After successful isolation of the most interfacially active subfraction of asphaltenes (IAA) reported in part one of this series of publications, comprehensive chemical analyses including ES-MS, elemental analysis, FTIR and NMR were used to determine how the molecular fingerprint features of IAA are different from those of the remaining asphaltenes (RA).Compared with RA, the IAA molecules were shown to have higher molecular weight and higher contents of heteroatoms (e.g., three times higher oxygen content). The analysis on the elemental content and FTIR spectroscopy suggested that IAA contained a higher content of high polarity sulfoxide groups which were not present in the RA. The results of ES-MS, NMR, FTIR and elemental analysis were used to construct average molecular representations of IAA and RA molecules. These structures were used in molecular dynamic (MD) simulation to study interfacial and aggregation behaviors of the proposed representative molecules. MD simulation study showed little affinity of representative RA molecules to the oil/water interface while the representative IAA molecules had a much higher interfacial activity, which corresponds to the extraction method. The aggregation of IAA molecules in the bulk oil phase and their adsorption at oil/water interface were not directly related to the ring system but rather to the associations between or including sulfoxide groups. The IAA molecules self-assembled in solvent, forming supramolecular structures and a porous network at the oil/water interface as suggested in our previous work. The results obtained in this study provide a better understanding of the role of asphaltenes in stabilizing petroleum emulsions.
The application of the direct electrospray ionization mass spectrometry (ESI-MS) assay to quantify interactions between bovine β-lactoglobulin (Lg) and a series of fatty acids (FA), CH 3 (CH 2 ) x COOH, where x=6 (caprylic acid, CpA), 8 (capric acid, CA), 10 (lauric acid, LA), 12 (myristic acid, MA), 14 (palmitic acid, PA) and 16 (stearic acid, SA), is described. Control ESI-MS binding measurements performed on the Lg-PA interaction revealed that both the protonated and deprotonated gas phase ions of the (Lg + PA) complex are prone to dissociate in the ion source, which leads to artificially small association constants (K a ). The addition of imidazole, a stabilizing solution additive, at high concentration (10 mM) increased the relative abundance of (Lg + PA) complex measured by ESI-MS in both positive and negative ion modes. The K a value measured in negative ion mode and using sampling conditions that minimize insource dissociation is in good agreement with a value determined using a competitive fluorescence assay. The K a values measured by ESI-MS for the Lg interactions with MA and SA are also consistent with values expected based on the fluorescence measurements. However, the K a values measured using optimal sampling conditions in positive ion mode are significantly lower than those measured in negative ion mode for all of the FAs investigated. It is concluded that the protonated gaseous ions of the (Lg + FA) complexes are kinetically less stable than the deprotonated ions. In-source dissociation was significant for the complexes of Lg with the shorter FAs (CpA, CA, and LA) in both modes and, in the case of CpA, no binding could be detected by ESI-MS. The affinities of Lg for CpA, CA, and LA determined using the reference ligand ESI-MS assay, a method for quantifying labile protein-ligand complexes that are prone to in-source dissociation, were found to be in good agreement with reported values.
The use of gas phase additives to stabilize noncovalent protein complexes in electrospray ionization mass spectrometry (ES-MS) is demonstrated for two protein-ligand interactions, an enzyme-small molecule inhibitor complex, and a protein-disaccharide complex. It is shown that the introduction of gas phase imidazole into the ES ion source effectively protects gas phase protein-ligand complexes against in-source dissociation. The stabilizing effect of imidazole vapor is comparable to that observed upon addition of imidazole to the ES solution. The introduction of sulfur hexafluoride, at high partial pressure, into the source region also effectively suppresses in-source dissociation of protein complexes. It is proposed that evaporative cooling is the primary mechanism responsible for the stabilizing effects observed for the gas phase additives.
ABSTRACT:Carboxylesterases hydrolyze numerous endogenous and foreign compounds with diverse structures. Humans and rodents express multiple forms of carboxylesterases, which share a high degree of sequence identity (ϳ70%). Alignment analyses locate in carboxylesterases several functional subsites such the catalytic triad as seen in acetylcholinesterase. The aim of this study was to determine among human and rodent carboxylesterases the immunorelatedness, overlapping substrate specificity, differential sensitivity to serine enzyme inhibitors, tissue distribution, and tumor-related expression. Six antibodies against whole carboxylesterases or synthetic peptides were tested for their reactivity toward 11 human or rodent recombinant carboxylesterases. The antibodies against whole proteins generally exhibited a broader cross-reactivity than the anti-peptide antibodies. All carboxylesterases hydrolyzed para-nitrophenylacetate and para-nitrophenylbutyrate. However, the relative activity varied markedly from enzyme to enzyme (>20-fold), and some carboxylesterases showed a clear substrate preference. Carboxylesterases with the same functional subsites had a similar profile on substrate specificity and sensitivity toward phenylmethylsulfonyl fluoride (PMSF) and paraoxon, suggesting that these subsites play determinant roles in the recognition of substrates and inhibitors. Among three human carboxylesterases, HCE-1 hydrolyzed both substrates to a similar extent, whereas HCE-2 and HCE-3 showed an opposite substrate preference. All three enzymes were inhibited by PMSF and paraoxon, but they showed a marked difference in relative sensitivities. Based on immunoblotting analyses, HCE-1 was present in all tissues examined, whereas HCE-2 and HCE-3 were expressed in a tissue-restricted pattern. Colon carcinomas expressed slightly higher levels of HCE-1 and HCE-2 than the adjacent normal tissues, whereas the opposite was true with HCE-3.
A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying proteinligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (L ref ), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein-ligand complex in the gas phase, is added to the solution. The fraction of P bound to L ref , which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. The assay is based on the direct detection and quantification of the abundance (Ab) of ligand-bound and unbound protein ions in the gas phase, e.g., PL nϩ and P nϩ , respectively. A key assumption is that the measured abundance ratio (R) is equivalent to the equilibrium concentration ratio of ligand-bound and free protein in solution, eq 1: The direct ES-MS assay has been used to measure affinities for a range of protein-ligand complexes, including antibody-antigen, lectin-carbohydrate, enzymesubstrate/inhibitor complexes and, in many instances, the K a values agree well with constants obtained by other analytical methods, including isothermal titration calorimetry (ITC), surface plasmon resonance, and frontal affinity chromatography MS [4 -10]. However, there have also been reports of protein-ligand complexes that could not be detected by ES-MS or, if detected, the relative abundance of ligand-bound and unbound protein ions did not match the distribution expected in solution, with less binding observed in the gas phase [11][12][13][14]. These anomalous results are often due the occurrence of in-source dissociation, whereby the gaseous complexes undergo partial or complete dissociation during ES-MS analysis. If the gas-phase PL ions are kinetically labile and undergo dissociation during analysis, the magnitude of the measured R value and, correspondingly, the K a value will be artificially low. In the extreme case, where no PL ions survive, in-source dissociation will result in a false negative. Recently, it was shown that solution or gas-phase additives can, in some instances, protect complexes from in-source dissociation [12,15]. However, this approach does have its limitations and the detection of very labile gas-phase complexes, which rapidly dissociate at ambient temperature, by ES-MS remains problematic. Here, we describe an indirect ES-MS approach to quantify protein-ligand interactions that are highly Address reprint requests to Dr.
The first quantitative comparison of the thermal dissociation rate constants measured for protein-ligand complexes in their hydrated and dehydrated states is described. Rate constants, measured using surface plasmon resonance spectroscopy, are reported for the dissociation of the 1:1 complexes of bovine β-lactoglobulin (Lg) with the fatty acids (FA), palmitic acid (PA), and stearic acid (SA), in aqueous solution at pH 8 and at temperatures ranging from 5 to 45 °C. The rate constants are compared to values determined from time-resolved blackbody infrared radiative dissociation measurements for the gaseous deprotonated (Lg+FA)(n-) ions, where n = 6 and 7, at temperatures ranging from 25 to 66 °C. Notably, the hydrated (Lg+PA) complex is kinetically less stable than the corresponding gas phase (Lg+PA)(n-) ions at all temperatures investigated; the hydrated (Lg+SA) complex is kinetically less stable than the gaseous (Lg+SA)(n-) ions at temperatures <45 °C. The greater kinetic stability of the gaseous (Lg+FA)(n-) ions originates from significantly larger, by 11-12 kcal mol(-1), E(a) values. It is proposed that the differences in the dissociation E(a) values measured in solution and the gas phase reflect the differential hydration of the reactant and the dissociative transition state.
Cancer can invade or spread to almost all parts of the body. The increasing morbidity and high mortality of cancer create a great demand for the development of novel anticancer drugs. Coumarin derivatives are ubiquitous in nature and can readily interact with diverse enzymes and receptors in cancer cells via weak bond interactions; hence, coumarin is a highly privileged pharmacophore for the development of novel anticancer agents. This review will focus on the recent development of coumarin hybrids as potential anticancer agents covering articles published from 2019 to 2020.
Environmental problems in China are intensifying and it is vital to evaluate the environmental knowledge, attitudes and behaviors of the generation poised to inherit their management. This study examines a survey of environmental awareness among Chinese students (aged between 16 and 20 years). Considering the contrasting levels of regional economic development and environmental problems in the eastern/coastal and western/inland regions of China, we examine how environmental differences affect university students' environmental awareness. Data were analyzed statistically using nonparametric tests to compare a population of urban residents from a developed region against a similar population of urbanites from a less-developed region. Students in the samples possessed rather low levels of environmental knowledge, but had positive environmental attitudes and were willing to commit to environment-friendly behaviors. Students growing up in developed versus less-developed settings had significantly different levels of general environmental awareness despite their shared exposure to institutionalized environmental education.
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