treatment with low concentrations of N-ethylmaleimide results in inhibition of thiol binding but has little effect on the nonthiol binding of these acyl groups. Prior treatment of the synthetase with iodoacetamide has no effect on the binding of acetyl and malonyl groups to the 4'-phosphopantetheine sulfhydryl but inhibits acetyl binding to the sulfhydryl of the cysteine residue. From these studies the order of acetyl binding to the synthetase appears to be binding first at the nonthiol site with successive transfers to the 4'-phosphopantetheine sulfhydryl and the cysteine sulfhydryl. The binding of malonyl groups follows the same sequence, except that malonyl groups are not transferred from the 4'-phosphopantetheine sulfhydryl to the cysteine sulfhydryl.