Home About us Contact
|
Home About us Contact | ||
|
|
||
Schiff Base Formation (schiff + base_formation)
Selected AbstractsIdentification and quantification of in vitro adduct formation between protein reactive xenobiotics and a lysine-containing model peptideENVIRONMENTAL TOXICOLOGY, Issue 1 2003Peter Reichardt Abstract Formation of in vitro adducts between different classes of xenobiotics and the lysine-containing peptide Lys-Tyr was monitored by high-performance liquid chromatography and electrospray ionization mass spectrometry. The molecular structures of the main resulting products could be sensitively analyzed by mass spectrometry (flow injection analysis), enabling the detection of characteristic binding formations. Aldehydes such as formaldehyde, acetaldehyde, and benzaldehyde were shown to form stable linkages to lysine amino groups via Schiff bases. Other electrophilic substances (e.g., toluene-2,4-diisocyanate, 2,4-dinitro-1-fluorobenzene, 2,4,6-trinitrobenzene sulfonic acid, dansyl chloride, and phthalic acid anhydride) also formed covalent adducts with lysine residues. The reactivity of the compounds was quantified by measuring the amount of peptide that remained unchanged after incubation for a certain period with the xenobiotic. Although reactivity levels within this group of aldehydes varied only to a small extent, as would be expected, extreme differences were seen among the structurally heterogeneous group of nonaldehyde xenobiotics. These results support the hypothesis that simple chemical reactions may lead to the adduction of nucleophilic macromolecules such as peptides or proteins. Such reactions, in particular, Schiff base formation of aldehydes, have previously been shown to be capable of specifically interfering with costimulatory signaling on T cells. Our results suggest that electrophilic xenobiotics of other classes may also inherit the capacity to exert similar effects. Forming covalent linkage to peptides may represent a possible molecular mechanism of electrophilic xenobiotics in vivo, yielding immunotoxic effects. The model utilized in this study is appropriate for monitoring the adduction of xenobiotics to basic peptides and for analyzing the resulting molecular structures. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 29,36, 2003. [source] Influence of carbohydrates on stability of papain in aqueous tetrahydrofuran mixtureJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 1 2009András Szabó Abstract BACKGROUND: The use of enzymes in organic solvents has extended the scale of their practical applications. Papain has been widely used in chemical syntheses because of its broad substrate specificity. The aim of the present study was to improve the stability of papain in aqueous tetrahydrofuran (THF) by using different saccharides. The effects of these carbohydrates on the structure of papain were followed by means of circular dichroism (CD) and fluorescence spectroscopic measurements. RESULTS: In contrast with most organic solvents, 60% (i.e. 600 mL L,1) THF practically inactivated the enzyme within 30 min. Sugars protected papain from THF-induced inactivation in the sequence D -ribose > D -fructose > D -glucose > D -saccharose > D -raffinose. Ribose at 1.6 mol L,1 proved the most effective stabiliser: in 60% THF in the presence of ribose, papain preserved about 55% of its initial activity after 2 h. Fluorescence and near-UV CD spectroscopic measurements revealed local changes in the papain conformation. With decrease in the free amino group content of the enzyme, protein-carbohydrate interactions (Schiff base formation) were detected. CONCLUSION: These results demonstrate that the catalytic activity and stability of papain may be increased in aqueous THF by using different carbohydrates, when a more compact structure of the enzyme is formed. Copyright © 2008 Society of Chemical Industry [source] QSARs for the skin sensitization potential of aldehydes and related compoundsMOLECULAR INFORMATICS, Issue 2 2003Grace Patlewicz Abstract Although not all aldehydes are skin sensitizers, many of them, covering a diverse range of structures, show varying degrees of sensitization potential. Based on consideration of their reaction chemistry, it is possible to identify structural features associated with sensitization potential or the lack of it. Many aldehydes, including several fragrance allergens, can sensitize by Schiff base formation. A QSAR based on reactivity and hydrophobicity parameters has been developed for these aldehydes. The QSAR can be extended to include 1,2-diketones, which can also react by Schiff base formation. The findings indicate that for skin sensitization, as for several other areas of toxicology, chemicals are better classified in terms of their reaction chemistry rather than in terms of their functional groups, i.e., based on mechanisms of action as opposed to chemical class. [source] Rhodopsin Regeneration is Accelerated via Noncovalent 11- cis Retinal,Opsin Complex,A Role of Retinal Binding Pocket of Opsin,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Hiroyuki Matsumoto The regeneration of bovine rhodopsin from its apoprotein opsin and the prosthetic group 11- cis retinal involves the formation of a retinylidene Schiff base with the , -amino group of the active lysine residue of opsin. The pH dependence of a Schiff base formation in solution follows a typical bell-shaped profile because of the pH dependence of the formation and the following dehydration of a 1-aminoethanol intermediate. Unexpectedly, however, we find that the formation of rhodopsin from 11- cis retinal and opsin does not depend on pH over a wide pH range. These results are interpreted by the Matsumoto and Yoshizawa (Nature258 [1975] 523) model of rhodopsin regeneration in which the 11- cis retinal chromophore binds first to opsin through the , -ionone ring, followed by the slow formation of the retinylidene Schiff base in a restricted space. We find the second-order rate constant of the rhodopsin formation is 6100 ± 300 mol,1 s,1 at 25°C over the pH range 5,10. The second-order rate constant is much greater than that of a model Schiff base in solution by a factor of more than 107. A previous report by Pajares and Rando (J Biol Chem264 [1989] 6804) suggests that the lysyl ,-NH2 group of opsin is protonated when the , -ionone ring binding site is unoccupied. The acceleration of the Schiff base formation in rhodopsin is explained by stabilization of the deprotonated form of the lysyl ,-NH2 group which might be induced when the , -ionone ring binding site is occupied through the noncovalent binding of 11- cis retinal to opsin at the initial stage of rhodopsin regeneration, followed by the proximity and orientation effect rendered by the formation of noncovalent 11- cis retinal,opsin complex. [source] Real-time reaction monitoring by probe electrospray ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 11 2010Zhan Yu Probe electrospray ionization (PESI) is a modified version of the electrospray ionization (ESI), where the capillary for sampling and spraying is replaced by a solid needle. High tolerance to salts and direct ambient sampling are major advantages of PESI compared with conventional ESI. In this study, PESI-MS was used to monitor some biological and chemical reactions in real-time, such as acid-induced protein denaturation, hydrogen/deuterium exchange (HDX) of peptides, and Schiff base formation. By using PESI-MS, time-resolved mass spectra and ion chromatograms can be obtained reproducibly. Real-time PESI-MS monitoring can give direct and detailed information on each chemical species taking part in reactions, and this is valuable for a better understanding of the whole reaction process and for the optimization of reaction parameters. PESI-MS can be considered as a potential tool for real-time reaction monitoring due to its simplicity in instrumental setup, direct sampling with minimum sample preparation and low sample consumption. Copyright © 2010 John Wiley & Sons, Ltd. [source] Structural, Functional and Calorimetric Investigation of MosA, a Dihydrodipicolinate Synthase from Sinorhizobium meliloti L5,30, does not Support Involvement in Rhizopine BiosynthesisCHEMBIOCHEM, Issue 10 2008Christopher P. Phenix Dr. Abstract MosA is an enzyme from Sinorhizobium meliloti L5,30, a beneficial soil bacterium that forms a symbiotic relationship with leguminous plants. MosA was proposed to catalyze the conversion of scyllo -inosamine to 3- O -methyl- scyllo -inosamine (compounds known as rhizopines), despite the MosA sequence showing a strong resemblance to dihydrodipicolinate synthase (DHDPS) sequences rather than to methyltransferases. Our laboratory has already shown that MosA is an efficient catalyst of the DHDPS reaction. Here we report the structure of MosA, solved to 1.95 Ĺ resolution, which resembles previously reported DHDPS structures. In this structure Lys161 forms a Schiff base adduct with pyruvate, consistent with the DHDPS mechanism. We have synthesized both known rhizopines and investigated their ability to interact with MosA in the presence and absence of methyl donors. No MosA-catalyzed methyltransferase activity is observed in the presence of scyllo -inosamine and S -adenosylmethionine (SAM). 2-Oxobutyrate can form a Schiff base with MosA, acting as a competitive inhibitor of MosA-catalyzed dihydrodipicolinate synthesis. It can be trapped on the enzyme by reaction with sodium borohydride, but does not act as a methyl donor. The presence of rhizopines does not affect the kinetics of dihydrodipicolinate synthesis. Isothermal titration calorimetry (ITC) shows no apparent interaction of MosA with rhizopines and SAM. Similar experiments with pyruvate as titrant demonstrate that the reversible Schiff base formation is largely entropically driven. This is the first use of ITC to study Schiff base formation between an enzyme and its substrate. [source] Electrochemical Characterization of In Situ Functionalized Gold Cysteamine Self-Assembled Monolayer with 4-Formylphenylboronic Acid for Detection of DopamineELECTROANALYSIS, Issue 5 2008Karimi Shervedani Abstract Functionalization of gold cysteamine (AuCA) self-assembled monolayer with 4-formylphenylboronic acid (BA) via Schiff's base formation, through in situ method to fabricate Au-CA-BA electrode is presented and described. The fabricated electrode was used as a novel sensor for accumulation and determination of dopamine (DA). The accumulation of DA as a diol on the topside of Au-CA-BA as a Lewis acid, was performed via esterification (AuCABADA), and followed for determination of DA. Functionalization, characterization, and determination steps were probed by electrochemical methods like cyclic voltammetry and electrochemical impedance spectroscopy. The data will be presented and discussed from which a new sensor for DA is introduced. [source] Cassette mutagenesis of lysine 130 of human glutamate dehydrogenaseFEBS JOURNAL, Issue 11 2001An essential residue in catalysis It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH). There are, however, conflicting views as to whether the lysine residues are involved in Schiff's base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2-oxoglutarate during catalysis, or some other function. We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5,-phosphate (PLP). For these studies, a 1557-bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild-type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues. Despite an approximately 400-fold decrease in the respective apparent Vmax of the Lys130 mutant enzymes, apparent Km values for NADH and 2-oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate. Unlike the wild-type GDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding. The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH. [source] | ||||||||||